Indexing & AbstractingRobust HPLC–MS/MS method for levofloxacin and ciprofloxacin determination in...

Indexing & Abstracting Of

Journal of Pharmaceutical and Biomedical Analysis

(JPBA)

YEAR 2017

Published by

LIBRARY & INFORMATION CENTRE

INDIAN PHARMACOPOEIA COMMISSION MINISTRY OF HEALTH & FAMILY WELFARE

GOVERNMENT OF INDIA

GHAZIABAD (UP)

i

INDEX

S.No. Contents Page No.

Volume 132, January 2017

1. From chemical consistency to effective consistency in precise quality discrimination of Sophora

flower-bud and Sophora flower: Discovering efficacy-associated markers by fingerprint-activity

relationship modelling 1

2. Impact of mono- and poly-ester fractions on polysorbate quantitation using mixed-mode HPLC-

CAD/ELSD and the fluorescence micelle assay 2

3. Quality assessment of marketed chamomile tea products by a validated HPTLC method

combined with multivariate analysis 3

4. Simultaneous quantification and identification of flavonoids, lignans, coumarin and amides in

leaves of Zanthoxylum armatum using UPLC-DAD-ESI-QTOF–MS/MS 3

5. Combination of HPLC–MS and QAMS as a new analytical approach for determination of

saponins in ginseng containing products 4

6. Simultaneous determination of phenolic acids and flavonoids in Chenopodium formosanum

Koidz (djulis) by HPLC-DAD-ESI–MS/MS 4

7. Design of a strong cation exchange methodology for the evaluation of charge heterogeneity in

glatiramer acetate 5

8. Development and characterization of the voriconazole loaded lipid-based nanoparticles 5

9. Selective functional activity measurement of a PEGylated protein with a modification-dependent

activity assay 6

10. Investigation of the effect of mobile phase composition on selectivity using a solvent-triangle

based approach in achiral SFC 6

11. NQO1 and CYP450 reductase decrease the systemic exposure of rifampicin-quinone and

mediate its redox cycle in rats 7

12. Study the influence of licorice and pomegranate drinks on nicotine metabolism in human urine

by LC-orbitrap MS 7

13. NMR-based metabonomics and correlation analysis reveal potential biomarkers associated with

chronic atrophic gastritis 8

14. Metabolomic profiling of doxycycline treatment in chronic obstructive pulmonary disease 8

15. Analysis of fenretinide and its metabolites in human plasma by liquid chromatography–tandem

mass spectrometry and its application to clinical pharmacokinetics 9

16. Poor and enantioselective bioavailability of naftopidil enantiomers is due to extensive and

stereoselective metabolism in rat liver 9

17. Robust HPLC–MS/MS method for levofloxacin and ciprofloxacin determination in human

prostate tissue 10

18. Pharmacokinetics, excretion of 8-cetylberberine and its main metabolites in rat urine 10

19. An UPLC–MS/MS method for the quantitation of alectinib in rat plasma 11

ii

20. Development and validation of a rapid and sensitive UPLC–MS/MS method for quantification of

kukoamine B in human plasma: Application to a clinical pharmacokinetic study 11

21. Quantitative determination of xanthorrhizol in rat plasma by HPLC–MS/MS and its application

to a pharmacokinetic study 12

22. Development and validation of a stability-indicating HPLC-UV method for the determination of

Thiocolchicoside and its degradation products 12

23. Dried blood spot analysis of gabapentin as a valid alternative for serum: a bridging study 13

24. SPR-based assays enable the full functional analysis of bispecific molecules 13

25. Angiotensin converting enzyme immobilized on magnetic beads as a tool for ligand fishing 14

26. MALDI-MS analysis and theoretical evaluation of olanzapine as a UV laser desorption

ionization (LDI) matrix 14

27. A simple and rapid UHPLC–MS/MS method for the quantitation of the dual aurora kinase A/B

inhibitor SCH-1473759 in murine plasma 15

28. Immobilized aptamer paper spray ionization source for ion mobility spectrometry 15

29. Urine metabolomics of high-fat diet induced obesity using UHPLC-Q-TOF-MS 16


30. Psidium guajava L. leaves as source of proanthocyanidins: Optimization of the extraction

method by RSM and study of the degree of polymerization by NP-HPLC-FLD-ESI-MS 16

31. Identification, synthesis and structural characterization of process related and degradation

impurities of acrivastine and validation of HPLC method 17

32. Preparation of a monoclonal antibody against amantadine and rimantadine and development of

an indirect competitive enzyme-linked immunosorbent assay for detecting the same in chicken

muscle and liver 17

33. Determination of pharmaceutical residues in wastewater using high performance liquid

chromatography coupled to quadrupole-Orbitrap mass spectrometry 18

34. High-capacity hollow porous dummy molecular imprinted polymers using ionic liquid as

functional monomer for selective recognition of salicylic acid 18

35. Demonstration of the IgG antibody repertoire against the bacteria Escherichia coli in Chinese

intravenous immunoglobulins 19

36. Metabolites profiling reveals for antimicrobial compositional differences and action mechanism

in the toothbrushing stick ―miswak‖ Salvadora persica 19

37. The investigation of anti-inflammatory activity of Yi Guanjian decoction by serum

metabonomics approach 20

38. Quantitative profiling of 4'-geranyloxyferulic acid and its conjugate with l-nitroarginine methyl

ester in mononuclear cells by high-performance liquid chromatography with fluorescence

detection 20

39. A serum nuclear magnetic resonance-based metabolomic signature of antiphospholipid

syndrome 21

40. Development of a multi-matrix LC–MS/MS method for urea quantitation and its application in

human respiratory disease studies 21

iii

41. Determination of five potential genotoxic impurities in dalfampridine using liquid

chromatography 22

42. Isolation and structural characterization of novel photolytic degradation impurities of

Deflazacort using Q-TOF, 2D-NMR and FTIR 22

Volume 134, February 2017

43. LC–ESI–MS/MS evaluation of forced degradation behaviour of silodosin: In vitro anti cancer

activity evaluation of silodosin and major degradation products 23

44. Quality by Design in the development of hydrophilic interaction liquid chromatography method

with gradient elution for the analysis of olanzapine 23

45. Lipophilicity estimation and characterization of selected steroid derivatives of biomedical

importance applying RP HPLC 24

46. Comparison of the chemical consituents and immunomodulatory activity of ophiopogonis radix

from two different producing areas 24

47. Physicochemical analysis in the evaluation of reconstituted dry emulsion tablets 25

48. Cleaning verification: Exploring the effect of the cleanliness of stainless steel surface on sample

recovery 25

49. Raman spectroscopy and capillary zone electrophoresis for the analysis of degradation processes

in commercial effervescent tablets containing acetylsalicylic acid and ascorbic acid 26

50. RP-HPLC determination of dissociation constant using solely aqueous mobile phase 26

51. Quantitative determination of salbutamol sulfate impurities using achiral supercritical fluid

chromatography 27

52. Hydrogen/deuterium exchange, a unique and effective method for MS fragmentation behavior

elucidation of ginkgolides and its application to systematic research in Ginkgo biloba 27

53. Identification and characterization of a new dapoxetine impurity by NMR: Transformation of N-

oxide by Cope elimination 28

54. A new approach to the rapid separation of isomeric compounds in a Silybum marianum extract

using UHPLC core-shell column with F5 stationary phase 28

55. A comparison report of three advanced methods for drug-cyclodextrin interaction measure-

ments 29

56. Portable near-infrared instruments: Application for quality control of polymorphs in

pharmaceutical raw materials and calibration transfer 29

57. Characterization and quantitation of the polyphenolic compounds detected in methanol extracts

of Pistacia atlantica Desf fruits from the Guelmim region of Morocco 30

58. Quantification of biologically active O-prenylated and unprenylated phenylpropanoids in dill

(Anethum graveolens), anise (Pimpinella anisum), and wild celery (Angelica archangelica) 30

59. Microfluidic-based G-quadruplex ligand displacement assay for alkaloid anticancer drug

screening 31

60. Chrysin cocrystals: Characterization and evaluation 31

iv

61. Stability behaviour of antiretroviral drugs and their combinations 5: Characterization of novel

degradation products of abacavir sulfate by mass and nuclear magnetic resonance spectro-

metry 32

62. Comparison of miRNA signature versus conventional biomarkers before and after off-pump

coronary artery bypas graft 32

63. Microfluidic device for label-free quantitation and distinction of bladder cancer cells from the

blood cells using micro machined silicon based electrical approach; suitable in urinalysis

assays 33

64. Simultaneous determination of anemoside B4, phellodendrine, berberine, palmatine, obakunone,

esculin, esculetin in rat plasma by UPLC–ESI–MS/MS and its application to a comparative

pharmacokinetic study in normal and ulcerative colitis rats 33

65. Simultaneous determination and pharmacokinetic study of four phenolic acids in rat plasma

using UFLC–MS/MS after intravenous administration of salvianolic acid for injection 34

66. A rapid and sensitive UHPLC–MS/MS method for quantification of 83b1 in plasma and its

application to bioavailability study in rats 34

67. Accurate quantitation of choline and ethanolamine plasmalogen molecular species in human

plasma by liquid chromatography–tandem mass spectrometry 35

68. The profiling of the metabolites of hirsutine in rat by ultra-high performance liquid

chromatography coupled with linear ion trap Orbitrap mass spectrometry: An improved strategy

for the systematic screening and identification of metabolites in multi-samples in vivo 35

69. Metabolic fate and detectability of the new psychoactive substances 2-(4-bromo-2,5-

dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25B-NBOMe) and 2-(4-chloro-

2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25C-NBOMe) in human and

rat urine by GC–MS, LC–MSn, and LC–HR–MS/MS approaches 36

70. Liquid chromatography-tandem mass spectrometric determination of propofol in rat serum and

hair at attogram level after derivatization with 3-bromomethyl-propyphenazone 36

71. Investigation of the metabolites of the HIF stabilizer FG-4592 (roxadustat) in five different in

vitro models and in a human doping control sample using high resolution mass spectro-

metry 37

72. A quantitative LC–MS/MS method for simultaneous determination of cocaine and its

metabolites in whole blood 37

73. Use of FTIR spectroscopy and PCA-LDC analysis to identify cancerous lesions within the

human colon 38

74. Antibody-free detection of infectious bacteria using quantum dots-based barcode assay 38

75. A simple and sensitive liquid chromatography–tandem mass spectrometry method for trans-ε-

viniferin quantification in mouse plasma and its application to a pharmacokinetic study in

mice 39

76. LC–MS/MS assay for the quantitation of the tyrosine kinase inhibitor neratinib in human

plasma 39

v

77. Comparative tissue distribution and excretion study of alkaloids from Herba Ephedrae-Radix

Aconiti Lateralis extracts in rats 40

78. Deep eutectic solvents as green media for extraction of flavonoid glycosides and aglycones from

Platycladi Cacumen 40

79. Testosterone and its dimers alter tRNA morphology 41

80. Development and validation of a simple and robust HPLC method with UV detection for

quantification of the hepatitis C virus inhibitor daclatasvir in human plasma 41

81. Evaluation of ion mobility spectrometry for the detection of mitragynine in kratom pro-

ducts 42

82. A single MCR-ALS model for drug analysis in different formulations: Application on diazepam

commercial preparations 42

83. Selective recognition of cis-trans-isomers of platinum drugs and the detection of triplex DNA

based on fluorescence reversible model of quantum dots 43

84. Drug-protein binding of Danhong injection and the potential influence of drug combination with

aspirin: Insight by ultrafiltration LC–MS and molecular modelling 43

85. The importance of vial composition in HPLC analysis: An unusual case of phosphorous

pseudorotation 44

86. Supercritical fluid chromatography for separation and preparation of tautomeric 7-epimeric spiro

oxindole alkaloids from Uncaria macrophylla 44


87. Renewal of an old European Pharmacopoeia method for Terazosin using modeling with mass

spectrometric peak tracking 45

88. A revisited structure for nitrosoprodenafil from NMR, mass spectrometry, X-ray and hydrolysis

data 45

89. The importance of system band broadening in modern size exclusion chromatography 46

90. Development of a multi-residue method for the determination of human and veterinary

pharmaceuticals and some of their metabolites in aqueous environmental matrices by SPE-

UHPLC–MS/MS 46

91. Development of new efficient method for isolation of phenolics from sea algae prior to their

rapid resolution liquid chromatographic–tandem mass spectrometric determination 47

92. Physico-chemical profiling of semisynthetic opioids 47

93. Interaction of anticancer drug clofarabine with human serum albumin and human α-1 acid

glycoprotein Spectroscopic and molecular docking approach 48

94. A novel method for the determination of chemical purity and assay of menaquinone-7

Comparison with the methods from the official USP monograph 48

95. Structure and pharmaceutical formulation development of a new long-acting recombinant human

insulin analog studied by NMR and MS 49

vi

96. Simultaneous HPLC assay for pretomanid (PA-824), moxifloxacin and pyrazinamide in an

inhaler formulation for drug-resistant tuberculosis 49

97. Levothyroxine sodium revisited: A wholistic structural elucidation approach of new impurities

via HPLC-HRMS/MS, on-line H/D exchange, NMR spectroscopy and chemical synthesis 50

98. Verification of the authenticity of drugs by means of NMR relaxometry—Viagra® as an

example 50

99. Metabolomics study of Populus type propolis 51

100. Hydrophilic interaction liquid chromatography method development and validation for the assay

of HEPES zwitterionic buffer 51

101. Simultaneous analysis of glucocorticosteroid fluticasone propionate and its metabolite

fluticasone propionate 17β-carboxylic acid in human plasma by UPLC–MS/MS at sub pg/mL

level 52

102. Fast Screening of Tissue Samples for Glycogen 52

103. Urinary metabolic profiling of cisplatin nephrotoxicity and nephroprotective effects of

Orthosiphon stamineus leaves elucidated by 1H NMR spectroscopy 53

104. Preparation, characterization and in vivo evaluation of a formulation of dantrolene sodium with

hydroxypropyl-β-cyclodextrin 54

105. Promotion of classic neutral bile acids synthesis pathway is responsible for cholesterol-lowing

effect of Si-miao-yong-an decoction: Application of LC–MS/MS method to determine 6 major

bile acids in rat liver and plasma 54

106. Analysis of amino acid and monoamine neurotransmitters and their metabolites in rat urine of

Alzheimer‘s disease using in situ ultrasound-assisted derivatization dispersive liquid-liquid

microextraction with UHPLC–MS/MS 55

107. Rapid determination of alkaloids in Macleaya cordata using ionic liquid extraction followed by

multiple reaction monitoring UPLC–MS/MS analysis 55

108. Volumetric absorptive microsampling (VAMS) as an alternative to conventional dried blood

spots in the quantification of miltefosine in dried blood samples 56

109. The integration of GC–MS and LC–MS to assay the metabolomics profiling in Panax ginseng

and Panax quinquefolius reveals a tissue- and species-specific connectivity of primary

metabolites and ginsenosides accumulation 56

110. Hierarchical identification of bioactive components in a medicinal herb by preparative high-

performance liquid chromatography and selective knock-out strategy 57

Volume 136, March 2017

111. Metabolomics: A potential way to know the role of vitamin D on multiple sclerosis 57

112. Impact of space environment on stability of medicines: Challenges and prospects 58

113. Evaluation of size-exclusion chromatography for the analysis of phosphorothioate

oligonucleotides 58

vii

114. Molecular insight into atypical instability behavior of fixed-dose combination containing

amlodipine besylate and losartan potassium 59

115. Qualification of HSQC methods for quantitative composition of heparin and low molecular

weight heparins 59

116. Selecting optimal columns for clarithromycin impurity analysis according to the quantitative

relationship of hydrophobic subtraction model 60

117. Efficacy of metformin in human single hair fibre by ATR-FTIR spectroscopy coupled with

statistical analysis 60

118. Primer design for SNP genotyping based on allele-specific amplification—Application to organ

transplantation pharmacogenomics 61

119. Development and validation of a liquid chromatography–mass spectrometric assay for

simultaneous determination of tacrolimus and 13-O-desmethyl tacrolimus in rat kidney

tissue 61

120. Towards interference free HPLC-SERS for the trace analysis of drug metabolites in biological

fluids 62

121. 1H NMR-based metabolomics study of liver damage induced by ginkgolic acid (15:1) in

mice 62

122. Dose-response characteristics of Clematis triterpenoid saponins and clematichinenoside AR in

rheumatoid arthritis rats by liquid chromatography/mass spectrometry-based serum and urine

metabolomics 63

123. Enhancing analysis throughput, sensitivity and specificity in LC/ESI–MS/MS assay of plasma

25-hydroxyvitamin D3 by derivatization with triplex 4-(4-dimethylaminophenyl)-1,2,4-

triazoline-3,5-dione (DAPTAD) isotopologues 63

124. A highly sensitive quantum dots-DNA nanobiosensor based on fluorescence resonance energy

transfer for rapid detection of nanomolar amounts of human papillomavirus 18 64

125. Development and validation of stability indicating HPLC methods for related substances and

assay analyses of amoxicillin and potassium clavulanate mixtures 64

126. Rapid analysis of drug dissolution by paper spray ionization mass spectrometry 65

127. Development and validation of a liquid chromatography-MS/MS method for simultaneous

quantification of tenofovir and efavirenz in biological tissues and fluids 65

128. Isolation, characterization using LC-ESI-QTOF, NMR and in vitro cytotoxicity assay of

niclosamide forced degradation products 66

129. Quantitative determinations using portable Raman spectroscopy 66

130. Screening active compounds from Corydalis yanhusuo by combining high expression VEGF

receptor HEK293 cell membrane chromatography with HPLC - ESI - IT - TOF - MSn

method 67

viii

Volume 137, April 2017

131. Powerful combination of analytical and chemometric methods for the photodegradation of 5-

Fluorouracil 67

132. Separation of antibody drug conjugate species by RPLC: A generic method development

approach 68

133. Time domain NMR as a new process monitoring method for characterization of pharmaceutical

hydrates 68

134. LC-method development for the quantification of neuromedin-like peptides Emphasis on column

choice and mobile phase composition 69

135. 2D-LC as an on-line desalting tool allowing peptide identification directly from MS unfriendly

HPLC methods 69

136. Quantification of EC-18, a synthetic monoacetyldiglyceride (1-palmitoyl-2-linoleoyl-3-acetyl-

rac-glycerol), in rat and mouse plasma by liquid-chromatography/tandem mass spect-

rometry 70

137. Compatibility study of a parenteral microdose polyethylene glycol formulation in medical

devices and identification of degradation impurity by 2D-LC/MS 70

138. Use of mixture design in drug-excipient compatibility determinations: Thymol nanoparticles

case study 71

139. Rapid analysis of Aurantii Fructus Immaturus (Zhishi) using paper spray ionization mass

spectrometry 71

140. Rapid determination of 30 bioactive constituents in XueBiJing injection using ultra high

performance liquid chromatography-high resolution hybrid quadrupole-orbitrap mass

spectrometry coupled with principal component analysis 72

141. Generic DART-MS platform for monitoring the on-demand continuous-flow production of

pharmaceuticals: Advancing the quantitative protocol for caffeates in microfluidic bioca-

talysis 72

142. Potential impurities of anxiolytic drug, clobazam: Identification, synthesis and characterization

using HPLC, LC-ESI/MSn and NMR 73

143. Simultaneous quantification of twenty-one ginsenosides and their three aglycones in rat plasma

by a developed UFLC–MS/MS assay: Application to a pharmacokinetic study of red

ginseng 73

144. Metabolic profiles of exudates from chronic leg ulcerations 74

145. Development and validation of a sensitive and fast UPLC–MS/MS method for simultaneous

determination of seven bioactive compounds in rat plasma after oral administration of Guizhi-

gancao decoction 74

146. Universal efavirenz determination in transport study, rat placenta perfusion and placenta lysate

by HPLC-UV 75

ix

147. Metabolite characterization of a novel sedative drug, remimazolam in human plasma and urine

using ultra high-performance liquid chromatography coupled with synapt high-definition mass

spectrometry 75

148. Chiral separation of new sulfonamide derivatives and evaluation of their enantioselective affinity

for human carbonic anhydrase II by microscale thermophoresis and surface plasmon

resonance 76

149. Ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS)

for determination of GHB, precursors and metabolites in different specimens: Application to

clinical and forensic cases 76

150. Impedimetric nanostructured genosensor for detection of schistosomiasis in cerebrospinal fluid

and serum samples 77

151. LC–MS/MS method for the simultaneous determination of Lys-MCC-DM1, MCC-DM1 and

DM1 as potential intracellular catabolite of the antibody-drug conjugate trastuzumab emtansine

(T-DM1) 77

152. Quantification of hydroxyurea in human plasma by HPLC–MS/MS and its application to

pharmacokinetics in patients with chronic myeloid leukaemia 78

153. Determination of rodenticides and related metabolites in rabbit liver and biological matrices by

liquid chromatography coupled to Orbitrap high resolution mass spectrometry 78

154. Comparative pharmacokinetic profiles of selected irreversible tyrosine kinase inhibitors,

neratinib and pelitinib, with apigenin in rat plasma by UPLC–MS/MS 79

155. LC–MS/MS determination of d-mannose in human serum as a potential cancer biomarker 79

156. Assessment of in vitro cardiotoxicity of extract fractions and diterpene alkaloids from Aconitum

leucostomum Worosch: A short communication 80

157. Development and validation of a liquid chromatography–tandem mass spectrometry method for

the assay of tafamidis in rat plasma: Application to a pharmacokinetic study in rats 80

158. Isolation and characterization of a novel dithio-carbodenafil analogue from a health suppl-

ement 81

159. Preparation and 68Ga-radiolabeling of porous zirconia nanoparticle platform for PET/CT-

imaging guided drug delivery 81

160. Determination of panduratin A in rat plasma by HPLC–MS/MS and its application to a

pharmacokinetic study 82

161. Simultaneous determination of creatinine and acetate by capillary electrophoresis with

contactless conductivity detector as a feasible approach for urinary tract infection

diagnosis 82

162. Analytical method development and validation for the analysis of verapamil hydrochloride and

its related substances by using ultra perfomance liquid chromatography 83

163. Evaluation of in silico pharmacokinetic properties and in vitro cytotoxic activity of selected

newly synthesized N-succinimide derivatives 83

x

164. Biopharmaceutical characterization of praziquantel cocrystals and cyclodextrin complexes

prepared by grinding 84

165. Development of an enzyme-linked immunosorbent assay for the detection of isomiroestrol, an

identical marker, in White Kwao Krua using a monoclonal antibody 84

Volume 138, May 2017

166. Fabrication of a novel hemin-based monolithic column and its application in separation of

protein from complex bio-matrix 85

167. High-throughput NIR-chemometric methods for chemical and pharmaceutical characterization of

sustained release tablets 85

168. Optimized multi-step NMR-crystallography approach for structural characterization of a stable

quercetin solvate 86

169. Study on the forced degradation behaviour of ledipasvir: Identification of major degradation

products using LC–QTOF–MS/MS and NMR 86

170. Raman scattering-based multiconformational analysis for probing the structural differences

between acetylcholine and acetylthiocholine 87

171. Characterization of metabolites in different kiwifruit varieties by NMR and fluorescence

spectroscopy 87

172. Comparison of bioactive components and pharmacological activities of ophiopogon japonicas

extracts from different geographical origins 88

173. Tiered analytics for purity assessment of macrocyclic peptides in drug discovery: Analytical

consideration and method development 88

174. Enantiomeric separation of the antiuremic drug colchicine by electrokinetic chromatography

Method development and quantitative analysis 89

175. Integrative hepatoprotective efficacy comparison of raw and vinegar-baked Radix Bupleuri using

nuclear magnetic resonance-based metabolomics 89

176. Systematic identification of flavonols, flavonol glycosides, triterpene and siraitic acid glycosides

from Siraitia grosvenorii using high-performance liquid chromatography/quadrupole-time-of-

flight mass spectrometry combined with a screening strategy 90

177. Rapid discrimination and determination of antibiotics drugs in plastic syringes using near

infrared spectroscopy with chemometric analysis: Application to amoxicillin and pen-

icillin 90

178. Identification of UV-absorbing extractables from rubber closures used in containers of injectable

powder and safety assessment of leachables in the drug 91

179. Solid state characterization of azelnidipine–oxalic acid co-crystal and co-amorphous complexes:

The effect of different azelnidipine polymorphs 91

180. Biorelevant physicochemical profiling of (E) - and (Z)-resveratrol determined from isomeric

mixtures 92

xi

181. An improved size exclusion-HPLC method for molecular size distribution analysis of

immunoglobulin G using sodium perchlorate in the eluent 92

182. Assessment of the structure of pegylated-recombinant protein therapeutics by the NMR

fingerprint assay 93

183. Prediction of the hydroxypropyl cellulose—poly (vinyl alcohol) ratio in aqueous solution

containing papaverine hydrochloride in terms of drug loaded electrospun fiber formation 93

184. Optimization of dispersive liquid-phase microextraction based on solidified floating organic drop

combined with high-performance liquid chromatography for the analysis of glucocorticoid

residues in food 94

185. A novel LCMSMS method for quantitative measurement of short-chain fatty acids in human

stool derivatized with 12C- and 13C-labelled aniline 94

186. Mixed-mode reversed phase/positively charged repulsion chromatography for intact protein

separation 95

187. Quantitative determination of five metabolites of aspirin by UHPLC–MS/MS coupled with

enzymatic reaction and its application to evaluate the effects of aspirin dosage on the metabolic

profile 95

188. HPLC–MS/MS method for the simultaneous quantification of desmethylmebeverine acid,

mebeverine acid and mebeverine alcohol in human plasma along with its application to a

pharmacokinetics study 96

189. The impact of ion-pairing reagents on the selectivity and sensitivity in the analysis of modified

oligonucleotides in serum samples by liquid chromatography coupled with tandem mass

spectrometry 96

190. A novel LC–MS/MS method for the simultaneous quantification of topiramate and its main

metabolites in human plasma 97

191. UPLC–MS/MS method for the simultaneous quantification of three new antiretroviral drugs,

dolutegravir, elvitegravir and rilpivirine, and other thirteen antiretroviral agents plus cobicistat

and ritonavir boosters in human plasma 97

192. Concentrations of antibodies against β-amyloid 40/42 monomer and oligomers in Chinese

intravenous immunoglobulins 98

193. Simultaneous quantification of estrogens, their precursors and conjugated metabolites in human

breast cancer cells by LC–HRMS without derivatization 98

194. Quantification and clinical application of carboplatin in plasma ultrafiltrate 99

195. An LC–MS/MS method for the determination of digitoxigenin in skin samples and its application

to skin permeation and metabolic stability studies 99

196. Stress-induced changes of neurosteroid profiles in rat brain and plasma under immobilized

condition 100

197. Identification, characterization and in silico ADMET prediction of Roflumilast degradation

products 100

xii

198. A rapid and robust UHPLC-DAD method for the quantification of amphotericin B in human

plasma 101

199. Quantitative analysis of mycosporine-like amino acids in marine algae by capillary

electrophoresis with diode-array detection 101

200. LC–MS/MS assay for the simultaneous quantitation of the ATM inhibitor AZ31 and the ATR

inhibitor AZD6738 in mouse plasma 102

201. The atypical excretion profile of meldonium: Comparison of urinary detection windows after

single- and multiple-dose application in healthy volunteers 102

202. Simultaneous quantitation of abiraterone, enzalutamide, N-desmethyl enzalutamide, and

bicalutamide in human plasma by LC–MS/MS 103

203. New enantioselective LC method development and validation for the assay of modafinil 103

204. Identification of a novel low-level impurity in fungicide pyraclostrobin by high-performance

liquid chromatography/tandem mass spectrometry 104

205. Paeonifiorin sulfonate as a characteristic marker for specifically inspecting Chinese patent

medicine Liu-Wei-Di-Huang-Wan contained sulfur-fumigated Moutan Cortex 104

206. Simple on-line pretreatment of column-switching coupled with ion chromatography for the

determination of lactic acid in lobaplatin 105

207. Discovery of discriminatory quality control markers for Chinese herbal medicines and related

processed products by combination of chromatographic analysis and chemometrics methods:

Radix Scutellariae as a case study 105

208. New and cost effective cell-based assay for Dialyzed Leukocyte Extract (DLE)-induced Jurkat

cells proliferation under azathioprine treatment 106

209. Spectroscopy analysis and molecular dynamics studies on the binding of penicillin V and

sulbactam to beta-lactamase II from Bacillus cereus 106

210. Variations in gut microbiota and fecal metabolic phenotype associated with depression by 16S

rRNA gene sequencing and LC/MS-based metabolomics 107

211. Chemical profiling of Fufang-Xialian-Capsule by UHPLC-Q-TOF-MS and its antioxidant

activity evaluated by in vitro method 107

Volume 139, May 2017

212. Development of an UPLC–MS/MS method for quantification of Avitinib (AC0010) and its five

metabolites in human cerebrospinal fluid: Application to a study of the blood-brain barrier

penetration rate of non-small cell lung cancer patients 108

213. The sodium salt of the enantiomers of ricobendazole: Preparation, solubility and chiroptical

properties 108

214. Application of design space optimization strategy to the development of LC methods for

simultaneous analysis of 18 antiretroviral medicines and 4 major excipients used in various

pharmaceutical formulations 109

xiii

215. Modeling and optimizing inhibitory activities of Nelumbinis folium extract on xanthine oxidase

using response surface methodology 109

216. Comparative preclinical evaluation of 68Ga-NODAGA and 68Ga-HBED-CC conjugated

procainamide in melanoma imaging 110

217. Development and validation of a HILIC-ELSD method for simultaneous analysis of non-

substituted and acetylated xylo-oligosaccharides 110

218. Enantioselective recognition of radezolid by cyclodextrin modified capillary electrokinetic

chromatography and electronic circular dichroism 111

219. Identification of leachables observed in the size exclusion chromatograms of a low concentration

product stored in prefilled syringes 111

220. Isolation and characterization of bioactive polyacetylenes Panax ginseng Meyer roots 112

221. Purity assessment of ginsenoside Rg1 using quantitative 1H nuclear magnetic resonance 112

222. Identification of forced degradation products of tedizolid phosphate by liquid

chromatography/electrospray ionization tandem mass spectrometry 113

223. Evaluation of automated Wes system as an analytical and characterization tool to support

monoclonal antibody drug product development 113

224. Pharmacokinetics and tissue distribution of 4, 5-dimethoxycanthin-6-one and its major

metabolites in rats 114

225. Development and validation of an ELISA method for the quantification of nivolumab in plasma

from non-small-cell lung cancer patients 114

226. Bioanalysis of Pseudomonas aeruginosa alkyl quinolone signalling molecules in infected mouse

tissue using LC–MS/MS; and its application to a pharmacodynamic evaluation of MvfR

inhibition 115

227. Metabolites identification of berberine in rats using ultra-high performance liquid

chromatography/quadrupole time-of-flight mass spectrometry 115

228. Quantitative chiral and achiral determination of ketamine and its metabolites by LC–MS/MS in

human serum, urine and fecal samples 116

229. Irinotecan binds to the internal cavity of beta-lactoglobulin: A multi-spectroscopic and

computational investigation 116

230. Determination of drugs in plasma samples by disposable pipette extraction with C18-BSA phase

and liquid chromatography–tandem mass spectrometry 117

231. Differentiation of protein secondary structure in clear and opaque human lenses: AFM – IR

studies 117

232. Introduction of a carbon paste electrode based on nickel carbide for investigation of interaction

between warfarin and vitamin K1 118

233. An integrated strategy using UPLC–QTOF-MSE and UPLC–QTOF-MRM (enhanced target) for

pharmacokinetics study of wine processed Schisandra Chinensis fructus in rats 118

xiv

234. Simultaneous determination of glipizide and its four hydroxylated metabolites in human urine

using LC–MS/MS and its application in urinary phenotype study 119

235. Plasma pharmacokinetics and bioavailability of verticillin A following different routes of

administration in mice using liquid chromatography tandem mass spectrometry 119

236. Robust quantitation of basic-protein higher-order aggregates using size-exclusion chromate-

graphy 120

237. A metabolomic approach shows sphingosine 1-phosphate and lysophospholipids as mediators of

the therapeutic effect of liver growth factor in emphysema 120

238. Hepatic and renal metabolism of genistein: An individual-based model to predict glucuronidation

behavior of genistein in different organs 121

239. Determination of free polysaccharide in Vi glycoconjugate vaccine against typhoid fever 121

240. HPLC–MS/MS method for quantification of paclitaxel from keratin containing samples 122

241. Development of an enzyme-linked immunosorbent assay for detection of CDCP1 shed from the

cell surface and present in colorectal cancer serum specimens 122

Volume 140, June 2017

242. Capillary blood collected on volumetric absorptive microsampling (VAMS) device for monit-

oring hydroxychloroquine in rheumatoid arthritis patients 123

243. Isolation and characterization of novel degradation products of Doxofylline using HPLC, FTIR,

LCMS and NMR 123

244. Enantiomers of triclabendazole sulfoxide: Analytical and semipreparative HPLC separation,

absolute configuration assignment, and transformation into sodium salt 124

245. Separation and characterization of unknown impurities and isomers in flomoxef sodium by LC-

IT-TOF MS and study of their negative-ion fragmentation regularities 124

246. Heparin and homogeneous model heparin oligosaccharides form distinct complexes with

protamine: Light scattering and zeta potential analysis 125

247. Quantitative analysis of binary polymorphs mixtures of fusidic acid by diffuse reflectance FTIR

spectroscopy, diffuse reflectance FT-NIR spectroscopy, Raman spectroscopy and multivariate

calibration 125

248. Mesoporous silica nanoparticles incorporated hybrid monolithic stationary phase immobilized

with pepsin for enantioseparation by capillary electrochromatography 126

249. Host-guest kinetic interactions between HP-β-cyclodextrin and drugs for prediction of bitter taste

masking 126

250. Crystal structures and physicochemical properties of amisulpride polymorphs 127

251. A UHPLC method for the rapid separation and quantification of phytosterols using tandem

UV/Charged aerosol detection – A comparison of both detection techniques 127

252. Analysis of chemical constituents in an herbal formula Jitong Ning Tablet 128

xv

253. Molecular recognition of pseudodistamine isomeric precursor trans-3(4)-aminopiperidin-4(3)-ols

by EI mass spectrometry 128

254. Development and validation of a general derivatization HPLC method for the trace analysis of

acyl chlorides in lipophilic drug substances 129

255. An isocratic hydrophilic interaction liquid chromatographic method for simultaneous determi-

nation of iodixanol and its related impurities in drug substance 129

256. Electrochemical and optical study of metallothionein interactions with prion proteins 130

257. Development of matrix effect-free MISPE-UHPLC–MS/MS method for determination of

lovastatin in Pu-erh tea, oyster mushroom, and red yeast rice 130

258. Facile preparation of fibrin coated open tubular column for characterization of monoclonal

antibody variants by capillary electrochromatography 131

259. Development and validation of a fast SFC method for the analysis of flavonoids in plant

extracts 131

260. A multi-matrix HILIC-MS/MS method for the quantitation of endogenous small molecule

neurological biomarker N-acetyl aspartic acid (NAA) 132

261. Chemotaxonomic studies of nine Paris species from China based on ultra-high performance

liquid chromatography tandem mass spectrometry and Fourier transform infrared spec-

troscopy 132

262. Rapid profiling and pharmacokinetic studies of major compounds in crude extract from

Polygonum multiflorum by UHPLC-Q-TOF-MS and UPLC–MS/MS 133

263. Metabolic profiling of nuciferine in rat urine, plasma, bile and feces after oral administration

using ultra-high performance liquid chromatography-diode array detection-quadrupole time-of-

flight mass spectrometry 133

264. Development and validation of liquid chromatography tandem mass spectrometry method

quantitative determination of polymyxin B1, polymyxin B2, polymyxin B3 and isoleucine-

polymyxin B1 in human plasma and its application in clinical studies 134

265. Human exposure to Bisphenol A and liver health status: Quantification of urinary and circulating

levels by LC–MS/MS 134

266. Determination of prodrug treosulfan and its biologically active monoepoxide in rat plasma, liver,

lungs, kidneys, muscle, and brain by HPLC–ESI–MS/MS method 135

267. Profiles of amino acids and biogenic amines in the plasma of Cri-du-Chat patients 135

268. Online microdialysis-ultra performance liquid chromatography–mass spectrometry method for

comparative pharmacokinetic investigation on iridoids from Gardenia jasminoides Ellis in rats

with different progressions of type 2 diabetic complications 136

269. Optimization of a new methodology for trace determination of elements in biological fluids:

Application for speciation of inorganic selenium in children‘s blood 136

270. Detailed analysis of cortisol, cortisone and their tetrahydro- and allo-tetrahydrometabolites in

human urine by LC–MS/MS 137

xvi

271. Urinary metabonomics study of the hepatoprotective effects of total alkaloids from Corydalis

saxicola Bunting on carbon tetrachloride-induced chronic hepatotoxicity in rats using 1H NMR

analysis 137

272. Pharmacokinetic properties of the synthetic cannabinoid JWH-018 and of its metabolites in

serum after inhalation 138

273. Charged derivatization and on-line solid phase extraction to measure extremely low cortisol and

cortisone levels in human saliva with liquid chromatography–tandem mass spectrometry 138

274. Comparability study of Rituximab originator and follow-on biopharmaceutical 139

275. Whole blood microsampling for the quantitation of estetrol without derivatization by liquid

chromatography-tandem mass spectrometry 139

276. Meropenem, levofloxacin and linezolid in human plasma of critical care patients: A fast semi-

automated micro-extraction by packed sorbent UHPLC-PDA method for their simultaneous

determination 140

277. Investigation on the combined effect of cocaine and ethanol administration through a liquid

chromatography–mass spectrometry metabolomics approach 140

278. Validation of a dried blood spot method for therapeutic drug monitoring of citalopram,

mirtazapine and risperidone and its active metabolite 9-hydroxyrisperidone using

HPLC–MS 141

279. Characterization of an unknown impurity in doxofylline using LC–MS and NMR 141

280. Determination of AB-CHMINACA and its metabolites in human hair and their deposition in hair

of abusers 142

281. Development of a new chlorogenic acid certified reference material for food and drug

analysis 142

282. Automation of plasma protein binding assay using rapid equilibrium dialysis device and Tecan

workstation 143

283. ―Ghost peaks‖ of ezetimibe: Solution degradation products of ezetimibe in acetonitrile induced

by alkaline impurities from glass HPLC vials 143

284. Metabolite quantification by NMR and LC-MS/MS reveals differences between unstimulated,

stimulated, and pure parotid saliva 144

285. Determination of AZD3759 in rat plasma and brain tissue by LC–MS/MS and its application in

pharmacokinetic and brain distribution studies 144

286. Studying the effects of natural extracts with metabolomics: A longitudinal study on the

supplementation of healthy rats with Polygonum cuspidatum Sieb et Zucc. 145

287. Identification and characterization of a thermally cleaved fragment of monoclonal antibody-A

detected by sodium dodecyl sulfate-capillary gel electrophoresis 145

288. Synchronous determination with double-wavelength by RP-HPLC-UV and optimization of

ultrasound-assisted extraction of phenolic acids from Caragana species using response surface

methodology 146

xvii

289. Untargeted metabolite analysis-based UHPLC-Q-TOF-MS reveals significant enrichment of p-

hydroxybenzyl dimers of citric acids in fresh beige-scape Gastrodia elata (Wutianma) 146

Volume 141, July 2017

290. Matrix-assisted laser-desorption/ionization mass spectrometric imaging of olanzapine in a single

hair using esculetin as a matrix 147

291. A novel liquid chromatography/tandem mass spectrometry (LC–MS/MS) based bioanalytical

method for quantification of ethyl esters of Eicosapentaenoic acid (EPA) and Docosahexaenoic

acid (DHA) and its application in pharmacokinetic study 147

292. Identification of a host cell protein impurity in therapeutic protein, P1 148

293. Simultaneous separation and determination of four uncaria alkaloids by capillary electrophoresis

using dual cyclodextrin system 148

294. CE method for the in-process control of the synthesis of active substances conjugated with gold

nanoparticles 149

295. Achievable separation performance and analysis time in current liquid chromatographic practice

for monoclonal antibody separations 149

296. Identification and characterization of process-related substances and degradation products in

apremilast: Process optimization and degradation pathway elucidation 150

297. Antiproliferative hydroxy-fatty acids from the fodder legume Stylosanthes guianensis 150

298. Development and validation of a liquid chromatographic method for the analysis of squaric acid

dibutyl ester and its impurities 151

299. Macro-Raman spectroscopy for bulk composition and homogeneity analysis of multi-component

pharmaceutical powders 151

300. Isolation, identification and characterization of potential impurities of anidulafungin 152

301. Dialkyl anionic surfactant in field-amplified sample injection and sweeping-micellar

electrokinetic chromatography for determination of eight leanness-promoting β-agonists in

animal feeds 152

302. Isolation and structural characterization of glucosylceramides from Ethiopian plants by

LC/APCI-MS/MS 153

303. Drug-protein binding mechanism of juglone for early pharmacokinetic profiling: Insights from

ultrafiltration, multi-spectroscopic and molecular docking methods 153

304. Quantification of alprenolol and propranolol in human plasma using a two-dimensional liquid

chromatography (2D-LC) 154

305. An LC–MS/MS method for quantification of AC0010, a novel mutant-selective epidermal

growth factor receptor (EGFR) inhibitor, and its metabolites in human plasma and the application

to a pharmacokinetic study 154

306. Validation and application of an ultrahigh-performance liquid chromatographic-Orbitrap mass

spectrometric method for the simultaneous detection and quantification of volatile and non-

volatile organic acids in human faecal samples 155

xviii

307. Validation of a SPE HPLC–UV method for the quantification of a new ER-specific

photosensitizer OR-141 in blood serum using total error concept 155

308. Determination of a novel anticancer AMPK activator hernandezine in rat plasma and tissues with

a validated UHPLC–MS/MS method: Application to pharmacokinetics and tissue distribution

study 156

309. LC–MS/MS determination of tranexamic acid in human plasma after phospholipid

clean-up 156

310. Metabolic profiles of neotuberostemonine and tuberostemonine in rats by high performance

liquid chromatography/quadrupole time-of-flight mass spectrometry 157

311. Comprehensive profiling and characterization of coumarins from roots, stems, leaves, branches,

and seeds of Chimonanthus nitens Oliv using ultra-performance liquid chromatography/

quadrupole-time-of-flight mass spectrometry combined with modified mass defect filter 157

312. New analytical method for determination of epimer metabolites in rat plasma after oral

administration of Paeoniflorin by UPLC-TOF-MS following picolinoyl derivatization 158

313. Application of 2D-NMR with room temperature NMR probes for the assessment of the higher

order structure of filgrastim 158

314. Influence of sulfur fumigation on the chemical profiles of Atractylodes macrocephala Koidz

evaluated by UFLC–QTOF–MS combined with multivariate statistical analysis 159

315. A sandwich immunoassay for brucellosis diagnosis based on immune magnetic beads and

quantum dots 159

316. Systematically characterize the absorbed effective substances of Wutou Decoction and their

metabolic pathways in rat plasma using UHPLC-Q-TOF-MS combined with a target network

pharmacological analysis 160

317. Nontargeted metabolomics approach for the differentiation of cultivation ages of mountain

cultivated ginseng leaves using UHPLC/QTOF-MS 160

318. Studies on the metabolites difference of psoralen/isopsoralen in human and six mammalian liver

microsomes in vitro by UHPLC–MS/MS 161

319. Al cation induces aggregation of serum proteins 161

Volume 142, August 2017

320. Fighting falsified medicines: The analytical approach 162

321. Lipophilicity estimation of statins as a decisive physicochemical parameter for their hepato-

selectivity using reversed-phase thin layer chromatography 162

322. Metabolite profiling of flavonols and in vitro antioxidant activity of young shoots of wild

Humulus lupulus L. (hop) 163

323. Development of assay for determination of eletriptan hydrobromide in loaded PLGA

nanoparticles 163

xix

324. Characterization of flavonol mono-, di-, tri- and tetra-O-glycosides by ultra-performance liquid

chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry and its

application for identification of flavonol glycosides in Viola tianschanica 164

325. Development and validation of a rapid reversed-phase HPLC method for the quantification of

monoclonal antibody bevacizumab from polyester-based nanoparticles 164

326. Quantification of active ingredients in semi-solid pharmaceutical formulations by near infrared

spectroscopy 165

327. Detection of regulated herbs and plants in plant food supplements and traditional medicines using

infrared spectroscopy 165

328. UV-induced electron transfer between triethylamine and 5-bromo-2′-deoxyuridine A puzzle

concerning the photochemical debromination of labeled DNA 166

329. Development of an in vivo-relevant drug product performance method for an amorphous solid

dispersion 166

330. Ultrasound-assisted low-density solvent dispersive liquid–liquid microextraction for the

simultaneous determination of 12 new antidepressants and 2 antipsychotics in whole blood by

gas chromatography–mass spectrometry 167

331. Application of protein A-modified capillary-channeled polymer polypropylene fibers to the

quantitation of IgG in complex matrices 167

332. Simple and rapid quantification of vancomycin in serum, urine and peritoneal/pleural effusion

via UHPLC–MS/MS applicable to personalized antibiotic dosing research 168

333. Biphenyl based stationary phases for improved selectivity in complex steroid assays 168

334. Ultra-sensitive and selective quantification of endothelin-1 in human plasma using ultra-

performance liquid chromatography coupled to tandem mass spectrometry 169

335. Serum metabolomics reveals the mechanistic role of functional foods and exercise for obesity

management in rats 169

336. Systematic screening and characterization of prototype constituents and metabolites of total

astragalosides using HPLC-ESI-IT-TOF-MSn after oral administration to rats 170

337. Development, validation and application of a novel liquid chromatography tandem mass

spectrometry assay measuring uracil, 5,6-dihydrouracil, 5-fluorouracil, 5,6-dihydro-5-

fluorouracil, α-fluoro-β-ureidopropionic acid and α-fluoro-β-alanine in human plasma 170

338. Multi-platform metabolomics and a genetic approach support the authentication of agarwood

produced by Aquilaria crassna and Aquilaria malaccensis 171

339. Apoptosis induction activity and molecular docking studies of survivin siRNA carried by Fe3O4-

PEG-LAC-chitosan-PEI nanoparticles in MCF-7 human breast cancer cells 171

340. Exploring the neuroprotective effects of ginkgolides injection in a rodent model of cerebral

ischemia–reperfusion injury by GC–MS based metabolomic profiling 172

341. Quantitative LC–HRMS determination of selected cardiovascular drugs, in dried blood spots, as

an indicator of adherence to medication 172

xx

342. Determination of thiopurine S-methyltransferase activity by hydrophilic interaction liquid

chromatography hyphenated with mass spectrometry 173

343. Workflow methodology for rat brain metabolome exploration using NMR, LC–MS and GC–MS

analytical platforms 173

344. Bioelectrical impedimetric sensor for single cell analysis based on nanoroughened quartz

substrate; suitable for cancer therapeutic purposes 174

345. Highly sensitive UHPLC–MS/MS method for the simultaneous estimation of propafenone and its

metabolites 5-hydroxypropafenone and N-depropylpropafenone on human dried blood spots

technique and application to a pharmacokinetic study 174

346. Simple determination of L-hydroxyproline in idiopathic pulmonary fibrosis lung tissues of rats

using non-extractive high-performance liquid chromatography coupled with fluorescence

detection after pre-column derivatization with novel synthetic 9-acetylimidazol-

carbazole 175

347. A simple method for assaying colistimethate sodium in pharmaceutical aerosol samples using

high performance liquid chromatography 175

348. Quantification of apigenin trimethyl ether in rat plasma by liquid chromatography–tandem mass

spectrometry: Application to a pre-clinical pharmacokinetic study 176

349. Simultaneous analysis of regorafenib and sorafenib and three of their metabolites in human

plasma using LC–MS/MS 176

350. Stability behavior of antiretroviral drugs and their combinations 7: Comparative degradation

pathways of lamivudine and emtricitabine and explanation to their differential degradation

behavior by density functional theory 177

351. An atmospheric pressure ionization source using a high voltage target compared to electrospray

ionization for the LC/MS analysis of pharmaceutical compounds 177

352. UHPLC–MS/MS method with sample dilution to test therapeutic adherence through

quantification of ten antihypertensive drugs in urine samples 178

353. A novel carbohydrate labeling method utilizing transfer hydrogenation-mediated reductive

amination 178

354. Non-volatile extractable analysis of prefilled syringes for parenteral administration of drug

products 179

355. A rapid microextraction by packed sorbent − liquid chromatography tandem mass spectrometry

method for the determination of dexamethasone disodium phosphate and dexamethasone in

aqueous humor of patients with uveitis 179

356. Isotope-coded derivatization based LC/ESI-MS/MS methods using a pair of novel reagents for

quantification of hydroxycinnamic acids and hydroxybenzoic acids in fermented brown rice

product 180

357. Rapid discovery of cyclopamine analogs from Fritillaria and Veratrum plants using LC-Q-TOF-

MS and LC-QqQ-MS 180

xxi

358. Development and validation of a HS/GC–MS method for the simultaneous analysis of diacetyl

and acetylpropionyl in electronic cigarette refills 181

359. Structural characterization and discrimination of the Paris polyphylla var. yunnanensis and Paris

vietnamensis based on metabolite profiling analysis 181

Volume 143, September 2017

360. Reliability of point-of-collection testing devices for drugs of abuse in oral fluid: A systematic

review and meta-analysis 182

361. High-throughput thermofluor-based assays for inhibitor screening of STAT SH2 domains 182

362. Trace level determination of 5-hydroxytryptamine and its related indoles in amniotic fluid by gas

chromatography–mass spectrometry 183

363. Identification of related substances in tofacitinib citrate by LC-MS techniques for synthetic

process optimization 183

364. Macro- and microstructural tracking of ageing-related changes of papaverine hydrochloride-

loaded electrospun nanofibrous buccal sheets 184

365. A simplified guide for charged aerosol detection of non-chromophoric compounds—Analytical

method development and validation for the HPLC assay of aerosol particle size distribution for

amikacin 184

366. Proton dissociation properties of arylphosphonates: Determination of accurate Hammett equation

parameters 185

367. Sub–1 min separation in sequential injection chromatography for determination of synthetic

water-soluble dyes in pharmaceutical formulation 185

368. Impurity profiling of liothyronine sodium by means of reversed phase HPLC, high resolution

mass spectrometry, on-line H/D exchange and UV/Vis absorption 186

369. A UHPLC method for the rapid separation and quantification of anthocyanins in acai berry and

dry blueberry extracts 186

370. Combined computational-experimental approach to predict blood–brain barrier (BBB)

permeation based on ―green‖ salting-out thin layer chromatography supported by simple

molecular descriptors 187

371. Development of a new extraction technique and HPLC method for the analysis of non-

psychoactive cannabinoids in fibre-type Cannabis sativa L. (hemp) 187

372. Analytical profiling of selected antioxidants and total antioxidant capacity of goji (Lycium spp.)

berries 188

373. Characterization and inhibition studies of tissue nonspecific alkaline phosphatase by

aminoalkanol derivatives of 1,7-dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-

3,5,10-trione, new competitive and non-competitive inhibitors, by capillary electrophoresis 188

374. A validated UHPLC-QTOF-MS method for quantification of metformin and teneligliptin in rat

plasma: Application to pharmacokinetic interaction study 189

xxii

375. Overcoming interference with the detection of a stable isotopically labeled microtracer in the

evaluation of beclabuvir absolute bioavailability using a concomitant microtracer approach 189

376. Ex-vivo measurement of scalp follicular infundibulum delivery of zinc pyrithione and climbazole

from an anti-dandruff shampoo 190

377. Pooled human liver preparations, HepaRG, or HepG2 cell lines for metabolism studies of new

psychoactive substances? A study using MDMA, MDBD, butylone, MDPPP, MDPV, MDPB, 5-

MAPB, and 5-API as examples 190

378. Evaluation of pharmacokinetics and blood-brain barrier permeability of mitragynine using in

vivo microdialysis technique 191

379. 1H NMR spectral identification of medication in cerebrospinal fluid of pediatric meningitis 191

380. Therapeutic drug monitoring of beta-lactam antibiotics – Influence of sample stability on the

analysis of piperacillin, meropenem, ceftazidime and flucloxacillin by HPLC-UV 192

381. Development and validation of an ultra-performance liquid chromatography–tandem mass

spectrometry method for quantification of SR1001, an inverse agonist of retinoid-related orphan

receptors, and its application to pharmacokinetic studies in streptozotocin-induced diabetic

mice 193

382. Quantum dot-linked immunosorbent assay (QLISA) using orientation-directed antibodies 193

383. Validation of liquid and gaseous calibration techniques for quantification of propofol in breath

with sorbent tube Thermal Desorption System GC–MS 194

384. Development and validation of a bioanalytical method based on LC–MS/MS analysis for the

quantitation of CIGB-814 peptide in plasma from Rheumatoid Arthritis patients 194

385. Application of volumetric absorptive microsampling device for quantification of tacrolimus in

human blood as a model drug of high blood cell partition 195

386. Fast and efficient zirconia-based reversed phase chromatography for selective determination of

triptans in rat plasma 195

387. Monitoring breast cancer treatment using a Fourier transform infrared spectroscopy-based

computational model 196

388. Enhancement in recovery of drugs with high protein binding efficiency from human plasma

using magnetic nanoparticles 196

389. Simultaneous quantification of endothelin receptor antagonists and phosphodiesterase

5 inhibitors currently used in pulmonary arterial hypertension 197

390. Application of 1H NMR spectroscopy to the metabolic phenotyping of rodent brain extracts: A

metabonomic study of gut microbial influence on host brain metabolism 197

391. A re-investigation of the phytochemical composition of the edible herb Amaranthus retro-

flexus L 198

392. A vote for robustness: Monitoring serum enzyme activity by thin-layer chromatography of

dabsylated bradykinin products 198

xxiii

393. Salting-out assisted liquid–liquid extraction combined with gas chromatography-mass

spectrometry for the determination of pyrethroid insecticides in high salinity and biological

samples 199

394. HPLC–MS/MS method for troventol determination in human plasma and its application to

biological samples 199

395. 1,4-Anthraquinone: A new useful pre-column reagent for the determination of N-acetylcysteine

and captopril in pharmaceuticals by high performance liquid chromatography 200

396. Development a validated highly sensitive LC–MS/MS method for simultaneous quantification of

Ledipasvir, sofosbuvir and its major metabolite GS-331007 in human plasma: Application to a

human pharmacokinetic study 200

397. Chemometrics and chromatographic fingerprints to classify plant food supplements according to

the content of regulated plants 201

398. Surrogate CD16-expressing effector cell lines for determining the bioactivity of therapeutic

monoclonal antibodies 201

399. Dual-target screening of bioactive components from traditional Chinese medicines by hollow

fiber-based ligand fishing combined with liquid chromatography–mass spectrometry 202

Volume 144, September 2017

400. Circular dichroism analysis of the calicheamicin-DNA interaction revisited 202

401. Induced circularly polarized luminescence for revealing DNA binding with fluorescent

dyes 203

402. Analysis of stereoselective drug interactions with serum proteins by high-performance affinity

chromatography: A historical perspective 203

403. An indirect stereoselective analysis of nebivolol glucuronides in plasma by LC–MS/MS:

Application to clinical pharmacokinetics 204

404. N-Decyl-S-trityl-(R)-cysteine, a new chiral selector for ―green‖ ligand-exchange chromatography

applications 204

405. The role of chirality in a set of key intermediates of pharmaceutical interest, 3-aryl-substituted-γ-

butyrolactones, evidenced by chiral HPLC separation and by chiroptical spectroscopies 205

406. Determination of the absolute configuration of a novel tetrasubstituted isoindolinone by

vibrational circular dichroism 205

407. GC–MS based Gestational Diabetes Mellitus longitudinal study: Identification of 2-and 3-

hydroxybutyrate as potential prognostic biomarkers 206

408. Development and validation of a quantification method for cucurbitacins E and I in rat plasma:

Application to population pharmacokinetic studies 206

409. Ultra-fast quantitation of voriconazole in human plasma by coated blade spray mass

spectrometry 207

410. Pharmacokinetic profile of bilberry anthocyanins in rats and the role of glucose transporters: LC–

MS/MS and computational studies 207

411. Comparative pharmacodynamic analysis of imidazoline compounds using rat model of ocular

mydriasis with a test of quantitative structure–activity relationships 208

xxiv

412. Characterization of oxycodone in vitro metabolism by human cytochromes P450 and UDP-

glucuronosyltransferases 208

413. Structural and functional integrity of human serum albumin: Analytical approaches and clinical

relevance in patients with liver cirrhosis 209

414. Targeted proteomics of cannabinoid receptor CB1 and the CB1b isoform 209

415. Application of an ESI-QTOF method for the detailed characterization of GSK-3β

inhibitors 210

416. Quantitative estimation of cholinesterase-specific drug metabolism of carbamate inhibitors

provided by the analysis of the area under the inhibition-time curve 210

417. A new method to characterize the kinetics of cholinesterases inhibited by carbamates 211

418. Cyclodextrins as inhibitors of the precipitation of riboflavin-5‘-phosphate due to presence of zinc

chloride: A NMR investigation 211

419. Monitoring drug–serum protein interactions for early ADME prediction through Surface

Plasmon Resonance technology 212

420. Capillary electrophoresis in the context of drug discovery 212

421. Simultaneous analysis of nucleobases, nucleosides and ginsenosides in ginseng extracts using

supercritical fluid chromatography coupled with single quadrupole mass spectrometry 213

422. Combined approach using capillary electrophoresis, NMR and molecular modeling for

ambrisentan related substances analysis: Investigation of intermolecular affinities, complexation

and separation mechanism 213

423. Molecularly imprinted polymer for glutathione by modified precipitation polymerization and its

application to determination of glutathione in supplements 214

424. Efficacy of a titanium dioxide nanoparticles − based indoor anti-odor product as assessed by

electronic nose and gaschromatography–mass spectrometry 214

425. Comprehensive study on the effects of sodium and potassium additives in size exclusion

chromatographic separations of protein biopharmaceuticals 215

426. Application of a rapid HILIC-UV method for synthesis optimization and stability studies of

immunogenic neo-glycoconjugates 215

427. Simple and rapid LC–MS method for the determination of circulating albumin

microheterogeneity in veal calves exposed to heat stress 216

428. Molecular fingerprinting of principal neurons in the rodent hippocampus: A neuroinformatics

approach 216

Volume 145, October 2017

429. Two-dimensional liquid chromatography in pharmaceutical analysis Instrumental aspects, trends

and applications 217

430. Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A

generic method development approach 217

431. A new platform for serological analysis based on porous 3-dimensional polyethylene sinter

bodies 218

xxv

432. Designing a calibration set in spectral space for efficient development of an NIR method for

tablet analysis 218

433. On-line prediction of the glucose concentration of CHO cell cultivations by NIR and Raman

spectroscopy: Comparative scalability test with a shake flask model system 219

434. Conventional and accelerated-solvent extractions of green tea (camellia sinensis) for

metabolomics-based chemometrics 219

435. Phase separation of in situ forming poly (lactide-co-glycolide acid) implants investigated using a

hydrogel-based subcutaneous tissue surrogate and UV–vis imaging 220

436. Mid-infrared and near-infrared spectroscopy for rapid detection of Gardeniae Fructus by a liquid-

liquid extraction process 220

437. Quantitative analysis of a biopharmaceutical protein in cell culture samples using automated

capillary electrophoresis (CE) western blot 221

438. Synchronous characterization of carbohydrates and ginsenosides yields deeper insights into the

processing chemistry of ginseng 221

439. Development and validation of an ICP-MS method for the determination of elemental impurities

in TP-6076 active pharmaceutical ingredient (API) according to USP 〈232〉/〈233〉 222

440. Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1


441. Revisiting blood-brain barrier: A chromatographic approach 223

442. Liquid chromatographic enantioseparation of carbocyclic β-amino acids possessing limonene

skeleton on macrocyclic glycopeptide-based chiral stationary phases 223

443. On-line coupling of molecularly imprinted solid phase extraction with liquid chromatography for

the fast determination of coumarins from complex samples 224

444. Metabolic profiling analysis of Siraitia grosvenorii revealed different characteristics of green

fruit and saccharified yellow fruit 224

445. Comparison of α-glucosidase inhibitory effect and bioactive constituents of Anemarrhenae

Rhizoma and Fibrous Roots 225

446. Characterization of forced degradation products of torasemide through MS tools and explanation

of unusual losses observed during mass fragmentation of drug and degradation products through

density functional theory 225

447. Metabolic profiling of the traditional Chinese medicine formulation Yu Ping Feng San for the

identification of constituents relevant for effects on expression of TNF-α, IFN-γ, IL-1β and IL-4

in U937 cells 226

448. A comprehensive stability-indicating HPLC method for determination of chloroquine in active

pharmaceutical ingredient and tablets: Identification of oxidation impurities 226

449. Identification of impurities in macrolides by liquid chromatography–mass spectrometric

detection and prediction of retention times of impurities by constructing quantitative structure–

retention relationship (QSRR) 227

xxvi

450. The impact of ZnO and TiO2 on the stability of clotrimazole under UVA irradiation:

Identification of photocatalytic degradation products and in vitro cytotoxicity assessment 227

451. Chemometrically assisted development and validation of LC–MS/MS method for the analysis of

potential genotoxic impurities in meropenem active pharmaceutical ingredient 228

452. Identification and interconversion of isomeric 4,5-functionalized 1,2,3-thiadiazoles and 1,2,3-

triazoles in conditions of electrospray ionization 228

453. Dissolution assessment of allopurinol immediate release tablets by near infrared spect-

roscopy 229

454. Enhanced and green extraction polyphenols and furanocoumarins from Fig (Ficus carica L.)

leave using deep eutectic solvents 229

455. Site- and species-specific hydrolysis rates of cocaine 230

456. Comparative stability-indicating chromatographic methods for determination of 4-

hexylresorcinol in pharmaceutical formulation and shrimps 230

457. Enantiomeric separation of seven β-agonists by NACE—Study of chiral selectivity with

diacetone-d-mannitol–boric acid complex 231

458. An optimized HPLC-UV method for quantitatively determining sesquiterpenes in Nardostachyos

Radix et Rhizoma 231

459. Photostability testing using online reactor HPLC hyphenation and mass spectrometric compound

identification illustrated by ketoprofen as model compound 232

460. Optoelectronic iron detectors for pharmaceutical flow analysis 232

461. Isoconversional approach for non-isothermal decomposition of un-irradiated and photon-

irradiated 5-fluorouracil 233

462. Characterization of impurities in sodium cromoglycate drug substance and eye drops using LC-

ESI-ion trap MS and LC-ESI-QTOF MS 233

463. Method validation and nanoparticle characterization assays for an innovative amphothericin B

formulation to reach increased stability and safety in infectious diseases 234

464. Surfactants enhance recovery of poorly soluble drugs during microdialysis sampling:

Implications for in vitro dissolution-/permeation-studies 234

465. Simultaneous identification and quantification of polymethoxyflavones, coumarin and phenolic

acids in Ageratum conyzoides by UPLC-ESI-QToF-MS and UPLC-PDA 235

466. Degradation and metabolite formation of estrogen conjugates in an agricultural soil 235

467. Chemical analysis and potential endocrine activities of aluminium coatings intended to be in

contact with cosmetic water 236

468. Identification and determination of related substances of ceftaroline fosamil in medicinal product

by high performance liquid chromatography with diode array detection and tandem mass

spectrometry 236

xxvii

469. Quantification of concentrated Chinese medicine granules by quantitative polymerase chain

reaction 237

470. Chiral separation of terbutaline and non-steroidal anti-inflammatory drugs by using a new

lysine–bridged hemispherodextrin in capillary electrophoresis 237

471. Separation and characterization of allergic polymerized impurities in cephalosporins by 2D-

HPSEC × LC-IT-TOF MS 238

472. Phytochemical analysis and anti-inflammatory evaluation of compounds from an aqueous extract

of Croton cajucara Benth 238

473. Supercritical fluid chromatography approach for a sustainable manufacture of new

stereoisomeric anticancer agent 239

474. Use of ionic liquids as headspace gas chromatography diluents for the analysis of residual

solvents in pharmaceuticals 239

475. Adduct ion-targeted qualitative and quantitative analysis of polyoxypregnanes by ultra-high

pressure liquid chromatography coupled with triple quadrupole mass spectrometry 240

476. Quantitative determination of dobutamine in newborn pig plasma samples by HPLC–

MS/MS 240

477. An integrative investigation of the toxicity of Aconiti kusnezoffii radix and the attenuation effect

of its processed drug using a UHPLC-Q-TOF based rat serum and urine metabolomics

strategy 241

478. Calibration and validation of a MCC/IMS prototype for exhaled propofol online measu-

rement 241

479. Biochemical and functional analysis of corticotropin releasing factor purified from an aqueous

extract of human placenta used as wound healer 242

480. Toxic compounds from tobacco in placenta samples analyzed by UPLC-QTOF-MS 242

481. HPLC-HG-AFS determination of arsenic species in acute promyelocytic leukemia (APL) plasma

and blood cells 243

482. Quantification of IDP-73152, a novel antibiotic, in plasma from mice, rats and humans using an

ultra-high performance liquid chromatography/tandem mass spectrometry method for use in

pharmacokinetic studies 243

483. New approach for the diagnosis of histamine intolerance based on the determination of histamine

and methylhistamine in urine 244

484. Development and validation of sensitive LC–MS/MS method for the quantification of SUVN-

502 and its metabolite and its application for first in human pharmacokinetic study 244

485. Glycosylation patterns of selected proteins in individual serum and cerebrospinal fluid

samples 245

486. MIL-101(Cr)@GO for dispersive micro-solid-phase extraction of pharmaceutical residue in

chicken breast used in microwave-assisted coupling with HPLC–MS/MS detection 245

xxviii

487. Development of a robust reporter gene assay to measure the bioactivity of anti-PD-1/anti-PD-L1

therapeutic antibodies 246

488. LC–MS/MS-ESI method for simultaneous quantification of darolutamide and its active

metabolite, ORM-15341 in mice plasma and its application to a pharmacokinetic study 246

489. Metabolite identification of AZD8055 in Sprague-Dawley rats after a single oral administration

using ultra-performance liquid chromatography and mass spectrometry 247

490. Quantification of 16 β-lactams in chicken muscle by QuEChERS extraction and UPLC-Q-

Orbitrap-MS with parallel reaction monitoring 247

491. Characterization of the phase I and phase II metabolic profile of tolvaptan by in vitro studies and

liquid chromatography–mass spectrometry profiling: Relevance to doping control

analysis 248

492. An LC–MS/MS method for simultaneous determination of nine steroidal saponins from Paris

polyphylla var. in rat plasma and its application to pharmacokinetic study 248

493. N-glucuronidation catalyzed by UGT1A4 and UGT2B10 in human liver microsomes: Assay

optimization and substrate identification 249

494. A simple high performance liquid chromatography–mass spectrometry method for Therapeutic

Drug Monitoring of isavuconazole and four other antifungal drugs in human plasma

samples 249

495. Metabolic profiling of dehydrodiisoeugenol using xenobiotic metabolomics 250

496. Development and validation of a reliable method for thiopurine methyltransferase (TPMT)

enzyme activity in human whole blood by LC–MS/MS: An application for phenotypic and

genotypic correlations 250

497. Development and validation of a GC–MS method for the determination of hydroxyzine and its

active metabolite, cetirizine, in whole blood 251

498. An on-spot internal standard addition approach for accurately determining colistin A and colistin

B in dried blood spots using ultra high-performance liquid chromatography–tandem mass

spectrometry 251

499. Comparative study of single/combination use of Huang-Lian-Jie-Du decoction and berberine on

their protection on sepsis induced acute liver injury by NMR metabolic profiling 252

500. Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices:

Sample preparation methods and liquid chromatography tandem mass spectrometric

separations 252

501. Validation of an HPLC-UV method for analysis of Kaempferol-loaded nanoemulsion and its

application to in vitro and in vivo tests 253

502. Development of direct assays for Toxoplasma gondii and its use in genomic DNA

sample 253

503. Investigation and structural elucidation of a new impurity in bulk drug of cilostazol by

LC/MS/MS, FT-IR and NMR 254

xxix

504. Selective screening of glutaric acid acidurias by capillary electrophoresis-mass spect-

rometry 254

505. Online turbulent flow extraction coupled with liquid chromatography–tandem mass spectrometry

for high throughput screening of anabolic steroids in horse urine 255

506. Development of a mixed-mode chromatography with tandem mass spectrometry method for the

quantitative analysis of 23 underivatized amino acids in human serum 255

507. Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance

sensor 256

508. Liquid chromatography-tandem mass spectrometry assay to quantify plitidepsin in human

plasma, whole blood and urine 256

509. Rapid analysis of benzalkonium chloride using paper spray mass spectrometry 257

510. Rapid screening of non-steroidal anti-inflammatory drugs illegally added in anti-rheumatic

herbal supplements and herbal remedies by portable ion mobility spectrometry 257

511. Simultaneous quantitative analysis of polyethylene glycol (PEG), PEGylated paclitaxel and

paclitaxel in rats by MS/MSALL technique with hybrid quadrupole time-of-flight mass

spectrometry 258

512. Lyophilic matrix method for dissolution and release studies of nanoscale particles 258

513. Incorporation of 14C-cholesterol in human adrenal corticocarcinoma H295R cell line and online-

radiodetection of produced 14C-steroid hormone metabolites 259

514. Verification of the effectiveness of the Fourier transform infrared spectroscopy computational

model for colorectal cancer 259

515. Quantification of paracetamol and 5-oxoproline in serum by capillary electrophoresis:

Implication for clinical toxicology 260

516. Quantification of cyclocreatine in mouse and rat plasma using hydrophilic-interaction ultra-

performance liquid chromatography-tandem mass spectrometry 260

517. Volumetric adsorptive microsampling-liquid chromatography tandem mass spectrometry assay

for the simultaneous quantification of four antibiotics in human blood: Method development,

validation and comparison with dried blood spot 261

518. PLGA Ethionamide Nanoparticles for Pulmonary Delivery: Development and in vivo evaluation

of dry powder inhaler 261

519. Simultaneous determination and pharmacokinetics of danshensu, protocatechuic aldehyde, 4-

hydroxy-3-methyloxyphenyl lactic acid and protocatechuic acid in human plasma by LC–

MS/MS after oral administration of Compound Danshen Dripping Pills 262

520. LC–MS bioanalysis of Trastuzumab and released emtansine using nano-surface and molecular-

orientation limited (nSMOL) proteolysis and liquid–liquid partition in plasma of Trastuzumab

emtansine-treated breast cancer patients 262

521. Separation of furostanol saponins by supercritical fluid chromatography 263

xxx

522. Macroporous monoliths for biodegradation study of polymer particles considered as drug

delivery systems 263

523. A novel enantioseparation approach based on liposome electrokinetic capillary chroma-

tography 264

524. Comparative study on the anticancer activities and binding properties of a hetero metal binuclear

complex [Co(dipic)2Ni(OH2)5]·2H2O (dipic = dipicolinate) with two carrier proteins 264

525. Characterization and quantitative analysis of phenolic derivatives in Longxuetongluo Capsule by

HPLC-DAD-IT-TOF-MS 265

Volume 145, October 2017

526. Two-dimensional liquid chromatography in pharmaceutical analysis Instrumental aspects, trends

and applications 265

527. Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A

generic method development approach 266

528. A new platform for serological analysis based on porous 3-dimensional polyethylene sinter

bodies 266

529. Designing a calibration set in spectral space for efficient development of an NIR method for

tablet analysis 267

530. On-line prediction of the glucose concentration of CHO cell cultivations by NIR and Raman

spectroscopy: Comparative scalability test with a shake flask model system 267

531. Conventional and accelerated-solvent extractions of green tea (camellia sinensis) for

metabolomics-based chemometrics 268

532. Phase separation of in situ forming poly (lactide-co-glycolide acid) implants investigated using a

hydrogel-based subcutaneous tissue surrogate and UV–vis imaging 268

533. Mid-infrared and near-infrared spectroscopy for rapid detection of Gardeniae Fructus by a liquid-

liquid extraction process 269

534. Quantitative analysis of a biopharmaceutical protein in cell culture samples using automated

capillary electrophoresis (CE) western blot 269

535. Synchronous characterization of carbohydrates and ginsenosides yields deeper insights into the

processing chemistry of ginseng 270

536. Development and validation of an ICP-MS method for the determination of elemental impurities

in TP-6076 active pharmaceutical ingredient (API) according to USP 〈232〉/〈233〉 270

537. Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1


538. Revisiting blood-brain barrier: A chromatographic approach 271

539. Liquid chromatographic enantioseparation of carbocyclic β-amino acids possessing limonene

skeleton on macrocyclic glycopeptide-based chiral stationary phases 272

xxxi

540. On-line coupling of molecularly imprinted solid phase extraction with liquid chromatography for

the fast determination of coumarins from complex samples 272

541. Metabolic profiling analysis of Siraitia grosvenorii revealed different characteristics of green

fruit and saccharified yellow fruit 273

542. Comparison of α-glucosidase inhibitory effect and bioactive constituents of Anemarrhenae

Rhizoma and Fibrous Roots 273

543. Characterization of forced degradation products of torasemide through MS tools and explanation

of unusual losses observed during mass fragmentation of drug and degradation products through

density functional theory 274

544. Metabolic profiling of the traditional Chinese medicine formulation Yu Ping Feng San for the


in U937 cells 274

545. A comprehensive stability-indicating HPLC method for determination of chloroquine in active

pharmaceutical ingredient and tablets: Identification of oxidation impurities 275

546. Identification of impurities in macrolides by liquid chromatography–mass spectrometric


retention relationship (QSRR) 275

547. The impact of ZnO and TiO2 on the stability of clotrimazole under UVA irradiation:

Identification of photocatalytic degradation products and in vitro cytotoxicity assessment 276

548. Chemometrically assisted development and validation of LC–MS/MS method for the analysis of

potential genotoxic impurities in meropenem active pharmaceutical ingredient 276

549. Identification and interconversion of isomeric 4,5-functionalized 1,2,3-thiadiazoles and 1,2,3-

triazoles in conditions of electrospray ionization 277

550. Dissolution assessment of allopurinol immediate release tablets by near infrared spectr-

oscopy 277

551. Enhanced and green extraction polyphenols and furanocoumarins from Fig (Ficus carica L.)

leave using deep eutectic solvents 278

552. Site- and species-specific hydrolysis rates of cocaine 278

553. Comparative stability-indicating chromatographic methods for determination of 4-hexylre-

sorcinol in pharmaceutical formulation and shrimps 279

554. Enantiomeric separation of seven β-agonists by NACE—Study of chiral selectivity with

diacetone-d-mannitol–boric acid complex 279

555. An optimized HPLC-UV method for quantitatively determining sesquiterpenes in Nardostachyos

Radix et Rhizoma 280

556. Photostability testing using online reactor HPLC hyphenation and mass spectrometric compound

identification illustrated by ketoprofen as model compound 280

557. Optoelectronic iron detectors for pharmaceutical flow analysis 281

xxxii

558. Isoconversional approach for non-isothermal decomposition of un-irradiated and photon-

irradiated 5-fluorouracil 281

559. Characterization of impurities in sodium cromoglycate drug substance and eye drops using LC-

ESI-ion trap MS and LC-ESI-QTOF MS 282

560. Method validation and nanoparticle characterization assays for an innovative amphothericin B

formulation to reach increased stability and safety in infectious diseases 282

561. Surfactants enhance recovery of poorly soluble drugs during microdialysis sampling:

Implications for in vitro dissolution-/permeation-studies 283

562. Simultaneous identification and quantification of polymethoxyflavones, coumarin and phenolic

acids in Ageratum conyzoides by UPLC-ESI-QToF-MS and UPLC-PDA 283

563. Degradation and metabolite formation of estrogen conjugates in an agricultural soil 284

564. Chemical analysis and potential endocrine activities of aluminium coatings intended to be in

contact with cosmetic water 284

565. Identification and determination of related substances of ceftaroline fosamil in medicinal product

by high performance liquid chromatography with diode array detection and tandem mass

spectrometry 285

566. Quantification of concentrated Chinese medicine granules by quantitative polymerase chain

reaction 285

567. Chiral separation of terbutaline and non-steroidal anti-inflammatory drugs by using a new

lysine–bridged hemispherodextrin in capillary electrophoresis 286

568. Separation and characterization of allergic polymerized impurities in cephalosporins by 2D-

HPSEC × LC-IT-TOF MS 286

569. Phytochemical analysis and anti-inflammatory evaluation of compounds from an aqueous extract

of Croton cajucara Benth 287

570. Supercritical fluid chromatography approach for a sustainable manufacture of new

stereoisomeric anticancer agent 287

571. Use of ionic liquids as headspace gas chromatography diluents for the analysis of residual

solvents in pharmaceuticals 288

572. Adduct ion-targeted qualitative and quantitative analysis of polyoxypregnanes by ultra-high

pressure liquid chromatography coupled with triple quadrupole mass spectrometry 288

573. Quantitative determination of dobutamine in newborn pig plasma samples by HPLC–

MS/MS 289

574. An integrative investigation of the toxicity of Aconiti kusnezoffii radix and the attenuation effect

of its processed drug using a UHPLC-Q-TOF based rat serum and urine metabolomics

strategy 289

575. Calibration and validation of a MCC/IMS prototype for exhaled propofol online measu-

rement 290

xxxiii

576. Biochemical and functional analysis of corticotropin releasing factor purified from an aqueous

extract of human placenta used as wound healer 290

577. Toxic compounds from tobacco in placenta samples analyzed by UPLC-QTOF-MS 291

578. HPLC-HG-AFS determination of arsenic species in acute promyelocytic leukemia (APL) plasma

and blood cells 291

579. Quantification of IDP-73152, a novel antibiotic, in plasma from mice, rats and humans using an


pharmacokinetic studies 292

580. New approach for the diagnosis of histamine intolerance based on the determination of histamine

and methylhistamine in urine 292

581. Development and validation of sensitive LC–MS/MS method for the quantification of SUVN-

502 and its metabolite and its application for first in human pharmacokinetic study 293

582. Glycosylation patterns of selected proteins in individual serum and cerebrospinal fluid

samples 293

583. MIL-101(Cr)@GO for dispersive micro-solid-phase extraction of pharmaceutical residue in

chicken breast used in microwave-assisted coupling with HPLC–MS/MS detection 294

584. Development of a robust reporter gene assay to measure the bioactivity of anti-PD-1/anti-PD-L1

therapeutic antibodies 294

585. LC–MS/MS-ESI method for simultaneous quantification of darolutamide and its active

metabolite, ORM-15341 in mice plasma and its application to a pharmacokinetic study 295

586. Metabolite identification of AZD8055 in Sprague-Dawley rats after a single oral administration

using ultra-performance liquid chromatography and mass spectrometry 295

587. Confirmation of metabolites of the neuroleptic drug prothipendyl using human liver microsomes,

specific CYP enzymes and authentic forensic samples—Benefits for routine drug testing 296

588. Quantification of 16 β-lactams in chicken muscle by QuEChERS extraction and UPLC-Q-

Orbitrap-MS with parallel reaction monitoring 296

589. Characterization of the phase I and phase II metabolic profile of tolvaptan by in vitro studies and

liquid chromatography–mass spectrometry profiling: Relevance to doping control

analysis 297

590. An LC–MS/MS method for simultaneous determination of nine steroidal saponins from Paris

polyphylla var. in rat plasma and its application to pharmacokinetic study 297

591. N-glucuronidation catalyzed by UGT1A4 and UGT2B10 in human liver microsomes: Assay

optimization and substrate identification 298

592. A simple high performance liquid chromatography–mass spectrometry method for Therapeutic

Drug Monitoring of isavuconazole and four other antifungal drugs in human plasma

samples 298

593. Metabolic profiling of dehydrodiisoeugenol using xenobiotic metabolomics 299

xxxiv

594. Development and validation of a reliable method for thiopurine methyltransferase (TPMT)


genotypic correlations 299

595. Development and validation of a GC–MS method for the determination of hydroxyzine and its

active metabolite, cetirizine, in whole blood 300

596. An on-spot internal standard addition approach for accurately determining colistin A and colistin

B in dried blood spots using ultra high-performance liquid chromatography–tandem mass

spectrometry 300

597. Comparative study of single/combination use of Huang-Lian-Jie-Du decoction and berberine on

their protection on sepsis induced acute liver injury by NMR metabolic profiling 301

598. Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices:


separations 301

599. Validation of an HPLC-UV method for analysis of Kaempferol-loaded nanoemulsion and its

application to in vitro and in vivo tests 302

600. Development of direct assays for Toxoplasma gondii and its use in genomic DNA

sample 302

601. Investigation and structural elucidation of a new impurity in bulk drug of cilostazol by

LC/MS/MS, FT-IR and NMR 303

602. Selective screening of glutaric acid acidurias by capillary electrophoresis-mass spect-

rometry 303

603. Online turbulent flow extraction coupled with liquid chromatography–tandem mass spectrometry

for high throughput screening of anabolic steroids in horse urine 304

604. Development of a mixed-mode chromatography with tandem mass spectrometry method for the

quantitative analysis of 23 underivatized amino acids in human serum 304

605. Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance

sensor 305

606. Liquid chromatography-tandem mass spectrometry assay to quantify plitidepsin in human

plasma, whole blood and urine 305

607. Rapid analysis of benzalkonium chloride using paper spray mass spectrometry 306

608. Rapid screening of non-steroidal anti-inflammatory drugs illegally added in anti-rheumatic

herbal supplements and herbal remedies by portable ion mobility spectrometry 306

609. Simultaneous quantitative analysis of polyethylene glycol (PEG), PEGylated paclitaxel and


spectrometry 307

610. Lyophilic matrix method for dissolution and release studies of nanoscale particles 307

611. Incorporation of 14C-cholesterol in human adrenal corticocarcinoma H295R cell line and online-

radiodetection of produced 14C-steroid hormone metabolites 308

xxxv

612. Verification of the effectiveness of the Fourier transform infrared spectroscopy computational

model for colorectal cancer 308

613. Quantification of paracetamol and 5-oxoproline in serum by capillary electrophoresis:

Implication for clinical toxicology 309

614. Quantification of cyclocreatine in mouse and rat plasma using hydrophilic-interaction ultra-

performance liquid chromatography-tandem mass spectrometry 309

615. Volumetric adsorptive microsampling-liquid chromatography tandem mass spectrometry assay


validation and comparison with dried blood spot 310

616. PLGA Ethionamide Nanoparticles for Pulmonary Delivery: Development and in vivo evaluation

of dry powder inhaler 310

617. Simultaneous determination and pharmacokinetics of danshensu, protocatechuic aldehyde, 4-


MS/MS after oral administration of Compound Danshen Dripping Pills 311

618. LC–MS bioanalysis of Trastuzumab and released emtansine using nano-surface and molecular-


emtansine-treated breast cancer patients 311

619. Separation of furostanol saponins by supercritical fluid chromatography 312

620. Macroporous monoliths for biodegradation study of polymer particles considered as drug

delivery systems 312

621. A novel enantioseparation approach based on liposome electrokinetic capillary chroma-

tography 313

622. Comparative study on the anticancer activities and binding properties of a hetero metal binuclear

complex [Co(dipic)2Ni(OH2)5]·2H2O (dipic = dipicolinate) with two carrier proteins 313

623. Characterization and quantitative analysis of phenolic derivatives in Longxuetongluo Capsule by

HPLC-DAD-IT-TOF-MS 314

Volume 146, November 2017

624. Analytical characterization of human milk oligosaccharides – potential applications in

pharmaceutical analysis 314

625. Analysis of macrolide antibiotics in water by magnetic solid-phase extraction and liquid

chromatography–tandem mass spectrometry 315

626. Second harmonic generation microscopy as a tool for the early detection of crystallization in

spray dried dispersions 315

627. Sensitive analysis of bioactive secondary metabolites in lichen species using liquid

chromatography–mass spectrometry 316

628. Semiautomated determination of neonicotinoids and characteristic metabolite in urine samples

using TurboFlow™ coupled to ultra high performance liquid chromatography coupled to

Orbitrap analyzer 316

xxxvi

629. A new polymorph of ciprofloxacin saccharinate: Structural characterization and pharmaceutical

profile 317

630. Qualitative and quantitative measurement of cannabinoids in cannabis using modified

HPLC/DAD method 317

631. Quantification of gabapentin polymorphs in gabapentin/excipient mixtures using solid state 13C

NMR spectroscopy and X-ray powder diffraction 318

632. Accurate recognition and feature qualify for flavonoid extracts from Liang-wai Gan Cao by

liquid chromatography-high resolution-mass spectrometry and computational MS/MS

fragmentation 318

633. Sample preparation composite and replicate strategy case studies for assay of solid oral drug

products 319

634. Separation and characterization of chemical constituents in Ginkgo biloba extract by off-line

hydrophilic interaction × reversed-phase two-dimensional liquid chromatography coupled with

quadrupole-time of flight mass spectrometry 319

635. Double-Track Electrochemical Green Approach for Simultaneous Dissolution Profiling of

Naproxen Sodium and Diphenhydramine Hydrochloride 320

636. A workflow for column interchangeability in liquid chromatography using modeling software

and quality-by-design principles 320

637. Absolute quantification of poly(dl-lactide-co-glycolide) in microspheres using quantitative 1H

NMR spectroscopy 321

638. A novel LC-MS/MS method for the quantitative measurement of the acetate content in

pharmaceutical peptides 321

639. A quality by design-based approach to a capillary electrokinetic assay for the determination of

dextromepromazine and levomepromazine sulfoxide as impurities of levomepromazine 322

640. An HPLC–MS/MS method for quantitation of Gly-MCA in mouse plasma: Application to a

pharmacokinetic study 322

641. Development and validation of a sensitive LC–MS/MS method without derivatization/ion-pairing

agents for etimicin quantification in rat plasma, internal ear and kidney 323

642. Bioanalysis of monomethyl fumarate in human plasma by a sensitive and rapid LC–MS/MS

method and its pharmacokinetic application 323

643. Simultaneous determination of tenofovir alafenamide and its active metabolites tenofovir and

tenofovir diphosphate in HBV-infected hepatocyte with a sensitive LC–MS/MS method 324

644. LC–MS/MS method for preclinical pharmacokinetic study of QX-OH, a novel long-acting local

anesthetic, in sciatic nerve blockade in rats 324

645. Capillary electrophoresis study on the base-catalyzed formation of bioactive oxidized metabolites

of 20-hydroxyecdysone 325

646. Quantification of the antimalarial drug pyronaridine in whole blood using LC–MS/MS —

Increased sensitivity resulting from reduced non-specific binding 325

647. Simultaneous extraction of propofol and propofol glucuronide from hair followed by validated

LC–MS/MS analyses 326

648. 11-nor-9-carboxy-Δ9-tetrahydrocannabinol glucuronide exhibits acyl-migration isomers 326

xxxvii

649. Simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human

serum using an immobilized trypsin 327

650. Development of quantitative immunochromatographic assay for rapid and sensitive detection of

carbohydrate antigen 19-9 (CA 19-9) in human plasma 327

651. Simultaneous determination of pentoxifylline, metabolites M1 (lisofylline), M4 and M5, and

caffeine in plasma and dried blood spots for pharmacokinetic studies in preterm infants and

neonates 328

652. Pharmacokinetics and tissue distribution of five major triterpenoids after oral administration of

Rhizoma Alismatis extract to rats using ultra high-performance liquid chromatography–tandem

mass spectrometry 328

653. Solid-phase microextraction coupled to gas chromatography–mass spectrometry followed by

multivariate data analysis for the identification of volatile organic compounds as possible

biomarkers in lung cancer tissues 329

654. UPLC–MS/MS assay of riluzole in human plasma and cerebrospinal fluid (CSF): Application in

samples from spinal cord injured patients 329

655. Simultaneous determination and pharmacokinetic study of twelve bioactive compounds in rat

plasma after intravenous administration of Xuebijing injection by UHPLC-Q-Orbitrap

HRMS 330

656. A surrogate analyte-based liquid chromatography-tandem mass spectrometry method for the

determination of endogenous cyclic nucleotides in rat brain 330

657. Determination of ketamine and its main metabolites by liquid chromatography coupled to tandem

mass spectrometry in pig plasma: Comparison of extraction methods 331

658. Ketamine metabolites with antidepressant effects: Fast, economical, and eco-friendly

enantioselective separation based on supercritical-fluid chromatography (SFC) and single

quadrupole MS detection 331

659. A new method based on supercritical fluid extraction for polyacetylenes and polyenes from

Echinacea pallida (Nutt.) Nutt. Roots 332

660. Validated LC–MS/MS method for the simultaneous determination of rotigotine and its prodrug

in rat plasma and an application to pharmacokinetics and biological conversion in vitro 332

661. Determination of higenamine in dietary supplements by UHPLC/MS/MS method 333

662. Determination of genotoxic epoxide at trace level in drug substance by direct injection

GC/MS 333

663. LC–MS/MS assay for the quantitation of the ribonucleotide reductase inhibitor triapine in human

plasma 334

664. Development and validation of a liquid chromatography-tandem mass spectrometry method for

pharmacokinetic study of TM-53, a novel transcriptional coactivator with PDZ-binding motif

(TAZ) modulator 334

665. Development of a sensitive LC–MS/MS method for quantification of coniferyl ferulate and its

metabolite coniferyl alcohol in rat plasma: Application to a pharmacokinetic study 335

666. LC–MS/MS assay for the quantitation of the ATR kinase inhibitor VX-970 in human

plasma 335

xxxviii

667. Quantitative determination of carfilzomib in mouse plasma by liquid chromatography–tandem

mass spectrometry and its application to a pharmacokinetic study 336

668. Development and implementation of a pass/fail field-friendly method for detecting sildenafil in

suspect pharmaceutical tablets using a handheld Raman spectrometer and silver colloids 336

669. Multi-residue determination of 47 organic compounds in water, soil, sediment and fish—Turia

River as case study 337

670. Separation of bioactive chamazulene from chamomile extract using metal-organic frame-

work 337

671. 1H NMR based pharmacometabolomics analysis of urine identifies metabolic phenotype of

clopidogrel high on treatment platelets reactivity in coronary artery disease patients 338

672. Permeability through the Caco-2 cell monolayer of 42 bioactive compounds in the TCM formula

Gegen-Qinlian Decoction by liquid chromatography tandem mass spectrometry analysis 338

673. Simultaneous quantification of arginine, alanine, methionine and cysteine amino acids in

supplements using a novel bioelectro-nanosensor based on CdSe quantum dot/modified carbon

nanotube hollow fiber pencil graphite electrode via Taguchi method 339

674. Simultaneous optimization of pH and binary organic composition by grid form modeling of the

retention behavior in reversed-phase ultra high-performance liquid chromatography 339

675. On-Line two dimensional liquid chromatography based on skeleton type molecularly imprinted

column for selective determination of sulfonylurea additive in Chinese patent medicines or

functional foods 340

676. Impact of chemotherapy on metabolic reprogramming: Characterization of the metabolic profile

of breast cancer MDA-MB-231 cells using 1H HR-MAS NMR spectroscopy 340

677. An integrated strategy for rapid discovery and identification of the sequential piperine

metabolites in rats using ultra high-performance liquid chromatography/high resolution mass

spectrometery 341

***

1


From chemical consistency to effective consistency in precise quality discrimination of Sophora

flower-bud and Sophora flower: Discovering efficacy-associated markers by fingerprint-activity

relationship modeling

Fei Wang, Zi-Yue Xiong, Ping Li, Hua Yang, Hui-Jun Li

ABSTRACT

Chromatographic fingerprint has been extensively used as a comprehensive approach for quality

evaluation of herbal medicines (HMs). However, similar chemical profiles do not always mean similar

efficacies. The present work, taking Sophora flower-bud and Sophora flower as a typical case,

attempts to develop a rational strategy based on fingerprint-activity relationship modeling to realize

quality evaluation from chemical consistency to effective consistency. A total of 57 batches of

Sophora samples were collected and their antioxidant and hyaluronidase inhibitory activities were

measured. Chemical fingerprints were established by high performance liquid chromatography

(HPLC) coupled with photodiode array (PDA) detector and quadrupole time-of-flight mass

spectrometry (Q-TOF MS), and similarity analyses were calculated based on eight common

characteristic peaks. Subsequently, three principal bioactive markers were discovered by correlating

biological effects with chemical fingerprints via partial least squares regression (PLSR) and back

propagation-artificial neural network modeling (BP-ANN). The selected markers were quantified by

the ‗single standard to determine multi-components‘ method, and then the quantitative data as well as

their bioactive properties were subjected to principal component analysis to generate two clear-cut

groups. This study not only demonstrates the necessity of effective consistency besides chemical

consistency in the quality evaluation of HMs, but also provides an applicable strategy to screen out

efficacy-associated markers by fingerprint-activity relationship modeling.

2

Impact of mono- and poly-ester fractions on polysorbate quantitation using mixed-mode HPLC-

CAD/ELSD and the fluorescence micelle assay

Steffen Lippold, Stijn H.S. Koshari, Robert Kopf, Rudolf Schuller, Henning Koehn

ABSTRACT

Determination of excipient content in drug formulation is an important aspect of pharmaceutical

formulation development and for analytical testing of the formulation. In this study, the influence of

polysorbate subspecies, in particular mono- and poly-esters, for determining polysorbate (PS) content

were investigated by comparing three of the most widely used PS quantitation approaches, the

Fluorescence Micelle Assay (FMA) and Mixed-Mode High Performance Liquid Chromatography

coupled with Charged Aerosol Detection (MM-CAD) or Evaporative Light Scattering Detection (MM-

ELSD). FMA and MM-CAD were employed to investigate the quantitation behavior of PS20 and

PS80 subspecies and corresponding degradation products in placebo formulations using forced

degradation conditions at 40 °C for up to 12 weeks. While both methods allowed accurate and

comparable quantification of neat PS at the beginning of stress studies, pronounced differences in

content determination between the methods were observed at later time points, which were attributable

to substantial differences in the contribution of individual mono- and poly-esters to the overall

quantitation results. It was particularly surprising to find that the main component of PS20,

polyoxyethylene sorbitan monolaurate, did not show a signal at the studied concentration using FMA.

Moreover, the degradation of polysorbate poly-esters, was reflected much stronger in FMA than MM-

CAD results. Additional experiments employing chemical oxidation and base hydrolysis to degrade

PS20, quantified by FMA and MM-ELSD, also show preferential reduction in certain subspecies

depending on the degradation pathway involved. For PS20 degraded by chemical oxidation,

quantitation results were lower for FMA than MM-ELSD, while the opposite trend was observed with

base hydrolysis.

3

Quality assessment of marketed chamomile tea products by a validated HPTLC method

combined with multivariate analysis

Etil Guzelmeric, Petar Ristivojević, Irena Vovk, Duńanka Milojković-Opsenica, Erdem Yesilada

ABSTRACT

Chamomile tea composed of dried flower heads of Matricaria recutita L. (Asteraceae) is one of the

most popular single ingredient herbal teas. Tea industries, spice shops or public bazaars are mostly

supplied chamomile as a raw material via cultivation or through nature-picking. However, one of the

drawbacks of nature-picking is adulteration. This could be either due to false authentication of the

plant materials by ingenuous pickers or intentional/unintentional substitution with other flowers

resembling to chamomile in appearance during harvesting. Therefore, quality control of raw

chamomile materials before marketing should be carefully considered not only by quantification of

apigenin 7-O-glucoside (active marker) but also by fingerprinting of chemical composition. This work

presents both quantification of apigenin 7-O-glucoside and chemical fingerprinting of commercial

chamomile tea products obtained from different food stores and spice shops by a validated HPTLC

method. In addition, HPTLC profiles of investigated chamomile tea samples were compared with

HPLC method stated in the European Pharmacopoeia and it was found that HPTLC method was

superior to HPLC method in the field of adulteration confirmation. Therefore, fingerprint profiles

performed on the silica gel 60 NH2 F254s HPTLC plates combined with pattern recognition

techniques of these marketed products were comparatively evaluated with wild and cultivar chamomile

samples and also chamomile-like species from Asteraceae. Consequently, not chamomile tea bags but

crude flowers sold on market were found to be adulterated with other plant materials.

Simultaneous quantification and identification of flavonoids, lignans, coumarin and amides in

leaves of Zanthoxylum armatum using UPLC-DAD-ESI-QTOF–MS/MS

Vinod Bhatt, Sushila Sharma, Neeraj Kumar, Upendra Sharma, Bikram Singh

ABSTRACT

The current study presents isolation and characterization of twelve compounds including catechin (1),

isovitexin (2), hesperidin (3), psoralin (4), eudesmin (5), kobusin (6), fargesin (7), sesamin (8),

asarinin (9), planispine-A (10), α-sanshool (11) and vitexin (12), from the leaves of Zanthoxylum

armatum. Further, two rapid and simple ultra performance liquid chromatography-diode array

detection (UPLC-DAD) methods were developed for the simultaneous quantitative determination of

isolated compounds from Z. armatum leaves. These analytical methods were validated for linearity,

precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ). The LOD and LOQ

were in the range of 0.06–0.21 μg/mL and 0.19–0.69 μg/mL, respectively. The validated method was

linear (R2 ≥ 0.9906), precise in terms of peak area (intra-day RSDs <3.8% and inter-day RSDs

<2.7%), and accurate (109.6–92.5%). This is the first report on the isolation and quantification of 1, 2,

4 and 12 in Z. armatum and 3 in Zanthoxylum genus. The methods: were successfully applied to assess

the quality of samples collected from different locations of Himachal Pradesh during summer and

winter season. The results demonstrated that flavonoids and furofuran lignans were the major

constituents in Z. armatum leaves. The developed methods: were further applied for tandem

electrospray ionization-mass spectrometry (UPLC-DAD-ESI-MS/MS) and total eighteen compounds

were identified including phenolic acid, flavonoids, furofuran lignans, coumarin and isobutyl amides.

4

Combination of HPLC–MS and QAMS as a new analytical approach for determination of

saponins in ginseng containing products

Andrey Stavrianidi, Elena Stekolshchikova, Anna Porotova, Igor Rodin, Oleg Shpigun

ABSTRACT

Conventional liquid chromatographic methods coupled with ultraviolet detection with low-wavelength

range are lacking selectivity and sensitivity to determine both polar and less polar ginsenosides. Also

the lack of standard substances for such quality control methods is leading to development of the

approaches using single standard for quantitative analysis of multi-component system (QAMS). The

objective of present study was to establish and compare for the first time liquid chromatography–

ultraviolet detection and liquid chromatography–mass spectrometry QAMS methods for the

simultaneous determination of protopanaxatriol-type and protopanaxadiol-type ginsenosides in a

variety of ginseng products. Sixteen polar and less polar ginsenosides were separated on a reversed-

phase C18-column (150mm × 2.0 mm, 2.2 μm) with a mobile phase consisting of 0.1% formic acid in

water and acetonitrile. Components were then detected by means of ultraviolet and mass spectrometry

detection. Characteristic sapogenin fragmentation signals with m/z 423 and 425 for two major groups

of ginseng saponins allowed their simultaneous determination in a single chromatographic run, while

the use of ultraviolet detection tends to give overvalued results. Structural correlation between the

relative response factors and saponin structure was demonstrated. The method was linear (R2 >0.999)

and sensitive (LODs, 0.01–0.03 mg/mL) within the concentration range tested. Concentrations of

individual ginsenosides and several quality control parameters were determined in ginseng root

extracts and commercial ginseng products of different types (root slices, tablets and tea samples), and

results showed that ginsenoside content can be successfully measured by means of QAMS approach.

Simultaneous determination of phenolic acids and flavonoids in Chenopodium formosanum

Koidz (djulis) by HPLC-DAD-ESI–MS/MS

B.Y. Hsu, S.W. Lin, B. Stephen Inbaraj, B.H. Chen

ABSTRACT

A high performance liquid chromatography-diode array detection-tandem mass spectrometry method

(HPLC-DAD-MS/MS) was developed for simultaneous determination of phenolic acids and

flavonoids in djulis (Chenopodium formosanum Koidz.), a traditional Chinese herb reported to possess

vital biological activities. A high yield of phenolic acids and flavonoids was attained by employing

50% ethanol in water as the extraction solvent and shaking in a 60 °C water bath for 3 h. A total of 8

phenolic acids and 14 flavonoids were separated and identified within 55 min by using a Poroshell 120

EC-C18 column with detection at 280 nm, flow rate at 0.8 mL/min, column temperature at 35 °C, and

a gradient solvent system of 0.1% formic acid in water and acetonitrile. Two internal standards caffeic

acid and kaempferol-3-O-rutinoside were used for quantitation of phenolic acids and flavonoids in

djulis respectively. The amounts of phenolic acids ranged from 11.5 ± 0.8 μg/g (caffeoyl-putrescine-

derivative (2)) to 1855.3 ± 16.9 μg/g (hydroxylphenylacetic acid pentoside), while the flavonoids

ranged from 19.93 ± 2.29 μg/g (quercetin-3-O-(coumaryl)-rutinoside-pentoside (1)) to 257.3 ± 2.05

μg/g (rutin-O-pentoside (2)). A high recovery (89.68–97.20%) and high reproducibility was obtained

for both phenolic acids and flavonoids with the relative standard deviation (RSD) for the latter ranging

from 0.09–8.22% (intra-day variability) and 0.80–8.48% (inter-day variability). This method may be

applied to determination of both phenolic acids and flavonoids in food products and Chinese herbs.

5

Design of a strong cation exchange methodology for the evaluation of charge heterogeneity in

glatiramer acetate

Víctor R. Campos-García, Carlos A. López-Morales, Eleuterio Benites-Zaragoza, Armando Jiménez-

Miranda, E. Medina-Rivero

ABSTRACT

Complex pharmaceuticals are in demand of competent analytical methods able to analyze charge

heterogeneity as a critical quality attribute (CQA), in compliance with current regulatory expectations.

A notorious example is glatiramer acetate (GA), a complex polypeptide mixture useful for the

treatment of relapsing-remitting multiple sclerosis. This pharmaceutical challenges the current state of

analytical technology in terms of the capacity to study their constituent species. Thus, a strong cation

exchange methodology was designed under the lifecycle approach to support the establishment of GA

identity, trough the evaluation of its chromatographic profile, which acts as a charge heterogeneity

fingerprint. In this regard, a maximum relative margin of error of 5% for relative retention time and

symmetry factor was proposed for the analytical target profile. The methodology met the proposed

requirements after precision and specificity tests results, the former comprised of sensitivity and

selectivity. Subsequently, method validation was conducted and showed that the method is able to

differentiate between intact GA and heterogeneity profiles coming from stressed, fractioned or

process-modified samples. In summary, these results provide evidence that the method is adequate to

assess charge heterogeneity as a CQA of this complex pharmaceutical.

Development and characterization of the voriconazole loaded lipid-based nanoparticles

Petra Füredi, Zsófia Edit Pápay, Kristóf Kovács, Borbála Dalmadi Kiss, Imre Klebovich

ABSTRACT

The number of topical fungal infections is growing, mostly owing to immunosuppressive therapy.

Several topical fungal infections, such as eye mycoses, can be treated by local administration of

antimycotic drugs. One major group of the antifungal agents is triazole, such as voriconazole (VCZ),

which is used as the first line treatment of aspergillosis. A disadvantage of VCZ is its low water

solubility making the drug difficult to administer in a liquid preparation. The lipid-based nanoparticles

(LNP) have attracted increasing attention due to their advantageous properties. Contrarily to the

conventional carrier systems, LNP can improve the poor solubility of topically used drugs, such as

VCZ. Therefore, LNP represents promising alternatives to traditional carrier systems. The aim of the

study was to formulate VCZ loaded lipid-based nanoparticles (VCZ-LNP) by high pressure

homogenization (HPH). The developed LNPs were characterized by particle size analysis, IR

spectroscopy, differential scanning calorimetry, dialysis test and antifungal efficacy studies. The

particle size of the optimized nanoparticles from the selected lipid base, Witepsol® W35, was 182 ±

4.1 nm after five cycles of homogenization at 600 bars. The antifungal study confirmed that the

optimized VCZ-LNP inhibited the fungus reproduction.

6

Selective functional activity measurement of a PEGylated protein with a modification-dependent

activity assay

Alfred Weber, Andrea Engelmaier, Gabriele Mohr, Sonja Haindl, Peter L. Turecek

ABSTRACT

BAX 855 (ADYNOVATE) is a PEGylated recombinant factor VIII (rFVIII) that showed prolonged

circulatory half-life compared to unmodified rFVIII in hemophilic patients. Here, the development and

validation of a novel assay is described that selectively measures the activity of BAX 855 as cofactor

for the serine protease factor IX, which actives factor X. This method type, termed modification-

dependent activity assay, is based on PEG-specific capture of BAX 855 by an anti-PEG IgG

preparation, followed by a chromogenic FVIII activity assay. The assay principle enabled sensitive

measurement of the FVIII cofactor activity of BAX 855 down to the pM-range without interference by

non-PEGylated FVIII. The selectivity of the capture step, shown by competition studies to primarily

target the terminal methoxy group of PEG, also allowed assessment of the intactness of the attached

PEG chains. Altogether, the modification-dependent activity not only enriches, but complements the

group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex

biological matrices. In contrast to all other methods described so far, it allows measurement of the

biological activity of the PEGylated protein. Data obtained demonstrate that this new method principle

can be extended to protein modifications other than PEGylation and to a variety of functional activity

assays.

Investigation of the effect of mobile phase composition on selectivity using a solvent-triangle

based approach in achiral SFC

Charlene Muscat Galea, Debby Mangelings, Yvan Vander Heyden

ABSTRACT

Defining a method development methodology for achiral drug impurity profiling in SFC requires a

number of steps. Initially, diverse stationary phases are characterized and a small number of

orthogonal or dissimilar phases are selected for further method development. In this paper, we focus

on a next step which is the investigation of the modifier composition on chromatographic selectivity. A

solvent-triangle based approach is used in which blends of organic solvents, mainly ethanol (EtOH),

propanol (PrOH), acetonitrile (ACN) and tetrahydrofuran (THF) mixed with methanol (MeOH) are

tested as modifiers on six dissimilar stationary phases. The tested modifier blends were composed to

have equal eluotropic strengths as calculated on bare silica. The modifier leads to minor changes in

terms of elution order, retention and mixture resolution. However, varying only the modifier

composition on a given stationary phase does not lead to the creation of dissimilar systems. Therefore

the modifier composition is an optimization parameter, with the stationary phase being the factor

determining most the selectivity of a given mixture in achiral SFC.

7

NQO1 and CYP450 reductase decrease the systemic exposure of rifampicin-quinone and

mediate its redox cycle in rats

Fuguo Shi, Xiaobing Li, Hong Pan, Li Ding

ABSTRACT

Rifampicin (RIF) is used in regimens for infections caused by Mycobacteria accompanied by serious

adverse reactions. Rifampicin-quinone (RIF-Q) is a major autoxidation product of RIF. It is not clear

whether RIF-Q plays a role in RIF induced adverse reactions. Investigation of the systemic exposure of

RIF-Q is helpful to better understand the role of RIF-Q in RIF induced adverse reactions. In this study,

a simple and reproducible high performance liquid chromatography-mass spectrometry (LC–MS)

method involving a procedure to prevent the RIF from oxidation for simultaneous quantification of

RIF and RIF-Q in rat plasma has been developed and validated, and applied to elucidate the systemic

exposure of RIF-Q in rats. The pharmacokinetics data showed that the systemic exposure of RIF-Q

was very low (0.67% of RIF, AUC0-24) in rats after oral administration of RIF. However, RIF-Q may

undergo the redox cycle in vivo by the evidence that the majority of RIF-Q was reduced to RIF after

an oral dose of RIF-Q. Pretreatment with the NAD (P) H: quinone oxidoreductase 1 (NQO1) specific

inhibitor dicoumarol and/or cytochrome P450 reductase (CPR) inhibitor diphenyleneiodonium

suppressed the redox cycle and significantly increased the systemic exposure of RIF-Q. The inhibitors

also attenuated the redox cycle induced reactive oxygen species formation and cytotoxicity in RIF-Q-

treated HepG2 cells. These results indicate that NQO1 and CPR play an important role in redox cycle

of RIF-Q and may thus contribute to RIF-induced adverse reactions.

Study the influence of licorice and pomegranate drinks on nicotine metabolism in human urine

by LC-orbitrap MS

Ahmad Abu-awwad, Tawfiq Arafat, Oliver J. Schmitz

ABSTRACT

Nicotine-diet interactions have a particular importance on human health. Some food substances are

subject to change hepatic CYP2A6 metabolism rate for nicotine and its levels in smokers

consequently. This study investigates the effect of pomegranate and licorice drinks on nicotine

metabolism, by a new developed and validated method for simultaneous determination of nicotine with

its major metabolites (cotinine and nicotine N-oxide) in human urine, utilizing LC ESI-orbitrap-MS.

Twenty-four Jordanian healthy and smoker volunteers were participated in two equal groups,

crossover design for each of pomegranate and licorice test drink. In the study periods each group

assigned either to drink test juice three times a day or to be avoided from test drink for 7 successive

days, and then both groups switched their drink treatment in subsequent period. Early morning urine

samples were collected from all volunteers after each period. Nicotine metabolism rate was evaluated

from nicotine/cotinine and nicotine/nicotine N-oxide ratios in urine. A consistent trend of increase in

metabolism rate for nicotine was observed from urine analysis under pomegranate or licorice drink

conditions compared to control conditions. Pomegranate and licorice drinks are increasing the

metabolism rate for nicotine in terms of induction effect for hepatic cytochrome p450 enzymes.

8

NMR-based metabonomics and correlation analysis reveal potential biomarkers associated with

chronic atrophic gastritis

Jiajia Cui, Yuetao Liu, Yinghuan Hu, Jiayu Tong, Guanhua Du

ABSTRACT

Chronic atrophic gastritis (CAG) is one of the most important pre-cancerous states with a high

prevalence. Exploring of the underlying mechanism and potential biomarkers is of significant

importance for CAG. In the present work, 1H NMR-based metabonomics with correlative analysis was

performed to analyze the metabolic features of CAG. 19 plasma metabolites and 18 urine metabolites

were enrolled to construct the circulatory and excretory metabolome of CAG, which was in response

to alterations of energy metabolism, inflammation, immune dysfunction, as well as oxidative stress. 7

plasma biomarkers and 7 urine biomarkers were screened to elucidate the pathogenesis of CAG based

on the further correlation analysis with biochemical indexes. Finally, 3 plasma biomarkers (arginine,

succinate and 3-hydroxybutyrate) and 2 urine biomarkers (α-ketoglutarate and valine) highlighted the

potential to indicate risks of CAG in virtue of correlation with pepsin activity and ROC analysis. Here,

our results paved a way for elucidating the underlying mechanisms in the development of CAG, and

provided new avenues for the diagnosis of CAG and presented potential drug targets for treatment of

CAG.

Metabolomic profiling of doxycycline treatment in chronic obstructive pulmonary disease

Brajesh Singh, Saikat K. Jana, Nilanjana Ghosh, Soumen K. Das, Koel Chaudhury

ABSTRACT

Serum metabolic profiling can identify the metabolites responsible for discrimination between

doxycycline treated and untreated chronic obstructive pulmonary disease (COPD) and explain the

possible effect of doxycycline in improving the disease conditions. 1H nuclear magnetic resonance

(NMR)-based metabolomics was used to obtain serum metabolic profiles of 60 add-on doxycycline

treated COPD patients and 40 patients receiving standard therapy. The acquired data were analyzed

using multivariate principal component analysis (PCA), partial least-squares-discriminant analysis

(PLS-DA), and orthogonal projection to latent structure with discriminant analysis (OPLS-DA). A

clear metabolic differentiation was apparent between the pre and post doxycycline treated group. The

distinguishing metabolites lactate and fatty acids were significantly down-regulated and formate,

citrate, imidazole and l-arginine upregulated. Lactate and folate are further validated biochemically.

Metabolic changes, such as decreased lactate level, inhibited arginase activity and lowered fatty acid

level observed in COPD patients in response to add-on doxycycline treatment, reflect the anti-

inflammatory action of the drug. Doxycycline as a possible therapeutic option for COPD seems

promising.

9

Analysis of fenretinide and its metabolites in human plasma by liquid chromatography–tandem

mass spectrometry and its application to clinical pharmacokinetics

Hwang Eui Cho, H. Kang Min

ABSTRACT

A simple and accurate high-performance liquid chromatography–tandem mass spectrometry (LC–

MS/MS) method was developed for the determination of N-(4-hydroxyphenyl) retinamide (fenretinide,

4-HPR) and its metabolites, 4-oxo-N-(4-hydroxyphenyl) retinamide (4-oxo-4-HPR) and N-(4-

methoxyphenyl) retinamide (4-MPR), in human plasma. Plasma samples were prepared using protein

precipitation with ethanol. Chromatographic separation of the three analytes and N-(4-ethoxyphenyl)

retinamide (4-EPR), an internal standard, was achieved on a Zorbax SB-C18 column (3.5 μm, 50 × 2.1

mm) using gradient elution with the mobile phase of 0.1% formic acid in water and acetonitrile (pH*

2.4) at a flow rate of 0.5 mL/min. Electrospray ionization (ESI) mass spectrometry was operated in the

positive ion mode with multiple reaction monitoring (MRM). The calibration curves obtained were

linear over the concentration range of 0.2–50 ng/mL with a lower limit of quantification of 0.2 ng/mL.

The relative standard deviation of intra-day and inter-day precision was below 7.64%, and the

accuracy ranged from 94.92 to 105.43%. The extraction recoveries were found to be higher than

90.39% and no matrix effect was observed. The analytes were stable for the durations of the stability

studies. The validated method was successfully applied to the analyses of the pharmacokinetic study

for patients treated with 4-HPR in a clinical trial.

Poor and enantioselective bioavailability of naftopidil enantiomers is due to extensive and

stereoselective metabolism in rat liver

Xiawen Liu, Lijun Zhu, Biyun Huang, Junjun Huang, Mu Yuan

ABSTRACT

Racemic naftopidil (NAF) is used to treat benign prostatic hyperplasia (BPH) and prostatic cancer

(PCa). It exhibits greater efficacy but requires higher dose than other ɑ1-adrenoceptor blockers

because of its poor bioavailability. It was previously shown that bioavailability of S (−)-NAF (14.5%)

was twice that of R (+)-NAF (6.8%). The present study aimed to elucidate the major factors

contributing to the poor and enantioselective bioavailability of NAF. First, absorption of NAF

enantiomers was examined using a perfusated intestinal model. NAF enantiomers were found to be

equally and highly permeable in all segments of the intestine. Second, the metabolites formed in

different parts of the intestine and in bile were investigated. Glucuronidation of NAF enantiomers was

found to occur primarily in the liver. Third, a new method consisting of ultra performance liquid

chromatography coupled with triple-quadruple mass spectrometry (UPLC–MS/MS) was employed to

quantify and calculate the pharmacokinetic parameters of NAF enantiomers and their glucuronides

after the enantiomers were intravenously injected into rats. The amounts of R(+)-NAF glucuronide

(R(+)-NAF-G) and S(−)-NAF glucuronide (S(−)-NAF-G) were six-fold higher than that of R(+)-NAF,

and three-fold higher than that of S(−)-NAF. Glucuronidation of S(−)-NAF was faster than that of

R(+)-NAF, but the conjugated amount was half of that of R(+)-NAF. Thus, bioavailability of S(−)-

NAF was twice that of R(+)-NAF. In conclusion, extensive phase II metabolism in the liver

significantly contributes to the low bioavailability of NAF enantiomers. Glucuronidation is the most

important metabolic pathway for NAF enantiomers. Glucuronidation of S(−)-NAF is faster but occurs

to a lesser extent than that of R(+)-NAF.

10

Robust HPLC–MS/MS method for levofloxacin and ciprofloxacin determination in human

prostate tissue

O. Szerkus, J. Jacyna, A. Gibas, M. Sieczkowski, M.J. Markuszewski

ABSTRACT

Fluoroquinolones are the drugs of choice in the prevention of bacterial infections after transrectal

ultrasound guided prostate biopsy. In order to improve assessment of antibacterial efficacy in the target

tissue a simple, selective, rapid and robust HPLC-ESI–MS/MS method for the determination of

levofloxacin and ciprofloxacin concentrations in human prostate bioptates was developed and

validated. Preparation procedure for prostate samples (10 mg) was carried out using homogenization

and filtration steps. Analyses were performed within 3.5 min using RP column in the isocratic elution

mode with mobile phase composed of a mixture of 0.1% formic acid aqueous solution and 0.1%

formic acid methanol solution (v/v; 79:21). The method was linear between 0.3 μg/g and 15 μg/g for

levofloxacin and ciprofloxacin with coefficient of correlation (r) ≥0.999. The limit of detection and the

limit of quantification for levofloxacin were 0.06 μg/g and 0.2 μg/g and for ciprofloxacin were 0.04

μg/g and 0.13 μg/g, respectively. Average concentrations (±SD) of levofloxacin and ciprofloxacin

obtained from patient tissue were 5.4 ± 2.2 μg/g and 3.9 ± 1.5 μg/g, respectively. Additionally, during

validation procedure a novel, experimental design approach was applied for the robustness study. For

evaluation of analytical method robustness, Plackett-Burman design was employed and for sample

preparation method robustness Fractional Factorial design was used. The developed and validated

method was successfully applied to examine prostate tissue samples obtained from patients enrolled

into a clinical study. Up to now, there has been no other HPLC-ESI-MS/MS method reported for the

simultaneous determination of levofloxacin and ciprofloxacin in human prostatic tissue.

Pharmacokinetics, excretion of 8-cetylberberine and its main metabolites in rat urine

Yuli Hu, Shoujun Fan, Xiaobing Liao, Chao Chen, Xuegang Li

ABSTRACT

The Berberine (BBR) is a bioactive plant ingredient derived from the roots and barks of Berberis

aristata and Coptis chinensis and has a wide variety of pharmacological effects. 8-cetylberberine (8-

BBR-C16) is the berberine (BBR) derivative reconstructed from adding octadecyl at C-8 of BBR to

enhance its activity. This study presents a reliable method for the determination of BBR and 8-BBR-

C16 in rat plasma, urine and feces. BBR and 8-BBR-C16 were determined by HPLC-UV after liquid-

liquid extraction for plasma samples, and solid-phase extraction for urinary and fecal samples. The

method was linear over the concentration range of 10-300 ng·ml−1 for the plasma samples, 25-2000

ng·ml−1 for the urinary samples, and 100-2000 ng·g−1 for the fecal samples. Furthermore, a metabolic

investigation on urine was performed by LC/MS/MS analysis to identify the structures of 8-BBR-C16

metabolites by full scan and product ion scan. Adult Sprague-Dawley rats were divided into two

groups. In the control group, rats received 80 mg·kg−1 BBR, and in the drug-treated group, rats

received 80 mg·kg−1 8-BBR-C16. The results indicate that there were significant differences in the

pharmacokinetic parameters and in the accumulated excretion levels between the control group and the

drug-treated group. The Cmax and AUC0-t of 8-BBR-C16 were 2.8 and 12.9 times higher than those

of BBR, and the relative bioavailability of BBR to 8-BBR-C16 was 7.7%. The total excretion amount

through the urine and feces of 8-BBR-C16 was 76.9%, but that of BBR was only 20.5%. Additionally,

8-BBR-C16 was metabolized in rat urine with phase I demethylation and phase II glucuronidation or

sulfation.

11

An UPLC–MS/MS method for the quantitation of alectinib in rat plasma

Xiang-xin Huang, Yun-xuan Li, Xiang-yu Li, Xiao-xia Hu, Guo-xin Hu

ABSTRACT

Currently, crizotinib is the first generation drug, which has been used in the treatment of ALK-

rearranged non-small cell lung cancer (NSCLC). However, more and more patients are found in

crizotinib-resistance. In the last year, alectinib has been approved for treatment of patients with

crizotinib-resistance. In this study, we aim to develop and validate a simple, rapid and sensitive tandem

mass spectrometry (UHPLC–MS/MS) method for determination of alectinib in rat plasma. Diazepam

was chosen as an internal standard (IS). Protein precipitation by acetonitrile was utilized to prepare

plasma samples. Chromatographic separation was achieved on a RRHD Eclipse plus C18 (2.1 × 50

mm, 1.8 μ) column with a gradient mobile phase consisting of acetonitrile and water (containing 0.1%

formic acid). The analytes were detected by an electrospray ionization (ESI) source in positive mode.

A dynamic multiple reactions monitoring (MRM) method was developed to detect specific precursor

and product ions. The target fragment ions were m/z 483.2 → 396.1 for alectinib and m/z 285.0 →

192.9 for diazepam (IS). Linear calibration plots were achieved in the range of 1–500 ng/ml for

alectinib (R2 = 0.997) in rat plasma. Mean recoveries of alectinib in rat plasma ranged from 84.2% to

92.2%. The intra- and inter-day precision was below 9.3% and accuracy was from −1.4% to 12.1%. No

obvious matrix effect was found. This method shows a good performance: accuracy, precision and

stability. It has been fully validated and successfully applied to pharmacokinetic study of alectinib.

Development and validation of a rapid and sensitive UPLC–MS/MS method for quantification of

kukoamine B in human plasma: Application to a clinical pharmacokinetic study

Zhenlei Wang, Qian Zhao, Lili Li, Pei Hu, Ji Jiang

ABSTRACT

A rapid, accurate and robust method was firstly developed using ultra-performance liquid

chromatography tandem mass spectrometry (UPLC–MS/MS) assay to quantify kukoamine B, which is

a novel drug under clinical development for the treatment of sepsis, in human plasma. Solid-phase

extraction (SPE) was used to extract kukoamine B from human plasma. The extracts were separated on

a Waters Acquity HSS T3 column (2.1 × 50 mm i.d., 1.8 μm) with a gradient elution method, using

mobile phases of A (formic acid-water (1:1000, v/v)) and B(formic acid-methanol (1:1000, v/v)).

Kukoamine B and internal standard (5-deuterated isotope kukoamine B) were detected under the

multiple-reaction monitoring mode by an API 5500 triple quadrupole mass spectrometer with

electrospray ionization. The method showed good linearity from 0.100 to 50.0 ng/mL according to

1/x2 weighted linear regression analysis. Inter- and intra-batch precision of kukoamine B were less

than 15% and the accuracy was within 85–115%. The extraction recoveries and matrix effect of

kukoamine B at three concentration levels were consistent. The sensitivity, specificity and stabilities

under various conditions were validated. In conclusion, the validation results showed that this method

was rapid, accurate, robusts and can successfully fulfill the requirement of clinical pharmacokinetic

study of kukoamine B mesylate in Chinese healthy subjects.

12

Quantitative determination of xanthorrhizol in rat plasma by HPLC–MS/MS and its application

to a pharmacokinetic study

Seungmok Choi, Minsoo Kim, Changhee Kim, Jae-Kwan Hwang, Wonku Kang

ABSTRACT

Although xanthorrhizol, a sesquiterpenoid oil isolated from the rhizoma of Curcuma xanthorrhiza

Roxb. Known as Java turmeric, represents a variety of pharmacological activities, to date, there have

been no validated determination methods of xanthorrhizol in biological samples. Thus, we developed a

liquid chromatographic method using a tandem mass spectrometry for the determination of

xanthorrhizol in rat plasma. After simple protein precipitation with acetonitrile including diclofenac

(internal standard, IS), the analytes were chromatographed on a reversed-phased column with a mobile

phase of 20 mM ammonium acetate aqueous solution and acetonitrile (20:80, v/v). The ion transitions

of the precursor to the product ion were principally deprotonated ions [M−H]− at m/z 216.9 → 132.8

for xanthorrhizol and 296.1 → 251.7 for the IS. The accuracy and precision of the assay were in

accordance with FDA regulations for the validation of bioanalytical methods. This analytical method

was successfully applied to monitor plasma concentrations of xanthorrhizol over time following

intravenous administration in rats.

Development and validation of a stability-indicating HPLC-UV method for the determination of

Thiocolchicoside and its degradation products

Silvio Aprile, Rossana Canavesi, Michele Bianchi, Giorgio Grosa, Erika Del Grosso

ABSTRACT

A stability indicating high performance liquid chromatography method has been developed for the

determination of thiocolchicoside (TCC) and its main degradation products thiocolchicoside S-oxide

(D1SO) and 3-O-demethylthiocolchicine (D3) in liquid and solid formulations. The method was

developed based on a previous forced degradation study showing that TCC underwent chemical

degradation by acid/base catalyzed hydrolysis and oxidation being the main degradation products D3

and D1SO respectively. The analytes separation and quantification were achieved on a Synergi™ 4 μm

Polar-RP 80 Å, column 150 × 4.6 mm (Phenomenex) using the mobile phase constituted (flow rate 1

mL min−1) of eluant A: 20 mM sodium acetate buffer (pH 5.0) and eluant B: MeOH:CH3CN (20:80);

the elution was performed in gradient mode detecting the analytes at 254 nm. The method showed

linearity for TCC assay in the 5–15 μg mL−1, range and for unknown (TCCfu) and known (D1SO and

D3) degradation products assay, in the 0.5–10 μg mL−1 range: all the square of the correlation

coefficients were greater than 0.999. The precision, determined in terms of intra-day and inter-day

were expressed as RSDs and resulted to be 1.19, 1.10, 1.37 and 1.04% and 0.95, 0.83, 1.30 and 0.72

for TCC, TCCfu, D1SO and D3, respectively. The method demonstrated also to be accurate; indeed,

the average recoveries were 102.1/102.0% for TCC (ampoules and hard capsules respectively),

101.3/100.3% for TCCfu, 101.7/100.2% for D1SO, and 101.4/101.4% for D3. The robustness was also

evaluated by variations of mobile phase composition and pH. Finally, the applicability of the method

was evaluated by analysis of commercial liquid and solid dosage forms.

13

Dried blood spot analysis of gabapentin as a valid alternative for serum: a bridging study

Nele Sadones, Elien Van Bever, Luc Van Bortel, Willy E. Lambert, Christophe P. Stove

ABSTRACT

We evaluated the applicability of a validated GC–MS method for the determination of gabapentin in

dried blood spots (DBS). Important for the acceptance of DBS sampling as an alternative sampling

strategy is the possibility to base solid conclusions on the quantification. Therefore, bridging studies –

studies in which the correlation between both DBS and a reference matrix (e.g. serum) is evaluated

statistically- need to be conducted. To this end, a comparative study was set up to quantify gabapentin

in both blood (DBS) and serum samples. Statistically significant differences between DBS and serum

concentrations were found (p < 0.001). A mean blood-to-serum ratio of 0.85 was observed, which is in

line with expectations. Calculated serum concentrations (obtained by dividing the DBS concentrations

by 0.85) demonstrated a good correlation with measured serum concentrations, with 87% of samples

fulfilling the criterion for incurred sample reanalysis. Furthermore, our data indicate a good correlation

between capillary and venous concentrations. Conclusively, this study demonstrated that DBS are a

valid alternative to serum for the determination of gabapentin.

SPR-based assays enable the full functional analysis of bispecific molecules

W. Meschendoerfer, C. Gassner, F. Lipsmeier, J.T. Regula, J. Moelleken

ABSTRACT

The increasing complexity of novel biotherapeutics such as bispecific antibodies or fusion proteins

raises new challenges for functional characterization. When compared to standard antibodies, two

individual interactions and the inter-dependency of binding events need to be considered for bispecific

antibodies. We have previously described an SPR-based assay setup, which enables us to assess the

binding activity of a bivalent-bispecific molecule to both targets simultaneously and − in addition to

one individual target − in a single setup. However, there might be some pitfalls when applying the

bridging assay, e.g. change of antigen activity upon immobilization. Therefore, we have developed an

alternative SPR-based assay principle, which allows the individual assessment of both targets in

solution. Comparison of data between the assays showed that simultaneous binding can be calculated

based on both individual readouts, and revealed a good correlation. Hence, both SPR-based assay

principles allow a ―full‖ functional analysis of a bispecific CrossMab in only one assay. The assay

principles can be qualified and enable an efficient drug development.

14

Angiotensin converting enzyme immobilized on magnetic beads as a tool for ligand fishing

Fernando G. de Almeida, Kenia L. Vanzolini, Quezia B. Cass

ABSTRACT

Angiotensin converting enzyme (ACE) presents an important role in blood pressure regulation, since

that converts angiotensin I to the vasoconstrictor angiotensin II. Some commercially available ACE

inhibitors are captopril, lisinopril and enalapril; due to their side effects, naturally occurring inhibitors

have been prospected. In order to endorse this research field we have developed a new tool for ACE

ligand screening. To this end, ACE was extracted from bovine lung, purified and chemically

immobilized in modified ferrite magnetic beads (ACE-MBs). The ACE-MBs have shown a Michaelian

kinetic behavior towards hippuryl-histidyl-leucine. Moreover, as proof of concept, the ACE-MBs were

inhibited by lisinopril with a half maximal inhibitory concentration (IC50) of 10 nM. At the fishing

assay, ACE-MBs were able not only to fish out the reference inhibitor, but also one peptide from a

pool of tryptic digested BSA. In conclusion, ACE-MBs emerge as new straightforward tool for ACE

kinetics determination, inhibition and binder screening.

MALDI-MS analysis and theoretical evaluation of olanzapine as a UV laser desorption

ionization (LDI) matrix

Syed Ghulam Musharraf, Mariam Ameer, Arslan Ali

ABSTRACT

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) being soft ionization

technique, has become a method of choice for high-throughput analysis of proteins and peptides. In

this study, we have explored the potential of atypical anti-psychotic drug olanzapine (OLZ) as a matrix

for MALDI-MS analysis of peptides aided with the theoretical studies. Seven small peptides were

employed as target analytes to check performance of olanzapine and compared with conventional

MALDI matrix α-cyano-4-hydroxycinnamic acid (HCCA). All peptides were successfully detected

when olanzapine was used as a matrix. Moreover, peptides angiotensin Ι and angiotensin ΙΙ were

detected with better S/N ratio and resolution with this method as compared to their analysis by HCCA.

Computational studies were performed to determine the thermochemical properties of olanzapine in

order to further evaluate its similarity to MALDI matrices which were found in good agreement with

the data of existing MALDI matrices.

15

A simple and rapid UHPLC–MS/MS method for the quantitation of the dual aurora kinase A/B

inhibitor SCH-1473759 in murine plasma

Marco A. Ferraz Nogueira Filho, Cody J. Peer, Jeffers Nguyen, Amy McCalla, William D. Figg

ABSTRACT

The Aurora kinase family facilitates cell division through various processes and is overexpressed in a

wide variety of human cancers, leading to aneuploidy. For that reason, these enzymes are currently

targets of a rising class of anticancer drugs, with some molecules already in therapeutic use. In this

study, a new UHPLC–MS/MS method was developed and validated to quantitate a new pan Aurora

kinase inhibitor still in preclinical development, SCH-1473759. This bioanalytical method employed a

liquid–liquid extraction from plasma using ethyl acetate before evaporation. Calibration range

encompassed 0.5–2500 ng/mL. The inter- and intra-day accuracy and precision were assessed over

five quality control levels; all within limits required by the FDA guidelines. Assay applicability was

demonstrated in a first-in-animals study with oral administration, where the maximum plasma

concentration (34 ng/mL) occurred at 1 h, the half-life (1 h) was consistent with a previous IV study,

and oral bioavailability was poor (F = 0.002).

Immobilized aptamer paper spray ionization source for ion mobility spectrometry

Tahereh Zargar, Taghi Khayamian, Mohammad T. Jafari

ABSTRACT

A selective thin-film microextraction based on aptamer immobilized on cellulose paper was used as a

paper spray ionization source for ion mobility spectrometry (PSI-IMS), for the first time. In this

method, the paper is not only used as an ionization source but also it is utilized for the selective

extraction of analyte, based on immobilized aptamer. This combination integrates both sample

preparation and analyte ionization in a Whatman paper. To that end, an appropriate sample

introduction system with a novel design was constructed for the paper spray ionization source. Using

this system, a continuous solvent flow works as an elution and spray solvent simultaneously. In this

method, analyte is adsorbed on a triangular paper with immobilized aptamer and then it is desorbed

and ionized by elution solvent and applied high voltage on paper, respectively. The effects of different

experimental parameters such as applied voltage, angle of paper tip, distance between paper tip and

counter electrode, elution solvent type, and solvent flow rate were optimized. The proposed method

was exhaustively validated in terms of sensitivity and reproducibility by analyzing the standard

solutions of codeine and acetamiprid. The analytical results obtained are promising enough to ensure

the use of immobilized aptamer paper-spray as both the extraction and ionization techniques in IMS

for direct analysis of biomedicine.

16

Urine metabolomics of high-fat diet induced obesity using UHPLC-Q-TOF-MS

Lihui Men, Zifeng Pi, Yuan Zhou, Mengying Wei, Zhongying Liu

ABSTRACT

Obesity has become a global epidemic and public health challenge which associates with serious

health issues including diabetes, cardiovascular disease, stroke, arthritis, and some types of cancer. To

better understand obesity and obesity-related dysfunction, a high-fat diet (HFD) induced obese model

was developed on Sprague-Dawley rats. Metabolomics based on ultra high-performance liquid

chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was

untilized to identify and analyze obesity related metabolites in rat urine samples. Multivariate analyses

were applied to differentiate metabolite patterns between HFD group and normal group. The study

successfully identified 20 altered urine metabolites that correlated with obesity. These metabolites are

mainly involved in tryptophan metabolism, phenylalanine and tyrosine metabolism, gut microbiota

metabolism and insulin resistance related metabolism. They could serve as potential biomarkers to

diagnose the development of obesity.


Psidium guajava L. leaves as source of proanthocyanidins: Optimization of the extraction

method by RSM and study of the degree of polymerization by NP-HPLC-FLD-ESI-MS

Elixabet Díaz-de-Cerio, Federica Pasini, Vito Verardo, Alberto Fernández-Gutiérrez, Maria Fiorenza

Caboni

ABSTRACT

Due to the importance of the proanthocyanidins (PAs) bioactivity and its relationship with the PAs

degree of polymerization (DP), an experimental design was carried out to establish the best extraction

conditions in order to evaluate the proanthocyanidins content and their degree of polymerization in

Psidium guajava leaves at different oxidation state. Optimal conditions achieved by response surface

methodology were 50% acetone/water (v/v), 48 °C, 30 min, and 0% acetic acid (v/v). The highest DP

has been found in the low oxidized state (DP 13 plus the polymers). Medium and high oxidized state

leaves reported a DP 11 plus the polymers. The total amounts of proanthocyanidins (sum of PAs by

HPLC-FLD-ESI-MS) decreased when oxidation state of leaves increased (15.8 ± 0.4, 12.6 ± 0.4, and

10.5 ± 0.3 mg/g leaf dry weight (d.w.) in low, medium and high oxidized state leaves, respectively).

Guava leaves present an interesting source of low DP-PAs.

17

Identification, synthesis and structural characterization of process related and degradation

impurities of acrivastine and validation of HPLC method

Ajay Kumar, Subba Rao Devineni, Shailender Kumar Dubey, Pradeep Kumar, Pramod Kumar

ABSTRACT

Four impurities (Imp-I–IV) were detected using gradient HPLC method in few laboratory batches of

acrivastine in the level of 0.03–0.12% and three impurities (Imp-I–III) were found to be known and

one (Imp-IV) was unknown. In forced degradation study, the drug is degraded into four degradation

products under oxidation and photolytic conditions. Two impurities (Imp-III and -IV) were concurred

with process related impurities whereas Imp-V and -VI were identified as new degradation impurities.

Based on LC-ESI/MSn study, the chemical structures of new impurities were presumed as 1-[(2E)-3-

(4-methylphenyl)-3-{6-[(1E)-3-oxobut-1-en-1-yl]pyridin-2-yl}prop-2-en-1-yl]pyrrolidin-1-ium-1-olate

(Imp-IV),1-{[3-(4-methylphenyl)-3-{6-[(1E)-3-oxobut-1-en-1-yl]pyridin-2-yl}oxiran-2-yl]methyl} py

rrolidin-1-ium-1-olate (Imp-V) and 2-[2-(4-methylphenyl)-3-[(1-oxidopyrrolidin-1-ium-1-yl)methyl]

oxiran-2-yl]-6-[(1E)-3-oxobut-1-en-1-yl]pyridin-1-ium-1-olate (Imp-VI), and confirmed by their

synthesis followed by spectroscopic analysis, IR, NMR (1H, 13C) and mass. An efficient and selective

high-performance liquid chromatography method has been developed and resolved well the drug

related substances on a Phenomenex Gemini C-18 (250 × 4.6 mm, particle size 5 μm) column. The

mobile phase was composed of sodium dihydrogen phosphate (10 mM) and methanol, temperature at

25 °C, and a PDA detector set at 254 nm used for detection. The method was validated with respect to

specificity, linearity, precision, accuracy, and sensitivity and satisfactory results were achieved.

Identification, synthesis, characterization of impurities and method validation were first reported in

this paper.

Preparation of a monoclonal antibody against amantadine and rimantadine and development of

an indirect competitive enzyme-linked immunosorbent assay for detecting the same in chicken

muscle and liver

Dapeng Peng, Wei Wei, Yuanhu Pan, Yulian Wang, Zonghui Yuan

ABSTRACT

A monoclonal antibody (mAb) was produced in order to monitor the illegal use of amantadine and

rimantadine in animals. The produced mAb 2G3 exhibited an IC50 value of 15.8 μg L−1 for

amantadine and exhibited cross-reactivity to both amantadine (100%) and rimantadine (70.6%).

Standard curves ranged from 5 to 80 μg L−1 for 2G3. The limits of detection of the developed indirect

competitive enzyme-linked immunosorbent assay (ic-ELISA) ranged from 5.0 μg kg−1 to 5.4 μg kg−1

in chicken muscle and liver. The recoveries were 81.3% to 98.1% with a coefficient of variation less

than 15.7%. Good correlations were observed between the results of the ic-ELISA and liquid

chromatography-mass spectrometry in the incurred tissues. These results suggest that ic-ELISA is a

sensitive, accurate, and low-cost method that could be a useful tool for screening the residues of

amantadine and rimantadine in chicken muscle and liver.

18

Determination of pharmaceutical residues in wastewater using high performance liquid

chromatography coupled to quadrupole-Orbitrap mass spectrometry

Iveta Pugajeva, Janis Rusko, Ingus Perkons, Elsa Lundanes, Vadims Bartkevics

ABSTRACT

A multi-class method for the determination of 24 emerging pharmaceutical residues has been

developed and validated. The method is based on solid-phase extraction of wastewater samples using

Strata-X cartridges followed by high performance liquid chromatography coupled to hybrid

quadrupole − Orbitrap high resolution mass spectrometry (HPLC-Q-Orbitrap-HRMS). A single-

laboratory validation procedure showed satisfactory analytical performance. The analysis of 21

samples collected at the wastewater treatment plant in Riga revealed the occurrence of 20 compounds

of different therapeutic classes. The highest concentration was found for the central nervous system

stimulator caffeine − up to 12 μg L−1, the analgesic acetaminophen up to 4.2 μg L−1, the antibiotic

ciprofloxacin in the concentration range of 250–400 ng L−1, and the non-steroidal anti-inflammatory

drug (NSAID) ibuprofen at 100–325 ng L−1.

High-capacity hollow porous dummy molecular imprinted polymers using ionic liquid as

functional monomer for selective recognition of salicylic acid

Haiyan Xiang, Mijun Peng, Hui Li, Sheng Peng, Shuyun Shi

ABSTRACT

The existence of strong intramolecular hydrogen bond in salicylic acid (SA) weakens its

intermolecular hydrogen bonding with functional monomer, then it is a challenge work to fabricate

molecularly imprinted polymers (MIPs) for SA recognition with high capacity and good selectivity.

Here, hollow porous dummy MIPs (HPDMIPs) were prepared using benzoic acid (BA) as dummy

template, ionic liquid (i.e. 1-vinyl-3-methylimidazolium chloride) as functional monomer, and MCM-

48 as sacrificial support. Factors that affected adsorption, such as type of template and porogen, mole

ratio of template–functional monomer–cross-linker and type of binding solvent, were optimized in

detail. Multiple strong interactions between SA and ionic liquid in HPDMIPs deduced higher binding

capacity (29.75 mg/g), imprinting factor (5.61) and selectivity than any previously reported MIPs by

traditional or surface imprinting technology. The large surface area (543.9 m2/g) with hollow porous

structure resulted in faster kinetic binding (25 min). The equilibrium data fitted well to Freundlich

equation and the adsorption process could be described by pseudo-second order model. Finally,

HPDMIPs were successfully applied to selectively extract and enrich SA from Actinidia chinensis

with a relatively high recovery (84.6–94.5%).

19

Demonstration of the IgG antibody repertoire against the bacteria Escherichia coli in Chinese

intravenous immunoglobulins

Shengliang Ye, Min Lei, Peng Jiang, Fengjuan Liu, Changqing Li

ABSTRACT

Intravenous immunoglobulin (IVIg) is produced by pooling plasma from thousands of healthy blood

donors, and the diversity of the antibody is critical for the clinical efficacy of IVIg. This study

investigated the antibody diversity of Chinese IVIg. Firstly, 2-dimensional gel electrophoresis and

immunoblotting with protein extracts of Escherichia coli (E. coli) O157:H7 were used to study IgG

antibody repertoire of 8 IVIg preparations from different Chinese manufacturers. This was followed by

the identification of the antibody-reactive proteins of E. coli by mass spectrometry and the sequence

similarity of the proteins was aligned by bioinformatics analysis. The results showed that all IVIg

preparations expressed a large range of antibody reactivities against E. coli proteins. 94-238 antigens

were recognized by the 8 IVIg preparations. 33 interesting target antigens were selected and identified

as 29 different proteins, mainly including membrane proteins, molecular chaperones, metabolism

enzymes, and proteins involved in cell cycle processes. Additionally, these antigens were highly

conserved proteins which were found extensively in a variety of other pathogenic microorganisms. Our

study indicated that Chinese IVIg preparations recognized a large range of high conserved proteins

which play key roles in pathogenic microorganisms, and showed each IVIg had its own distinct

antibody repertoire.

Metabolites profiling reveals for antimicrobial compositional differences and action mechanism

in the toothbrushing stick ―miswak‖ Salvadora persica

Mohamed A. Farag, Sherifa Fahmy, Mouchira A. Choucry, Mariam O. Wahdan, Mahmoud Fahmi

Elsebai

ABSTRACT

Among many plant species suitable for preparing toothbrushing sticks, miswak (Salvadora persica,

family Salvadoraceae) is found the most effective tool for oral hygiene. S. persica possesses

antibacterial, antiviral and antifungal effects against oral microbes, mostly due to its benzyl

isothiocyanate content. To provide insight into S. persica chemical composition, volatile constituents

from roots and stems of S. persica grown in Egypt and Saudi Arabia were profiled using solid-phase

microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC–MS). A total of 21

volatiles were identified with sulfur compounds amounting for the major volatile class. Orthogonal

projection to latent structures-discriminant analysis (OPLS-DA) revealed for benzyl isothiocyanate

(BITC) enrichment in roots versus stems. Primary metabolites contributing to S. persica taste viz.

sugars and organic acids were profiled using GC–MS with silylation. Polyols (sugars) viz. arabitol,

meso-erythritol, and mannitol were found to predominate sugars composition in S. persica stems being

most enriched in meso-erythritol. The impact of saliva on S. persica aroma profile was further assessed

and revealing for no enhancement in BITC production with salivation, and further not being detected

in toothpaste preparation claimed to contain S. persica extract. This study provides the most complete

profile of volatiles, sugars, and organic acids in S. persica organs and more rationalizing its use as a

toothbrush.

20

The investigation of anti-inflammatory activity of Yi Guanjian decoction by serum

metabonomics approach

Sufang Shui, Xiaorong Cai, Rongqing Huang, Bingkun Xiao, Jianyun Yang

ABSTRACT

Yi Guanjian (YGJ), one of the Chinese herbal medicines most commonly used in western countries,

reported to possess significant anti-inflammatary effects that inhibit the process of inflammation.

However, the mechanisms underlying its anti-inflammation effects remain largely unresolved. This

study was aimed to investigate the anti-inflammatory activity of YGJ and to explore its potential anti-

inflammatory mechanisms by serum metabonomics approach. A xylene-induced mouse right-ear-

edema model was used as an inflammatory response in vivo model. Ear edema, prostaglandin E2

(PGE2) and Tumor-Necrosis-Factor-alpha (TNF-α) were detected. Then, serum metabolic profiling

was analyzed and pathway analysis performed on the biomarkers reversed after YGJ administration

and further integration of metabolic networks. The results showed that YGJ alleviated ear edema and

decreased serum PGE2 and TNF-α level. Fourteen biomarkers were screened, and the levels were all

reversed to different degrees after YGJ administration. These biomarkers were mainly related to

linoleic acid metabolism, taurine and hypotaurine metabolism, glyoxylate and dicarboxylate

metabolism, glycine, serine and threonine metabolism and citrate cycle (TCA cycle). In metabolic

networks, glycine and pyruvate were node molecules. This indicated that YGJ could significantly

inhibit inflammatory response triggered by acute local stimulation and exerted anti-inflammatory

activity mainly by regulating node molecules.

Quantitative profiling of 4'-geranyloxyferulic acid and its conjugate with l-nitroarginine methyl

ester in mononuclear cells by high-performance liquid chromatography with fluorescence

detection

Vito Alessandro Taddeo, Salvatore Genovese, Giuseppe Carlucci, Vincenzo Ferrone, Francesco

Epifano

ABSTRACT

Oxyprenylated natural products were shown to exert in vitro and in vivo remarkable anti-cancer and

anti-inflammatory effects. This paper describes a rapid, selective, and sensitive HPLC method with

fluorescence detection for determination of 4'-geranyloxyferulic acid (GOFA) and its conjugate with l-

nitroarginine methyl ester (GOFA-L-NAME) in mononuclear cells. Analytes were extracted from cells

using methanol and eluted on a GraceSmart RP18 analytical column (250 × 4.6 mm i.d., 5 μm particle

size) kept at 25 °C. A mixture of formic acid 1% in water (A) and methanol (B) were used as mobile

phase, at a flow-rate of 1.2 mL/min in gradient elution. A fluorescence detector (excitation/emission

wavelength of 319/398 nm for GOFA and GOFA-L-NAME), was used for the two analytes.

Calibration curves of GOFA and GOFA-L-NAME were linear over the concentration range of 1.0–50

μg/mL, with correlation coefficients (r2) ≥ 0.9995. Intra- and inter-assay precision do not exceed 6.8%.

The accuracy was from 94% to 105% for quality control samples (2.0, 25.0 and 40 μg/mL). The mean

(RSD%) extraction recoveries (n = 5) for GOFA and GOFA-L-NAME from spiked cells at 2.0, 25.0

and 40.0 μg/mL were 92.4 ± 1.5%, 94.7 ± 0.9% and 93.8 ± 1.1%, for GOFA and 95.3 ± 1.2%, 94.8 ±

1.0% and 93.9 ± 1.3%, for GOFA-L-NAME. The limits of detection and quantification were 0.3

μg/mL and 1.0 μg/mL for GOFA and GOFA-L-NAME. This method was successfully applied to

measure GOFA and GOFA-L-NAME concentrations in a mononuclear cell.

21

A serum nuclear magnetic resonance-based metabolomic signature of antiphospholipid

syndrome

Angelica Palisi, Manuela Grimaldi, Paola Sabatini, Paola Montoro, Anna Maria D‘Ursi

ABSTRACT

Antiphospholipid syndrome (APS) is a rheumatic inflammatory chronic autoimmune disease inducing

hypercoagulable state associated with vascular thrombosis and pregnancy loss in women. Cardiac,

cerebral and vascular strokes in these patients are responsible for reduction in life expectancy. Timely

diagnosis and accurate monitoring of disease are decisive to improve the accuracy of therapy. In the

present work, we present a NMR-based metabolomic study of blood sera of APS patients. Our data

show that individuals suffering APS have a characteristic metabolomic profile with abnormalities

associated to the metabolism of methyl group donors, ketone bodies and amino acids. We have

identified for the first time the metabolomic fingerprint characterizing APS disease having potential

application to improve APS timely diagnosis and appropriate therapeutic approaches.

Development of a multi-matrix LC–MS/MS method for urea quantitation and its application in

human respiratory disease studies

Jianshuang Wang, Yang Gao, Drew W. Dorshorst, Fang Cai, Xiao Ding

ABSTRACT

In human respiratory disease studies, liquid samples such as nasal secretion (NS), lung epithelial lining

fluid (ELF), or upper airway mucosal lining fluid (MLF) are frequently collected, but their volumes

often remain unknown. The lack of volume information makes it hard to estimate the actual

concentration of recovered active pharmaceutical ingredient or biomarkers. Urea has been proposed to

serve as a sample volume marker because it can freely diffuse through most body compartments and is

less affected by disease states. Here, we report an easy and reliable LC–MS/MS method for cross-

matrix measurement of urea in serum, plasma, universal transfer medium (UTM), synthetic absorptive

matrix elution buffer 1 (SAMe1) and synthetic absorptive matrix elution buffer 2 (SAMe2) which are

commonly sampled in human respiratory disease studies. The method uses two stable-isotope-labeled

urea isotopologues, [15N2]-urea and [13C,15N2]-urea, as the surrogate analyte and the internal

standard, respectively. This approach provides the best measurement consistency across different

matrices. The analyte extraction was individually optimized in each matrix. Specifically in UTM,

SAMe1 and SAMe2, the unique salting-out assisted liquid-liquid extraction (SALLE) not only

dramatically reduces the matrix interferences but also improves the assay recovery. The use of an

HILIC column largely increases the analyte retention. The typical run time is 3.6 min which allows for

high throughput analysis.

22

Determination of five potential genotoxic impurities in dalfampridine using liquid

chromatography

Mohit Jain, Vishal Srivastava, Rajesh Kumar, Vishal Dangi, Pramod Kumar

ABSTRACT

A sensitive and selective HPLC method was developed for identification and quantification of five

Potential genotoxic impurities (PGIs) viz. Impurity-I, Impurity-II, Impurity-III, Impurity-IV and

Impurity-V in Dalfampridine (Drug substance). The method utilizes Zorbax silica column (250 mm ×

4.6 mm, 5.0 μm) with UV detector in HILIC (Hydrophilic Interaction Liquid Chromatography) mode

for quantitation of five PGIs. It has been validated as per International Council for Harmonisation

(ICH) guidelines and is able to quantitate all PGIs at 75 ppm with respect to 20 mg/mL of sample

concentration. It is linear in the range of 22.5–112.5 ppm for all PGIs, which matches the range of

LOQ-150% of estimated permitted level (75 ppm). Its accuracy was established in the range from

88.14 to 107.65% for these PGIs. The correlation coefficient of each impurity was >0.999. It is a good

quality control tool for quantitation of PGIs in Dalfampridine at low level.

Isolation and structural characterization of novel photolytic degradation impurities of

Deflazacort using Q-TOF, 2D-NMR and FTIR

Ch Krishnam Raju, Avadhesh Kumar Pandey, Kaushik Ghosh, Arunima Pola, Koduru V.

Surendranath

ABSTRACT

Forced Degradation of Deflazacort drug substance in ultraviolet light condition resulted into a number

of significant degradation products. Two of these degradation products were found to be unknown

during the study and marked as DD-I and DD-II. Thus, the objective of this work is to investigate and

identify these two novel degradation products of DFZ. The isolation method for these new degradation

products were developed using a new reverse-phase high performance liquid chromatography (HPLC).

DD-I and DD-II, eluting at 0.53 and 1.57 relative retention times with respect to Deflazacort (DFZ)

peak respectively, were isolated from reaction mass using preparative HPLC and their structures were

elucidated using high resolution MS, multidimensional NMR and FTIR spectroscopic techniques. To

best of our knowledge, these two degradation products are novel impurities which are not discussed in

any form of publication yet.

Volume 96, Number 1, January 2017

23


VLC–ESI–MS/MS evaluation of forced degradation behaviour of silodosin: In vitro anti cancer

activity evaluation of silodosin and major degradation products

Chiguru Vishnuvardhan, Baikadi Saibaba, Lingesh Allakonda, Debasish Swain, N. Satheeshkumar

ABSTRACT

Silodosin (SLD) a novel α1-adrenoceptor antagonist was subjected to forced degradation involving

hydrolysis (acidic, alkaline and neutral), oxidative, photolysis and thermal stress, as per ICH specified

conditions. The drug underwent significant degradation under hydrolytic (acidic, alkaline and neutral)

and oxidative stress conditions whereas, it was found to be stable under other stress conditions. A

rapid, precise, accurate and robust chromatographic method for the separation of the drug and its

degradation products (DPs) was developed on a Fortis C18 analytical column (150 × 4.6 mm, 5 μm)

using 0.1% formic acid and acetonitrile as a mobile phase in gradient elution mode at a flow rate of 1.0

mL/ min. A total of 5 (DP 1 to DP 5) hitherto unknown DPs were identified by LC-ESI-TOF-MS/MS

experiments and accurate mass measurements. The most probable mechanisms for the formation of

DPs have been proposed based on a comparison of the fragmentation of the [M+H]+ ions of silodosin

and its DPs. The major DPs (DP 1 and DP 2) were isolated and evaluated for anticancer activity using

PC3 (human prostate cancer) cell lines by MTT assay. The results revealed that silodosin, DP 1 and

DP 2 have potential anticancer activity with IC50 values (μM) 72.74 (±4.51), 25.21 (±2.36), and,

114.07 (±11.90) respectively.

Quality by Design in the development of hydrophilic interaction liquid chromatography method

with gradient elution for the analysis of olanzapine

Anja Tumpa, Ana Stajić, Biljana Jančić-Stojanović, Mirjana Medenica

ABSTRACT

This paper deals with the development of hydrophilic interaction liquid chromatography (HILIC)

method with gradient elution, in accordance with Analytical Quality by Design (AQbD) methodology,

for the first time. The method is developed for olanzapine and its seven related substances. Following

step by step AQbD methodology, firstly as critical process parameters (CPPs) temperature, starting

content of aqueous phase and duration of linear gradient are recognized, and as critical quality

attributes (CQAs) separation criterion S of critical pairs of substances are investigated. Rechtschaffen

design is used for the creation of models that describe the dependence between CPPs and CQAs. The

design space that is obtained at the end is used for choosing the optimal conditions (set point). The

method is fully validated at the end to verify the adequacy of the chosen optimal conditions and

applied to real samples.

24

Lipophilicity estimation and characterization of selected steroid derivatives of biomedical

importance applying RP HPLC

Lidija R. Jevrić, Milica Ņ. Karadņić, Anamarija I. Mandić, Sanja O. Podunavac Kuzmanović, SrĎan Z.

Stojanović

ABSTRACT

The present paper deals with chromatographic lipophilicity determination of twenty-nine selected

steroid derivatives using reversed-phase high-performance liquid chromatography (RP HPLC)

combined with two mobile phase, acetonitrile-water and methanol-water. Chromatographic behavior

of four groups (triazole and tetrazole, toluenesulfonylhydrazide, nitrile and dinitrile and dione) of

selected steroid derivatives was studied. Investigated compounds were grouped using principal

component analysis (PCA) according to their logk values for both mobile phases. Grouping was in the

very good accordance with the polarity and lipophilicity of the investigated compounds. QSRR

(quantitative structure-retention relationship) approach was used to model chromatographic

lipophilicity behavior using molecular descriptors. Modeling was performed using linear regression

(LR) and multiple linear regression (MLR) methods. The most influential molecular descriptors were

lipophilicity descriptors that are important for molecules ability to pass through biological membranes

and geometrical descriptors. All established LR-QSRR and MLR-QSRR models were statistically

validated by standards, cross- and external validation parameters as well as with two graphical

methods. According to all these assessments, MLR models were better for chromatographic

lipophilicity prediction. It was shown that chromatographic systems with methanol-water were better

for modeling of logk than systems with acetonitrile-water, as well as the systems that contained lower

volume fractions of organic component in mobile phase. Modeling was performed in order to obtain

lipophilicity profiles of investigated compounds as future drug candidates of biomedical importance.

Comparison of the chemical consituents and immunomodulatory activity of ophiopogonis radix

from two different producing areas

Xiaoyan Lu, Wei Tong, Shufang Wang, Jinghui Li, Li Liu

ABSTRACT

Zhemaidong (ZMD) and Chuanmaidong (CMD) are the two genuine cultivation areas of

Ophiopogonis Radix which has been widely used as a traditional Chinese medicine in China for

treating cardiovascular and pulmonary diseases. Differences between ZMD and CMD in chemical

constituents and pharmacological effects have been reported, however, the details remain largely

unknown. The aim of this study was to comprehensively characterize the chemical composition of

Ophiopogonis Radix from the two producing areas and compare their immunomodulatory activities.

An approach of HPLC–MS coupled with multivariate statistical analysis was established to reveal the

characteristic constituents of ZMD and CMD. Furthermore, the effects of ZMD and CMD on the

macrophage phagocytosis and gastrointestinal peristalsis were also determined in zebrafish models for

assessing the immunomodulatory activities of these two strains. The results revealed that the chemical

constituents of ZMD and CMD were much different from each other, and 19 constituents could be

served as chemical markers to distinguish these two strains. Moreover, ZMD showed higher promoting

rates in macrophage phagocytosis and gastrointestinal motility than those of CMD, suggesting ZMD

might possess better immunomodulatory activities. Taken together, the results generated from this

study indicated that the herbs from different producing areas should be evaluated and considered in

preparing TCM prescriptions.

25

Physicochemical analysis in the evaluation of reconstituted dry emulsion tablets

Noémi Anna Niczinger, Barnabás Kállai-Szabó, Miléna Lengyel, Péter Gordon, István Antal

ABSTRACT

The aim of this study was to characterize the formation of emulsions by droplet size analysis and

turbidimetry during reconstitution from a solid dosage form, namely from dry emulsion systems,

which carry an oil phase for poorly soluble active ingredients. For the dry emulsion systems tablets

were prepared either from oil-in-water systems using a freeze-drying process or through direct

compression containing the same oil and excipients. The ratios of oil to emulgents and oil to xanthan

gum were equal in both methods. In the preparation methods applied, mannitol, erythritol and lactose

were used as excipients and mannitol was found to be the most effective excipient based on droplet

size reconstitution, turbidimetry and physical properties. Quality control involved testing the physical

properties of tablets and characterizing the reconstituted emulsions.

Cleaning verification: Exploring the effect of the cleanliness of stainless steel surface on sample

recovery

Imad A. Haidar Ahmad, James Tam, Xue Li, William Duffield, Andrei Blasko

ABSTRACT

The parameters affecting the recovery of pharmaceutical residues from the surface of stainless steel

coupons for quantitative cleaning verification method development have been studied, including active

pharmaceutical ingredient (API) level, spiking procedure, API/excipient ratio, analyst-to-analyst

variability, inter-day variability, and cleaning procedure of the coupons. The lack of a well-defined

procedure that consistently cleaned coupon surface was identified as the major contributor to low and

variable recoveries. Assessment of acid, base, and oxidant washes, as well as the order of treatment,

showed that a base-water-acid-water-oxidizer-water wash procedure resulted in consistent, accurate

spiked recovery (>90%) and reproducible results (Srel ≤ 4%). By applying this cleaning procedure to

the previously used coupons that failed the cleaning acceptance criteria, multiple analysts were able to

obtain consistent recoveries from day-to-day for different APIs, and API/excipient ratios at various

spike levels. We successfully applied our approach for cleaning verification of small molecules (MW

< 1000 Da) as well as large biomolecules (MW up to 50,000 Da). Method robustness was greatly

influenced by the sample preparation procedure, especially for analyses using total organic carbon

(TOC) determination.

26

Raman spectroscopy and capillary zone electrophoresis for the analysis of degradation processes

in commercial effervescent tablets containing acetylsalicylic acid and ascorbic acid

Sabine Neuberger, Kevin Jooß, Dirk Flottmann, Gerhard Scriba, Christian Neusüß

ABSTRACT

In order to ensure the stability of pharmaceutical products appropriate manufacturing and storage

conditions are required. In general, the degradation of active pharmaceutical ingredients (APIs) and

subsequent formation of degradation products affect the pharmaceutical quality. Thus, a fast and

effective detection and characterization of these substances is mandatory. Here, the applicability of

Raman spectroscopy and CZE for the characterization of the degradation of effervescent tablets

containing acetylsalicylic acid (ASA) and ascorbic acid (AA) was evaluated. Therefore, a degradation

study was performed analyzing tablets from two different manufacturers at varying conditions (relative

humidity (RH) 33%, 52% and 75% at 30 °C). Raman spectroscopy combined with principal

component analysis could be successfully applied for the fast and easy discrimination of non-degraded

and degraded effervescent tablets after a storage period of approximately 24 h (RH 52%).

Nevertheless, a clear identification or quantification of APIs and degradation products within the

analyzed tablets was not possible, i.a. due to missing reference materials. CZE-UV enabled the

quantification of the APIs (ASA, AA) and related degradation products (salicylic acid (SA); semi-

quantitative also mono- and diacetylated AA) within the complex tablet mixtures. The higher the RH,

the faster the degradation of ASA and AA as well as the formation of the degradation products. Mono-

and diacetylated AA are major primary degradation products of AA for the applied effervescent

tablets. A significant degradation of the APIs was detected earlier by CZE (6–12 h, RH 52%) than by

Raman spectroscopy. Summarized, Raman spectroscopy is well-suited as quick test to detect

degradation of these tablets and CZE can be utilized for further detailed characterization and

quantification of specific APIs and related degradation products.

RP-HPLC determination of dissociation constant using solely aqueous mobile phase

Tereza Volná, Kamil Motyka, Jan Hlaváč

ABSTRACT

The proposed HPLC method using solely or nearly 100% aqueous mobile buffer as mobile phase

offers fast determination of dissociation constant for compounds in relatively wide range of

lipophilicity (log P from −2.26 to 2.26). The dissociation constant value for simpler chemical

compounds can be determined via only 8 chromatographic runs. The number of needed

chromatographic separations depends on the structural complexity of the tested compound. Moreover,

the proposed method does not require a measurement of Yasuda-Shedlovsky extrapolation that

includes several pKa determinations in solutions with different methanol content which speeds up

considerably the procedure. The methodology is suitable for evaluation of large series of drug

candidates, which can be present as complex mixtures and in small amounts.

27

Quantitative determination of salbutamol sulfate impurities using achiral supercritical fluid

chromatography

Amandine Dispas, Vincent Desfontaine, Bertyl Andri, Pierre Lebrun, Philippe Hubert

ABSTRACT

In the last years, supercritical fluid chromatography has largely been acknowledged as a singular and

performing technique in the field of separation sciences. Recent studies highlighted the interest of SFC

for the quality control of pharmaceuticals, especially in the case of the determination of the active

pharmaceutical ingredient (API). Nevertheless, quality control requires also the determination of

impurities. The objectives of the present work were to (i) demonstrate the interest of SFC as a

reference technique for the determination of impurities in salbutamol sulfate API and (ii) to propose an

alternative to a reference HPLC method from the European Pharmacopeia (EP) involving ion-pairing

reagent. Firstly, a screening was carried out to select the most adequate and selective stationary phase.

Secondly, in the context of robust optimization strategy, the method was developed using design space

methodology. The separation of salbutamol sulfate and related impurities was achieved in 7 min,

which is seven times faster than the LC-UV method proposed by European Pharmacopeia (total run

time of 50 min). Finally, full validation using accuracy profile approach was successfully achieved for

the determination of impurities B, D, F and G in salbutamol sulfate raw material. The validated dosing

range covered 50 to 150% of the targeted concentration (corresponding to 0.3% concentration level),

LODs close to 0.5 μg/mL were estimated. The SFC method proposed in this study could be presented

as a suitable fast alternative to EP LC method for the quantitative determination of salbutamol

impurities.

Hydrogen/deuterium exchange, a unique and effective method for MS fragmentation behavior

elucidation of ginkgolides and its application to systematic research in Ginkgo biloba

Xingliang Niu, Jun Luo, Deran Xu, Hongyan Zou, Lingyi Kong

ABSTRACT

Ginkgolides, the main active constituents of Ginkgo biloba, possess significant selectively inhibition

on platelet-activating factor and pancreatic lipase and attract wide attention in pharmacological

research area. In our study, an effective hydrogen/deuterium (H/D) exchange method was developed

by exchanging the α-Hs of lactone groups in ginkgolides with Ds, which was very useful for the

elucidation of the fragmentation patterns of ginkgolides in Quadrupole Time-of-flight Mass

Spectrometry (Q-TOF-MS), especially in accurately distinguishing the type and position of substituent

in framework of ginkgolides. Then, a systematic research strategy for qualitative and quantitative

analysis of ginkgolides, based on H/D exchange, tandem solid-phase extraction and LC-Q-TOF-MS,

was developed, which was successfully applied in each medicinal part of G. biloba, which indicated

that ginkgolide B was the most abundant ginkgolide in the seeds of G. biloba (60.6 μg/g). This

research was the successful application of H/D exchange in natural products, and proved that H/D

exchange is a potential method for analysis research of complex TCMs active constituents.

28

Identification and characterization of a new dapoxetine impurity by NMR: Transformation of

N-oxide by Cope elimination

András Darcsi, Ákos Rácz, Szabolcs Béni

ABSTRACT

Unknown impurity associated with the degradation process of dapoxetine base was isolated. The

structure elucidation of this new compound using accurate mass data, IR and NMR spectroscopy is

presented herein. The unambiguous resonance assignment concluded to the formation of geometrical

isomers of cinnamyloxynaphtalenes via Cope elimination of dapoxetin-N-oxide, the major oxidative

and metabolic degradation product of dapoxetine. An efficient and simple synthetic approach has also

been developed for the synthesis of dapoxetine-N-oxide for the first time and cinnamyloxynaphtalene

in order to confirm the proposed degradation pathway and structures of the degradation products. It

was observed that the main degradation product of dapoxetine base when exposed to air is 1-(2E)-

cinnamyloxynaphthalene, while its Z isomer was also confirmed as a minor impurity.

A new approach to the rapid separation of isomeric compounds in a Silybum marianum extract

using UHPLC core-shell column with F5 stationary phase

Jakub Fibigr, Dalibor Ńatínský, Petr Solich

ABSTRACT

In this paper, a new ultra-high performance liquid chromatography (UHPLC) method using a core–

shell column with a pentafluorophenyl stationary phase for separation of seven active compounds of a

Silybum marianum extract was developed and validated. Silymarin, an extract of Silybum marianum,

is known for its abilities to protect the liver from toxic substances, hepatitis therapy, and anti-tumour

activity. Silymarin is currently being widely used in commercial preparations and herbal teas.

Separation of seven compounds contained in the Silybum marianum extract (taxifolin, silychristin,

silydianin, silybin A, silybin B, isosilybin A, isosilybin B) and other substances occurring in real

samples was performed on the Kinetex 1.7 μ F5 100A (150 × 2.1 mm), 1.7 μm particle size core–shell

column, with a mobile phase methanol/100 mM phosphate buffer pH 2.0 according to the gradient

program. A mobile phase 0.35 mL min−1 flow rate and 50 °C temperature was used for the separation.

The detection wavelength was set at 288 nm. Under optimal chromatographic conditions, good

linearity with a correlation coefficient of R2 >0.999 for all compounds was achieved. The available

commercial samples of herbal teas and food supplements were extracted with methanol using an

ultrasonic bath. After dilution with water and centrifugation, a 2 μL sample of the filtered supernatant

was directly injected into the UHPLC system. The use of a pentafluorophenyl stationary phase with

methanol as the organic component of the mobile phase showed new ways to effectively separate

isomeric compounds in herbal extracts, which could not be done with the conventional C18 stationary

phase.

29

A comparison report of three advanced methods for drug-cyclodextrin interaction

measurements

Vikramjeet Singh, Yaping He, Caifen Wang, Jianghui Xu, Jiwen Zhang

ABSTRACT

Three advanced methods, high performance affinity chromatography (HPAC), surface plasmon

resonance (SPR) and surface plasmon resonance imaging (SPRi) were compared and evaluated for

determining the drug-cyclodextrin (CD) interactions herein. In total, 18 sparingly soluble drugs were

selected for this comparative study. The three methods share a unique connection in the working

principles and strategies. The same strategies of CD fixation onto solid phase were used in HPAC and

SPR for the measurements, whereas, the SPR and SPRi share identical working principles. However,

whilst these relationships are evident, no strong correlation was found between kinetic constants

obtained from the three methods: Four drugs, namely, prednisolone, pseudolaric acid B, diazepam and

gramisetron failed to show any response on SPR, whereas, the kinetics parameters from SPRi and

HPAC were successfully measured. From a comparative review of all the kinetic data, random results

without any trends were observed (ka, kd and KA) regardless of the relationships between the three

methods: It is apparent that the measurement conditions (volume, flow rate, buffers), non-specific

adsorption and experimental procedures had a strong impact on the generated data. The relative

advantages and limitations of each method are critically presented on the basis of generated data. This

comparative study provides a basis to further upgrade these techniques for confident measurement of

drug-CDs interactions.

Portable near-infrared instruments: Application for quality control of polymorphs in

pharmaceutical raw materials and calibration transfer

Vitor Hugo da Silva, Jailson José da Silva, Claudete Fernandes Pereira

ABSTRACT

This work presents an evaluation of the analytical performance of three different portable near-infrared

(NIR) instruments (denominated Port.1, Port.2 and Port.3) for quantifying mebendazole polymorphs

(A, B and C) in pharmaceutical raw materials using multivariate calibration models. The performance

of the portable instruments was compared with a benchtop one (FT-NIR Frontier spectrometer). In

addition, calibration transfer between the benchtop and one of the portable instruments was also

performed. For polymorph A, the Port.1 presented the lowest RMSEP value (1.01% w/w) even when

compared to the FT-NIR instrument. For polymorphs B and C, the same Port.1 instrument presented

RMSEP values of 2.09% w/w and 2.41% w/w, respectively, which were statistically similar to those

obtained with the benchtop instrument. The LOD ranges (3.9–5.5 for polymorph A, 3.6–5.1 for

polymorph B and 5.7–7.7 for polymorph C) obtained with the Port.1 was higher than those achieved

with the benchtop NIR instrument, with high spectral resolution, signal-to-noise ratio and better

wavelength reproducibility. Calibration transfer was performed between the benchtop NIR and Port.1

instruments. According to the results, the transferability of models is possible. The results obtained for

complete recalibration of the portable instrument and those for the benchtop are comparable. The

methods developed demonstrated a flexible, easy, cheap and fast way for quality control of MBZ

polymorphs in incoming material, mainly in pharmaceutical laboratory chains.

30

Characterization and quantitation of the polyphenolic compounds detected in methanol extracts

of Pistacia atlantica Desf fruits from the Guelmim region of Morocco

Farid Khallouki, Andrea Breuer, Elzemzoumi Merieme, Cornelia M. Ulrich, Robert W. Owen

ABSTRACT

High performance liquid chromatography coupled with electrospray ionization mass spectrometry

(HPLC-ESI–MS) was used for the identification of the major phenolic compounds in mature P.

atlantica fruits from the Guelmim region (southeast of Morocco). In this study twenty seven

polyphenolic compounds are identified and quantitated. To date, this is the most comprehensive report

on the polyphenolic content of Pistacia fruits. The profiles comprise, three major polyphenolic classes,

namely gallates (18.76 g/kg; 63.92%), flavonoids (10.12 g/kg; 34.48%) and ellagic acid derivatives

(0.47 g/kg; 1.60%) with a total of 29.35 g/kg detected. The major gallate was pentagalloyl glucoside

(5.0 g/kg; 17.04% of total polyphenolics), the major flavonoid luteolin (3.18 g/kg; 10.83% of total

polyphenolics) and the major ellagic acid derivative ellagic acid (0.25 g/kg; 0.85% of total

polyphenolics). Identification of galloyl quinate, digalloyl quinates (x 2), galloyl glucoside, digalloyl

glucosides (x 2), trigalloyl glucoside, tetragalloyl glucosides (x 2), pentagalloyl glucoside, 2″-O-

galloyl-quercetin-3-O-galactoside, quercetin-3-O-rhamnogalactoside, quercetin-3-O-galactoside,

ellagic acid diglucoside, luteolin-4′-O-glucoside, 2″-O-galloyl-luteolin-4′-O-glucoside, quercetin-3-O-

glucuronide, kaempferol-3-O-glucoside, eriodictyol, apigenin, ellagic acid diglucoside, ellagic acid

glucoside, methyl ellagic acid glucoside, and ellagic acid are described as phytochemical components

of Pistacia fruits for the first time.

Quantification of biologically active O-prenylated and unprenylated phenylpropanoids in dill

(Anethum graveolens), anise (Pimpinella anisum), and wild celery (Angelica archangelica)

Vito Alessandro Taddeo, Salvatore Genovese, Philippe de Medina, Roberta Palmisano, Serena Fiorito

ABSTRACT

An analytical strategy based on different extraction methodologies and HPLC with spectrophotometric

(UV–vis) detection has been developed to investigate the presence of and to quantitate biologically

active selected unprenylated and O-prenylated phenylpropanoids, namely umbelliferone, 4′-

geranyloxyferulic acid, 7-isopentenyloxycoumarin, auraptene, and umbelliprenin in dill (Anethum

graveolens L.), anise (Pimpinella anisum L.), and wild celery (Angelica archangelica L.). Absolute

ethanol or 7:3 water/ethanol mixtures were seen to be the most powerful extraction solvents to perform

―classic―maceration or ultrasound-assisted one in terms of yields in secondary metabolites. For

anethum and anise, umbelliprenine was found to be the most abundant prenyloxy secondary

metabolite, while in wild celery 4′-geranyloxyferulic acid recorded the highest concentration. Our

experimental approach demonstrated to be efficient for the simultaneous identification and quantitation

of the above mentioned prenyloxyphenylpropanoids in the title plant species that is reported herein for

the first time in the literature.

31

Microfluidic-based G-quadruplex ligand displacement assay for alkaloid anticancer drug

screening

Haihui Shen, Bo Zhang, Huiyan Xu, Yue Sun, Yingchun Liu

ABSTRACT

Some natural heterocyclic alkaloids containing planar group show potential to complex with specific

promoter region of protooncogene for stabilizing the G-quadruplex (G4) structure which nowadays

promises to be a target in anticancer drug design. However, in view of the polymorphic characteristics

and structural complexity of heterocyclic alkaloids, it is desirable to develop high-throughput and low-

consumption approach for anticancer drug screening. In this paper, an intensive study on alkaloid

ligand/G4 DNA interaction has been conducted, demonstrating that the end-stacking interaction is the

favorable binding mode between the oncogene-related Pu22 G4 DNA and the heterocyclic alkaloid

ligand. Based on structural feasibility and energy minimization, a ligand displacement assay for

screening alkaloid ligand in stabilizing the oncogene target G4 has been developed, which also helps to

facilitate the assessment of drug specificity. Coupled with microfluidic-based DNAzyme-catalytic

chemiluminescence detection, the approach showed the advantages of high sensitivity, high throughput

with low sample and reagent consumptions.

Chrysin cocrystals: Characterization and evaluation

Renu Chadha, Yashika Bhalla, Avdesh Nandan, Kunal Chadha, Maninder Karan

ABSTRACT

Solvent free mechanochemical approach is utilized to synthesise new cocrystals of chrysin using

supramolecular chemistry based upon reliable synthons. Chrysin, a flavone nutraceutical with wide

range of beneficial effects has critically low bioavailability on account of its poor aqueous solubility

and consequently poor absorption from the gastrointestinal tract. The present study focuses on this

critical aspect and has exploited non covalent interactions to prepare its cocrystals with cytosine and

thiamine hydrochloride. Various techniques were used for characterization including Differential

Scanning Calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FT-IR), Solid State NMR

Spectroscopy (SSNMR) and Powder X-Ray Diffraction (PXRD). The molecules in the cocrystals

crystallized in neutral forms and assembled in a molecular layer by means of hydrogen bonding which

was confirmed by structural characterization. The cocrystals share a common supramolecular motif

being the OH⋯Narom interaction, involving phenolic moiety of C7 functionality of the parent

molecule. Approximately 3–4 fold increase in solubility and dissolution profile of cocrystals was

observed which was further corroborated by improved in vitro and in vivo activities including

antioxidant, antihaemolytic and anti-inflammatory thus, opening a new viable technique for the

exploitation of useful phytonutrients.

32

Stability behaviour of antiretroviral drugs and their combinations 5: Characterization of novel

degradation products of abacavir sulfate by mass and nuclear magnetic resonance spectrometry

Moolchand Kurmi, Archana Sahu, Saranjit Singh

ABSTRACT

In the present study, degradation behaviour of abacavir sulfate was evaluated in solution and solid

stress conditions. Solution state studies resulted in formation of eleven degradation products; of which

two were also formed on solid stress. The same were separated by high performance liquid

chromatography. They were characterized using liquid chromatography-high resolution mass

spectrometry, liquid chromatography-multistage mass spectrometry and hydrogen/deuterium exchange

mass spectrometry data. Additionally, seven degradation products were isolated and subjected to 1D

and 2D nuclear magnetic resonance studies for their structural confirmation.

Comparison of miRNA signature versus conventional biomarkers before and after off-pump

coronary artery bypas graft

Fatemeh Pourrajab, Fereshteh Torkian Velashani, Masoud Khanaghaei, Seyedhossein

Hekmatimoghaddam, Mohamad Reza Zare-Khormizi

ABSTRACT

Circulating levels of microRNAs (miRNAs) and their expression patterns are supposed to serve as

signatures for diagnosis or prognosis of cardiovascular events. The present study aimed at determining

if there is any correlation between the release pattern of 2 miRNAs and the plasma levels of

conventional biomarkers cardiac troponin I (cTnI), creatine kinase (CK) and uric acid (UA) in patients

undergoing their first off-pump coronary artery bypass graft (OCABG). Seventy OCABG patients

(69% men, aged 59.2 ± 8.2 years) were enrolled. Emergencies, re-operations, abnormal preoperative

serum cTnI and combined procedures were excluded from this study. Pre-operative mean ejection

fraction was 45.8 ± 8.6%, the average number of grafts was 3 ± 0.87/patient, and the internal

mammary artery was used for all. Beside conventional clinical assays, we performed real-time

quantitative PCR to analyze the circulating levels of miR-155, miR-126 and miR-499 at 1 day before

surgery as well as 4 days after surgery. Importantly, there was no report of myocardial infarction in our

patients, pre- or post-operatively. In contrast to conventional biomarkers cTnI and CK, circulating

levels of miRNAs decreased significantly (P < 0.01) after revascularization surgery. A significant

positive correlation was seen between the cTnI and miR-499 (r ∼ 0.53, P < 0.01) and between miR-

126 and UA (r ∼ 0.5, P < 0.01). Time course study of circulating miR-499, miR-126 and miR-155 in

cardiac surgery clarified their advantage and correlations to the traditional biomarkers cTnI, total CK,

CK-MB and UA. Our results suggest that this signature is a novel, early biomarker which indicates

myocardial ischemia in cardiac surgery. It could be postulated that the application of these miRNAs

may be considered for monitoring of response to pharmacological interventions aimed at reducing

cardiac ischemia, especially in OCABG candidates.

33

Microfluidic device for label-free quantitation and distinction of bladder cancer cells from the

blood cells using micro machined silicon based electrical approach; suitable in urinalysis assays

Seied Ali Hosseini, Somayeh Zanganeh, Elaheh Akbarnejad, Fatemeh Salehi, Mohammad Abdolahad

ABSTRACT

This paper introduces an integrated microfluidic chip as a promising tool to measure the concentration

of bladder cancer cells (BCC) in urine samples. Silicon microchannels were used as trapping gates for

both floated BCC and leukocytes which are found in the urine of patients. By the assistance of the gold

electrodes patterned at the bottom of the micro gates, the capacitance of captured cancerous and blood

cells were measured. Different membrane capacitance between BCC and leukocyte was the indicative

signal for diagnosing the nature of captured cells in urine like solution. The concentration range of the

target that could be detected was about 10 BCCs per one chip. Such response has been achieved

without applying any biochemical or florescent markers. Thus, it could be a simple and cheap

approach to support cytological and immune-fluorescent assays. The limit of detection was

approximately 1 cancerous cell/11 leukocytes in 1 ml of the urine like solution. The entire

measurement time was less than an hour. Consequently, this electrical microfluidic device promises

significant potential in urinalysis.

Simultaneous determination of anemoside B4, phellodendrine, berberine, palmatine, obakunone,

esculin, esculetin in rat plasma by UPLC–ESI–MS/MS and its application to a comparative

pharmacokinetic study in normal and ulcerative colitis rats

Lianrong Yang, Xin Meng, Xiaojin Yu, Haixue Kuang

ABSTRACT

A sensitive and rapid ultra-performance liquid chromatography-electrospray ionization-mass

spectrometry (UPLC–ESI–MS/MS) method was developed for the simultaneous analysis of anemoside

B4, phellodendrine, berberine, palmatine, obakunone, esculin, esculetin, toosendanin (IS1 of

anemoside B4), tetrahydropalmatine (IS2 of phellodendrine, berberine, palmatine and obakunone) and

scopoletin (IS3 of esculin and esculetin) and to compare the pharmacokinetics of these active

ingredients in normal and ulcerative colitis rats. After methanol deproteinization, solvents were

evaporated at 40 °C under a gentle stream of nitrogen. Chromatography was performed using a C18

column with a gradient elution of 0.1% aqueous formic acid and acetonitrile at 0.4 ml/min. Detection

and measurement were performed on a 4000 QTRAP UPLC–MS/MS system from AB Sciex in the

multiple reaction monitoring (MRM) mode. Phellodendrine, berberine, palmatine, obakunone, esculin,

esculetin, tetrahydropalmatine (IS2) and scopoletin (IS3) were monitored under positive ionization

conditions. Anemoside B4, and toosendanin (IS1) were monitored under negative ionization

conditions. The optimized mass transition ion-pairs (m/z) were 1221.1/750.7 for anemoside B4,

343.2/193.2 for phellodendrine, 337.1/321.0 for berberine, 353.0/336.9 for palmatine, 455.1/161.1 for

obakunone, 341.2/179.2 for esculin, 179.1/123.0 for esculetin, 573.4/531.4 for toosendanin (IS1),

356.2/192.2 for tetrahydropalmatine (IS2) and 193.0/133.1 for scopoletin (IS3).

34

Simultaneous determination and pharmacokinetic study of four phenolic acids in rat plasma

using UFLC–MS/MS after intravenous administration of salvianolic acid for injection

Xiuman Xie, Jingzhuo Miao, Wanyang Sun, Jingyi Huang, Guoxiang Sun

ABSTRACT

A simple, sensitive and selective ultra-fast liquid chromatography-tandem mass spectrometry (UFLC–

MS/MS) method was established for simultaneous determination and pharmacokinetic study of

rosmarinic acid (RA), salvianolic acid D (Sal D), lithospermic acid (LA) and salvianolic acid B (Sal B)

in rat plasma after intravenous administration of salvianolic acid for injection (SAFI). Three doses of

administration, containing 14, 28 and 56 mg/kg, were investigated in this study. Plasma samples were

pretreated using protein precipitation (PP) with pre-cooled acetonitrile. Chromatographic separation

was achieved on a CORTECS™ UPLC C18 column (1.6 μm, 2.1 × 100 mm) with a mobile phase

composed of 0.1% formic acid aqueous (V/V) and 0.1% formic acid acetonitrile (V/V). Analytes were

detected using electrospray ionization (ESI) source in negative ionization mode and quantified in

multiple reaction monitoring (MRM) mode. The validated method is stable and reliable. No significant

difference of half lives (t1/2) of four analytes at three doses was observed. Area under the curve

(AUC0-∞) and peak concentration (Cmax) of the four analytes demonstrated a linear increase in across

the doses with the linear correlation r of each analyte at three doses were greater than 0.95. It indicated

that the pharmacokinetic behavior of SAFI is positively related to dose at the range of 14–56 mg/kg.

A rapid and sensitive UHPLC–MS/MS method for quantification of 83b1 in plasma and its

application to bioavailability study in rats

Dingsheng Wen, Jing Guo, Fulin Jiang, Caishun Huang, Guoping Zhong

ABSTRACT

Great attentions have been drawn by quinoline for its broad bioactivity as anti-fungal, anti-bacterial

and anti-tumor activities. Compared with cisplatin, 83b1, a quinoline derivative, showed equal activity

in anti-tumor and lower cyctotoxicity in normal cell. In this study, a simple, rapid and sensitive method

for determination of 83b1 in rat plasma using UHPLC–MS/MS was developed for the first time.

Loratadine was used as an internal standard (IS). Separation was performed on an Xterra MS C18

column by isocratic elution using acetonitrile: water solution with 1‰ formic acid (90:10, v/v) as

mobile phase at a flow rate of 0.3 mL/min. A triple quadrupole mass spectrometer operating in the

positive ion-switching electron spray ionization mode with selection reaction monitoring (SRM) was

employed to determine 83b1 and IS transitions of m/z 321.82 → 147.84, 382.71 → 258.76 for 83b1

and Loratadine, respectively. The values of specificity, linearity and lower limit of quantification,

intra- and inter- day precision and accuracy, extraction recovery, matrix effect and stability for this

method satisfied the acceptable limits. The lower limit of quantification was 0.5 ng/mL with a linear

range of 0.5–1500 ng/mL. The validated method was employed to study the bioavailability of 83b1 in

rat by dosing with intravenous injection (1 mg/kg) and gavage (10 mg/kg), and the oral bioavailability

of 83b1 in rat was calculated as 20.9 ± 8.8%.

35

Accurate quantitation of choline and ethanolamine plasmalogen molecular species in human

plasma by liquid chromatography–tandem mass spectrometry

Yurika Otoki, Shunji Kato, Fumiko Kimura, Katsutoshi Furukawa, Kiyotaka Nakagawa

ABSTRACT

Concentration of both choline plasmalogen (PC-Pls) and ethanolamine Pls (PE-Pls) in human

plasma/serum has been getting attention to, since certain patients including those with

neurodegenerative disorders, have been reported to exhibit reduced levels of specific Pls species.

However, despite using liquid chromatography–tandem mass spectrometry (LC–MS/MS), accurate

quantitation of Pls is still difficult because of less product ion from PC-Pls and quantitative issues

(e.g., extraction recoveries and matrix effects). The present study aimed to develop a method for

accurate identification and quantitation of Pls molecular species using LC–MS/MS operated in the

multiple reaction monitoring modes. The LC–MS/MS conditions in the presence of sodium, and the

extraction method using methanol protein precipitation were optimized. Under the optimal condition,

Pls was detected at femtomole levels. The recoveries of Pls from human plasma were nearly 100%,

and matrix effects were not observed. The novel method enabled determination of each Pls species in

human plasma at the concentrations of 0.5–13.6 μM. Then the PC-Pls and PE-Pls species in the plasma

of both healthy subjects and patients with Alzheimer‘s disease were quantitated. The method

developed herein represents a powerful tool for analyzing Pls, which may provide a better

understanding of their physiological roles in vivo.

The profiling of the metabolites of hirsutine in rat by ultra-high performance liquid

chromatography coupled with linear ion trap Orbitrap mass spectrometry: An improved

strategy for the systematic screening and identification of metabolites in multi-samples in vivo

Jianwei Wang, Peng Qi, Jinjun Hou, Yao Shen, Dean Guo

ABSTRACT

Drug metabolites identification and construction of metabolic profile are meaningful work for the drug

discovery and development. The great challenge during this process is the work of the structural

clarification of possible metabolites in the complicated biological matrix, which often resulting in a

huge amount data sets, especially in multi-samples in vivo. Analyzing these complex data manually is

time-consuming and laborious. The object of this study was to develop a practical strategy for

screening and identifying of metabolites from multiple biological samples efficiently. Using hirsutine

(HTI), an active components of Uncaria rhynchophylla (Gouteng in Chinese) as a model and its

plasma, urine, bile, feces and various tissues were analyzed with data processing software (Metwork),

data mining tool (Progenesis QI), and HR-MSn data by ultra-high performance liquid

chromatography/linear ion trap-Orbitrap mass spectrometry (U-HPLC/LTQ-Orbitrap-MS). A total of

67 metabolites of HTI in rat biological samples were tentatively identified with established library, and

to our knowledge most of which were reported for the first time. The possible metabolic pathways

were subsequently proposed, hydroxylation, dehydrogenation, oxidation, N-oxidation, hydrolysis,

reduction and glucuronide conjugation were mainly involved according to metabolic profile. The result

proved application of this improved strategy was efficient, rapid, and reliable for metabolic profiling of

components in multiple biological samples and could significantly expand our understanding of

metabolic situation of TCM in vivo.

36

Metabolic fate and detectability of the new psychoactive substances 2-(4-bromo-2,5-

dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25B-NBOMe) and 2-(4-chloro-2,5-

dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25C-NBOMe) in human and rat

urine by GC–MS, LC–MSn, and LC–HR–MS/MS approaches

Achim T. Caspar, Simon D. Brandt, Andreas E. Stoever, Markus R. Meyer, Hans H. Maurer

ABSTRACT

25B-NBOMe and 25C-NBOMe are potent 5-HT2A receptor agonists that have been associated with

inducing hallucinogenic effects in drug users and severe intoxications. This paper describes the

identification of their metabolites in rat and human urine by liquid chromatography (LC)-high

resolution (HR)-MS/MS, the comparison of metabolite formation in vitro and in vivo and in different

species, the general involvement of human cytochrome-P450 (CYP) isoenzymes on their metabolism

steps, and their detectability by standard urine screening approaches (SUSAs) using GC–MS, LC–

MSn, or LC-HR-MS/MS. Both NBOMe derivatives were mainly metabolized by O-demethylation,

O,O-bis-demethylation, hydroxylation, and combinations as well as by glucuronidation and sulfation

of the main phase I metabolites. For 25B-NBOMe, 66 metabolites could be identified and 69 for 25C-

NBOMe. After application of low doses of both substances to rats, they were detectable mainly via

their metabolites by both LC-based SUSAs. In case of acute intoxication, it was possible to detect

25B-NBOMe and its metabolites in an authentic human urine sample when using the GC–MS SUSA

in addition to the LC-based SUSAs. Initial CYP activity screening revealed the involvement of

CYP1A2 and CYP3A4 in hydroxylation and CYP2C9 and CYP2C19 in O-demethylation. The

presented study demonstrated that 25B-NBOMe and 25C-NBOMe were extensively metabolized and

detectable by both LC-based SUSAs.

Liquid chromatography-tandem mass spectrometric determination of propofol in rat serum and

hair at attogram level after derivatization with 3-bromomethyl-propyphenazone

Alaa Khedr, Soad S.Abd El-Hay, Ahmed K. Kammoun

ABSTRACT

A sensitive, selective and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS)

method for determination of propofol (PRO) in rat serum and hair has been developed. 3-

Bromomethyl-propyphenazone was used as derivatization reagent forming propofol-methyl-

propyphenazone compound. The derivatization reaction was optimized and validated for maximum

MS sensitivity. The MS instrumental sensitivity reached to 10 attogram. The serum samples were

extracted by using Chromabond C8 columns, while hair samples extracted with methanol. The

tendency of volatility of PRO was minimized by adding triethylamine to the extract before the use of

nitrogen gas for evaporation of solvent. The limit of quantitation (LLOQ) was 0.01 pg/mL and the

assay was linear to 10000 pg/mL. The intra-and inter-day precision (RSD%) ranged from 0.33 to

3.44% while the accuracy (Er%, relative error) were −6.4 to 1.1%. The ionization suppression, due to

reagent, was minimized by reacting the excess reagent with methanol, and eluting to waste before MS

ionization source (2–4.5 min). The method was successfully applied for detection and determination of

PRO in rat serum and hair after 7–28 days from administration of only one dose of propofol (10

mg/kg).

37

Investigation of the metabolites of the HIF stabilizer FG-4592 (roxadustat) in five different in

vitro models and in a human doping control sample using high resolution mass spectrometry

Annelie Hansson, Mario Thevis, Holly Cox, Geoff Miller, Mikael Hedeland

ABSTRACT

FG-4592 is a hypoxia-inducible factor (HIF) stabilizer, which can increase the number of red blood

cells in the body. It has not been approved by regulatory authorities, but is available for purchase on

the Internet. Due to its ability to improve the oxygen transportation mechanism in the body, FG-4592

is of interest for doping control laboratories, but prior to this study, little information about its

metabolism was available. In this study, the metabolism of FG-4592 was investigated in a human

doping control sample and in five in vitro models: human hepatocytes and liver microsomes, equine

liver microsomes and S9 fraction and the fungus Cunninghamella elegans. By using liquid

chromatography coupled to a Q-TOF mass spectrometer operated in MSE and MSMS modes, twelve

different metabolites were observed for FG-4592. One monohydroxylated metabolite was detected in

both the human and equine liver microsome incubations. For the fungus Cunninghamella elegans

eleven different metabolites were observed of which the identical monohydroxylated metabolite had

the highest response. This rich metabolic profile and the higher levels of metabolites produced by

Cunninghamella elegans demonstrates its usefulness as a metabolite producing medium. In the doping

control urine sample, one metabolite, which was the result of a direct glucuronidation, was observed.

No metabolites were detected in neither the human hepatocyte nor in the equine liver S9 fraction

incubates.

A quantitative LC–MS/MS method for simultaneous determination of cocaine and its

metabolites in whole blood

Xiabin Chen, Xirong Zheng, Kai Ding, Ziyuan Zhou, Fang Zheng

ABSTRACT

As new metabolic pathways of cocaine were recently identified, a high performance liquid

chromatography tandem mass spectrometry (LC–MS/MS) method was developed to simultaneously

determine cocaine and nine cocaine-related metabolites in whole blood samples. One-step solid phase

extraction was used to extract all of the ten compounds and corresponding internal standards from

blood samples. All compounds and internal standards extracted were separated on an Atlantis T3 (100

Å, 3 μm, 2.1 mm × 150 mm I.D) column and detected in positive ion and high sensitivity mode with

multiple reaction monitoring. This method was validated for its sensitivity, linearity, specificity,

accuracy, precision, recovery, and stability. All of the ten compounds were quantifiable ranging from

the lower limit of quantification (LLOQs) of ∼10 nM (1.9–3.2 ng/ml) to ∼1000 nM (190–320 ng/ml)

without any interfering substance. Accuracy and precision were determined, and both of them were

within the acceptance criteria of the United States (US) Food and Drug Administration (FDA) and

European Medicines Agency (EMA) guidelines. The recovery was above 66.7% for all compounds.

Stability tests demonstrated the stability of compounds under different storage conditions in whole

blood samples. The method was successfully applied to a pharmacokinetic study with co-

administration of cocaine and alcohol in rats.

38

Use of FTIR spectroscopy and PCA-LDC analysis to identify cancerous lesions within the human

colon

E. Kaznowska, J. Depciuch, K. Szmuc, J. Cebulski

ABSTRACT

Colorectal cancer constitutes 33% of all cancer morbidity, so the research of the new methods for

colorectal cancer diagnosis and chemotherapy monitoring is gaining its momentum. Diagnostic

instruments are being sought, which enable the detection of single malignant cells based on the

analysis of tissue material potentially reusable at further stages of diagnostic management. The most

common approach to tissue specimen processing is paraffin-embedding. Yet, paraffin may cause

background noise in spectroscopic measurements with the wavenumber ranging between 900 cm−1

and 3500 cm−1. However, the study by Depciuch et al. (2016) proved that appropriate specimen

processing and paraffin-embedding technique as well as a strict measurement methodology may

eliminate paraffin vibrations. As a result, spectroscopic measurements may become a reliable and

precise method for the diagnosis and treatment monitoring in patients with colorectal cancer as long as

the high standards of specimen processing are maintained. Chemotherapy is the main medical

treatment in colorectal cancer. Unfortunately, the absence of tools which enable monitoring its efficacy

leads to the partial response or non-response frequently seen in affected patients. Hence, diagnostic

instruments are also being sought capable of monitoring treatment efficacy so as to enable early

changes of chemotherapy regimen thus increasing the chance of cure. The paper aims at comparing the

results of FTIR (Fourier Transform Infrared) spectroscopy in several types of colon tissue: healthy

colon, cancerous colon, post-chemotherapy colon and healthy surgical margin of colon cancer sample.

The obtained FTIR spectra along with the Principal Component Analysis-Linear Discriminant

Analysis (PCA-LDC) as well as bandwidth analysis of the primary amide region revealed some

differences between the spectra of healthy tissues as compared to cancerous tissues (pre- or post-

chemotherapy). Apart from confirming that FTIR spectroscopy is a good source of information on the

composition of analysed samples, this fact supports its application as a tool to facilitate understanding

the pathophysiology of various conditions and to monitor efficacy of chemotherapy in cancer patients.

Antibody-free detection of infectious bacteria using quantum dots-based barcode assay

Kristyna Cihalova, Dagmar Hegerova, Ana Maria Jimenez, Vedran Milosavljevic, Vojtech Adam

ABSTRACT

Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Klebsiella pneumoniae are the

most representative bacteria causing infectious diseases. Due to the increased application of

antibiotics, the bacterial resistance is growing causing severe complications. Therefore, a sensitive

determination of these pathogens is crucial for effective treatment. The aim of this study was to design

an effective method for multiplex detection of Staphylococcus aureus, methicillin-resistant

Staphylococcus aureus and Klebsiella pneumoniae taking advantage from properties of magnetic

particles as well as fluorescent nanoparticles (quantum dots). The method was able to detect as low

concentrations of bacteria as 102 CFU/mL using the bacteria-specific genes (fnbA, mecA and wcaG).

39

A simple and sensitive liquid chromatography–tandem mass spectrometry method for trans-ε-

viniferin quantification in mouse plasma and its application to a pharmacokinetic study in mice

Jiseon Kim, Jee Sun Min, Doyun Kim, Yu Fen Zheng, Soo Kyung Bae

ABSTRACT

In this study, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC–MS/MS)

method for the quantification of trans-ε-viniferin in small volumes (10 μl) of mouse plasma using

chlorpropamide as an internal standard was developed and validated. Plasma samples were

precipitated with acetonitrile and separated using an Eclipse Plus C18 column (100 × 4.6 mm, 1.8-μm)

with a mobile phase consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in water

(60:40 v/v) at a flow rate of 0.5 ml/min. A triple quadrupole mass spectrometer operating in positive

ion mode with selected reaction-monitoring mode was used to determine trans-ε-viniferin and

chlorpropamide transitions of 455.10 → 215.05 and 277.00 → 111.00, respectively. The lower limit of

quantification was 5 ng/ml with a linear range of 5–2500 ng/ml (r ≥ 0.9949). All validation data,

including the selectivity, precision, accuracy, recovery, dilution integrity, and stability, conformed to

the acceptance requirements. No matrix effects were observed. The developed method was

successfully applied to pharmacokinetic studies of trans-ε-viniferin following intravenous (2.5 mg/kg),

intraperitoneal (2.5, 5 and 10 mg/kg), and oral (40 mg/kg) administration in mice. This is the first

report on the pharmacokinetic properties of trans-ε-viniferin. The results provide a meaningful basis

for evaluating the pre-clinical or clinical applications of trans-ε-viniferin.

LC–MS/MS assay for the quantitation of the tyrosine kinase inhibitor neratinib in human

plasma

Brian F. Kiesel, Robert A. Parise, Alvin Wong, Kiana Keyvanjah, Jan H. Beumer

ABSTRACT

Neratinib is an orally available tyrosine kinase inhibitor targeting HER2 (ERBB2) and EGFR (ERBB).

It is being clinically evaluated for the treatment of breast and other solid tumors types as a single agent

or in combination with other chemotherapies. In support of several phase I/II clinical trials

investigating neratinib combinations, we developed and validated a novel LC–MS/MS assay for the

quantification of neratinib in 100 μL of human plasma with a stable isotopic internal standard.

Analytes were extracted from plasma using protein precipitation and evaporation of the resulting

supernatant followed by resuspension. Chromatographic separation was achieved using an Acquity

UPLC BEH Shield RP18 column and a gradient methanol-water mobile phase containing 10%

ammonium acetate. An ABI 4000 mass spectrometer and electrospray positive mode ionization were

used for detection. The assay was linear from 2 to 1,000 ng/mL and proved to be accurate (98.9–

106.5%) and precise (<6.2%CV), and met the FDA guidance for bioanalytical method validation. This

LC–MS/MS assay will be an essential tool to further define the pharmacokinetics of neratinib.

40

Comparative tissue distribution and excretion study of alkaloids from Herba Ephedrae-Radix

Aconiti Lateralis extracts in rats

Mengyue Ren, Shuai Song, Dedong Liang, Weiting Hou, Jiabo Luo

ABSTRACT

Herba Ephedrae-Radix Aconiti Lateralis, composed of Ephedrae (Mahuang in Chinese) and Radix

Aconiti Lateralis (Fuzi in Chinese), is a classical herbal combination proven to be effective in treating

common cold, asthma, and rheumatoid arthritis. Alkaloids, bioactive components of the herbal extract,

have been associated with many side effects. Nine alkaloids, including norephedrine,

norpseudoephedrine, ephedrine, pseudoephedrine, methylephedrine, hypaconitine, benzoylaconine,

benzoylmesaconine and benzoylhypaconine, were simultaneously quantified within 14.5 min, by a

validated ultra-performance liquid chromatography-tandem mass spectrometry method in various rat

tissues, urine, and feces after oral administration of Mahuang-Fuzi and single-herb extracts. The results

indicated that the alkaloids were widely distributed in the heart, liver, spleen, lung, kidney, and brain.

Lower bioavailability and higher clearance of some alkaloids were observed for the herbal

combination, but hypaconitine showed a longer residence time and lower clearance. Elimination

kinetics demonstrated that ephedra and aconitum alkaloids were mainly excreted in urine and feces,

respectively. The tissue distribution and excretion of ephedra and aconitum alkaloids are

comprehensively reported for the first time for the Mahuang-Fuzi combination. Compared with single-

herb extracts, lower extraction efficiencies of alkaloids in vitro were observed which may result in

their lower intake. However, the combination showed a prolonged residence time and delayed

elimination of aconitum alkaloids, which increases the risk of drug accumulation. The study

demonstrated potential risks of intoxication with aconitum alkaloids, associated with the use of Fuzi in

combination with Mahuang. Mahuang-Fuzi is a classical combination used in clinics, further

investigation is needed.

Deep eutectic solvents as green media for extraction of flavonoid glycosides and aglycones from

Platycladi Cacumen

Bo Zhuang, Li-Li Dou, Ping Li, E-Hu Liu

ABSTRACT

Deep eutectic solvents (DESs) are emerging as alternatives to conventional ionic liquids and organic

solvents due to their unique advantages. In the present study, the tuneability of DESs as tailor-made

solvents to efficiently extract polar and non-polar bioactive compounds from Platycladi Cacumen was

detailedly investigated. Totally 12 types of choline chloride-, betaine-, and l-proline-based DESs were

synthesized for initial screening, and extraction conditions was optimized by single-factor experiment.

Experiments with different DESs and principal components analysis demonstrated that the

extractability of both flavonoid glycosides and aglycones was greater with certain designed DESs than

conventional solvents. In addition, the water content in DESs led to significantly different extraction

yields of flavonoid compounds. The target compounds were recovered from DESs by macroporous

resin LX-38 with a satisfactory yield between 77.44% and 98.92%. The knowledge acquired in this

study could contribute to further DES application in extraction of bioactive compounds from natural

sources.

41

Testosterone and its dimers alter tRNA morphology

P. Chanphai, D. Agudelo, A.R. Vesper, G. Bérubé, H.A. Tajmir-Riahi

ABSTRACT

The morphology of tRNA was studied upon conjugation with testosterone and its aliphatic and

aromatic dimers, using multiple spectroscopic methods, transmission electron microscopy (TEM) and

molecular modeling. Structural analysis showed that testosterone binds tRNA through A62, A64, C60,

C61, C63, G51, U50 and U59 bases. The binding affinity was testosterone dimer-aromatic >

testosterone dimer-aliphatic > testosterone. The steroid loading efficacy was 35–45%. Transmission

electron microscopy showed major changes in tRNA morphology upon testosterone interaction with an

increase in the diameter of the tRNA aggregate, indicating encapsulation of testosterone by tRNA.

Development and validation of a simple and robust HPLC method with UV detection for

quantification of the hepatitis C virus inhibitor daclatasvir in human plasma

Giulio Nannetti, Lorenzo Messa, Marta Celegato, Silvana Pagni, Arianna Loregian

ABSTRACT

Daclatasvir is an inhibitor of hepatitis C virus NS5A protein that is used for the therapy of chronic

hepatitis. So far, published methods for analysis of daclatasvir in plasma are exclusively based on mass

spectrometry, which is not always available in standard clinical laboratories. Thus, we wished to

develop and validate a simple, but still reliable and sensitive high-performance liquid chromatography

(HPLC) assay with UV detection for the quantification of daclatasvir, feasible for a wide-spread

clinical routine use. The method consisted of solid-phase extraction of daclatasvir using Waters Oasis

HLB 1cc cartridges, reversed-phase liquid chromatography with a Waters XTerra RP18 (150 mm × 4.6

mm, 3.5 μm) column and a mobile phase of ammonium acetate buffer (pH 5.0, 10 mM) and

acetonitrile (56:44, v/v), and UV detection at 318 nm. This assay proved to be sensitive (lower limit of

quantification of 0.05 μg/mL), linear (correlation coefficients ≥0.997), specific (no interference with

various potentially co-administrated drugs), reproducible (both intra-day and inter-day coefficients of

variation ≤8.9%), and accurate (deviations ranged from −2.2 to 8.0% and from −6.5 to 9.2% for intra-

day and inter-day assays, respectively). The method was applied to therapeutic monitoring of patients

undergoing daclatasvir therapy for hepatitis C and showed to be reliable and robust. Thus, this method

provides a simple, sensitive, precise, and reproducible assay for dosing daclatasvir that can be readily

adaptable to routine use by clinical laboratories with standard equipment. In addition, the stability of

daclatasvir in plasma was evaluated under various conditions, including after the heating procedure

required for inactivation of infectious viruses and in different light exposure conditions. These studies

evidenced photo-instability of the compound under sunlight exposure over time. Thus, blood sampling

and the whole handling procedure have to be performed quickly and with minimal light exposure.

42

Evaluation of ion mobility spectrometry for the detection of mitragynine in kratom products

Nathan Fuenffinger, Melissa Ritchie, Ashley Ruth, Connie Gryniewicz-Ruzicka

ABSTRACT

An ion mobility spectrometry (IMS) method was developed for the rapid detection of mitragynine, the

most abundant alkaloid in Mitragyna speciosa also known as kratom. The peak corresponding to the

mitragynine protonated ion exhibited a reduced ion mobility of 0.95 ± 0.00014 cm2/(V s), and the

mitragynine limit of detection using IMS was 0.5 ng. The IMS method was applied to the analysis of

15 commercial samples suspected of containing kratom. IMS results were compared to those obtained

from liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis of the same samples.

Mitragynine was conclusively detected in 14 of 15 samples using LC–MS/MS and 13 of 15 samples

using IMS. The discrepancy between methods reflected the fact that one sample contained mitragynine

at a concentration below the IMS detection limit. This study demonstrates the utility of IMS for the

rapid screening of products containing kratom as well as the scientific reliability of the IMS screening

method, which was demonstrated by comparing the IMS results to the confirmatory results obtained

using LC–MS/MS.

A single MCR-ALS model for drug analysis in different formulations: Application on diazepam

commercial preparations

Michele De Luca, Giuseppina Ioele, Claudia Spatari, Gaetano Ragno

ABSTRACT

A multivariate curve resolution – alternating least squares (MCR-ALS) analysis was used to quantify

diazepam (DZP) in thirty commercial liquid formulations. MCR calibration was run on the UV

spectrophotometric data of the commercial DZP samples over the range 200–400 nm, allowing the

resolution of the drug signal and then the excipients contained in all the formulations. A single model

MCR for the determination of the drug in all samples was then built through the adoption of the

correlation constraint. This model was optimized by an appropriate selection of the most useful

wavelength ranges and then validated on external samples. DZP concentrations in the pharmaceutical

formulations were measured by HPLC-DAD analysis. The performance of the MCR model was

compared with that from application of classical partial least squares regression (PLSR). The results, in

terms of error of prediction, were very satisfactory, reaching a relative error below of 1.66% against

2.56%, respectively.

43

Selective recognition of cis-trans-isomers of platinum drugs and the detection of triplex DNA

based on fluorescence reversible model of quantum dots

Xiaoling Xu, Fang Gao, Xincai Xiao, Yan Hu, Dan Zhao

ABSTRACT

The identification of spatial structures of drugs and the researches on their interaction mechanism with

DNA are always attractive to the researchers. However, their realization is lack of simple and fast

method. This paper reports the establishment of multiple-functional detection platform based on the

―turn off-on‖ model of ZnCdSe quantum dots. In this system, ZnCdSe quantum dots work as the

fluorescent probe, platinum anti-cancer drugs as the quencher and triplex DNA as the trapping agent.

The seemingly similar cisplatin and transplatin exhibited different fluorescent recovery behaviors due

to their difference in structure, and thus realized the selective detection of cisplatin and transplatin with

the reaction time set at 10 min as well as the quantitation of cisplatin over the range of 2.5 × 10−8–100

× 10−8M. Based on this, the interactions between platinum anti-cancer drugs and ctDNA as well as

polymorphic DNA were further studied, and realized the recognition of triplex DNA. The multiple-

functional detection platform integrates the functions of the filtration of high-efficient platinum anti-

cancer drugs, the researches on interaction mechanism of drugs, and the recognition of polymorphic

DNA, meaningful to the future treatment of viral and cancers based on antisense gene strategy.

Drug-protein binding of Danhong injection and the potential influence of drug combination with

aspirin: Insight by ultrafiltration LC–MS and molecular modeling

Junfeng Zhu, Xiaojiao Yi, Peng Huang, Shuqing Chen, Yongjiang Wu

ABSTRACT

Danhong injection (DHI) is a widely used Chinese medicine injection (CMI) for the clinical treatment

of cardiovascular and cerebrovascular diseases. In this study, a simple and efficient in vitro method

based on ultrafiltration LC–MS and molecular modeling has been developed to study the human serum

albumin (HSA) binding of the compounds in DHI. Seven major components including protocatechuic

aldehyde, p-coumaric acid, salvianolic acid D, rosmarinic acid, salvianolic acid E, lithospermic acid

and salvianolic acid B were identified as HSA ligands and their binding degrees in the proposed non-

saturated model were 26.17, 37.69, 99.77, 91.78, 96.91, 99.42 and 98.10%, respectively. Considering

the drug-HSA binding property of the compounds in DHI may change during drug combination

therapy, competitive binding assay was carried out to evaluate the influence of aspirin on the DHI-

HSA binding. Experimental results revealed that the salvianolic acids in DHI had stronger binding

ability to HSA than sodium salicylate. To further verify the results above, molecular modeling and

probe displacement assay were conducted to investigate the optimum binding site and binding affinity

of the ligands on HSA. Our findings suggested that the established method could be a powerful tool to

study the drug-HSA binding property of CMIs.

44

The importance of vial composition in HPLC analysis: An unusual case of phosphorous

pseudorotation

Rebecca Arvary, Ian Mangion

ABSTRACT

Glass HPLC vials are ubiquitous in analytical laboratories and vendors have developed many varieties

to meet the various needs of scientists. As such there may be multiple types of vials being used

simultaneously in a single laboratory without much consideration as to which is best suited for

analytical method development and validation. This study highlights the possibility of vial

composition as a potential factor that impacts solution stability. Here we describe a case where the

type of HPLC vial used results in an interesting phosphorous pseudorotation driven by the mild

alkalinity of glass.

Supercritical fluid chromatography for separation and preparation of tautomeric 7-epimeric

spiro oxindole alkaloids from Uncaria macrophylla

Wenzhi Yang, Yibei Zhang, Huiqin Pan, Changliang Yao, Dean Guo

ABSTRACT

Increasing challenge arising from configurational interconversion in aqueous solvent renders it rather

difficult to isolate high-purity tautomeric reference standards and thus largely hinders the holistic

quality control of traditional Chinese medicine (TCM). Spiro oxindole alkaloids (SOAs), as the

markers for the medicinal Uncaria herbs, can easily isomerize in polar or aqueous solvent via a retro-

Mannich reaction. In the present study, supercritical fluid chromatography (SFC) is utilized to separate

and isolate two pairs of 7-epimeric SOAs, including rhynchophylline (R) and isorhynchophylline (IR),

corynoxine (C) and corynoxine B (CB), from Uncaria macrophylla. Initially, the solvent that can

stabilize SOA epimers was systematically screened, and acetonitrile was used to dissolve and as the

modifier in SFC. Then, key parameters of ultra-high performance SFC (ultra-performance

convergence chromatography, UPC2), comprising stationary phase, additive in modifier, column

temperature, ABPR pressure, and flow rate, were optimized in sequence. Two isocratic UPC2 methods

were developed on the achiral Torus 1-AA and Torus Diol columns, suitable for UV and MS detection,

respectively. MCI gel column chromatography fractionated the U. macrophylla extract into two

mixtures (R/IR and C/CB). Preparative SFC, using a Viridis Prep Silica 2-EP OBD column and

acetonitrile-0.2% diethylamine in CO2 as the mobile phase, was finally employed for compound

purification. As a result, the purity of four SOA compounds was all higher than 95%. Different from

reversed-phase HPLC, SFC, by use of water-free mobile phase (inert CO2 and aprotic modifier),

provides a solution to rapid analysis and isolation of tautomeric reference standards for quality control

of TCM.

45


Renewal of an old European Pharmacopoeia method for Terazosin using modeling with mass

spectrometric peak tracking

Róbert Kormány, Imre Molnár, Jenő Fekete

ABSTRACT

An older method for terazosin was reworked in order to reduce the analysis time from 90 min (2 × 45

min) to below 5 min. The method in European Pharmacopoeia (Ph.Eur.) investigates the specified

impurities separately. The reason of the different methods is that the retention of two impurities is not

adequate in reversed phase, not even with 100% water. Therefore ion-pair-chromatography has to be

applied and since that two impurities absorb at low UV-wavelength they had to be analyzed by

different method than the other specified impurities. In our new method we could improve the

retention with pH elevation using a new type of stationary phases available for high pH applications.

Also a detection wavelength could be selected that is appropriate for the detection and quantification

of all impurities. The method development is the bottleneck of liquid chromatography even today,

when more and more fast chromatographic systems are used. Expert knowledge with intelligent

programs is available to reduce the time of method development and offer extra information about the

robustness of the separation. Design of Experiments (DoE) for simultaneous optimization of gradient

time (tG), temperature (T) and ternary eluent composition (tC) requires 12 experiments. A good

alternative way to identify a certain peak in different chromatograms is the molecular mass of the

compound, due to its high specificity. Liquid Chromatography–Mass Spectrometry (LC–MS) is now a

routine technique and increasingly available in laboratories. In our experiment for the resolution- and

retention modeling the DryLab4 method development software (Version 4.2) was used. In recent

versions of the software the use of (m/z)-MS-data is possible along the UV-peak-area-tracking

technology. The modelled and measured chromatograms showed excellent correlations. The average

retention time deviations were ca. 0.5 s and there was no difference between the predicted and

measured Rs,crit −values.

A revisited structure for nitrosoprodenafil from NMR, mass spectrometry, X-ray and hydrolysis

data

Robert Martino, Christophe Menendez, Stéphane Balayssac, Nathalie Martins-Froment, Myriam

Malet-Martino

ABSTRACT

The sildenafil analogue adulterant previously identified as a nitroso derivative (nitrosoprodenafil) in a

dietary supplement (DS) marketed to increase sexual performance and sold in Europe in the early 2010

s is the same as that found in the same type of DS available in Japan whose structure was established

as a nitro derivative (mutaprodenafil or nitroprodenafil). Indeed, the compound isolated from the Man

Power DS has identical UV, IR, NMR and MS spectroscopic characteristics and hydrolysis behavior

than nitrosoprode-nafil. By revisiting its NMR assignments and MS and MS/MS data interpretation, it

is demonstrated that the compound is actually a nitrothioimidazole-methisosildenafil hybrid, i.e.

nitroprodenafil, whose structure is unequivocally confirmed by X-ray crystallography and synthesis

experiments. Because the product is converted to methisosildenafil by hydrolysis, it is named

nitropromethisosildenafil.

46

The importance of system band broadening in modern size exclusion chromatography

Alexandre Goyon, Davy Guillarme, Szabolcs Fekete

ABSTRACT

In the last few years, highly efficient UHP-SEC columns packed with sub–3 μm particles were

commercialized by several providers. Besides the particle size reduction, the dimensions of modern

SEC stationary phases (150 × 4.6 mm) was also modified compared to regular SEC columns (300 × 6

or 300 × 8 mm). Because the analytes are excluded from the pores in SEC, the retention factors are

very low, ranging from −1 < k < 0, resulting in very small column band variance. Therefore, the

contribution of the system itself to peak variance can become significant under UHP-SEC conditions.

The goal of this study was to evaluate the loss of efficiency observed with three different instruments

(regular HPLC, non-optimized UHPLC and fully optimized UHPLC) offering different system

variances. It appears that the new 150 × 4.6 mm, sub–3 μm SEC columns cannot be employed on a

regular HPLC instrument, since the efficiency loss was equal to 60–85%, when analyzing mAb

sample. Optimized UHPLC systems having very low extra-column volumes (typically Vec < 10 μL)

have therefore to be used to properly operate these columns. Due to the instrument contribution to

band broadening, the apparent efficiency of SEC columns packed with sub–2 μm particles can indeed

be hampered when using inappropriate system. Considering the extra-column band broadening

contribution of current UHPLC instruments, a further decrease of SEC column dimension is therefore

not desired.

Development of a multi-residue method for the determination of human and veterinary

pharmaceuticals and some of their metabolites in aqueous environmental matrices by SPE-

UHPLC–MS/MS

P. Paíga, L.H.M.L.M. Santos, C. Delerue-Matos

ABSTRACT

The aim of the present work was to develop and validate a multi-residue method for the analysis of 33

human and veterinary pharmaceuticals (non-steroidal anti-inflammatory drugs (NSAIDs)/analgesics,

antibiotics and psychiatric drugs), including some of their metabolites, in several aqueous

environmental matrices: drinking water, surface water and wastewaters. The method is based on solid

phase extraction (SPE) followed by ultra-high performance liquid chromatography-tandem mass

spectrometry (UHPLC–MS/MS) and it was validated for different aqueous matrices, namely bottled

water, tap water, seawater, river water and wastewaters, showing recoveries between 50% and 112%

for the majority of the target analytes. The developed analytical methodology allowed method

detection limits in the low nanograms per liter level. Method intra- and inter-day precision was under

8% and 11%, respectively, expressed as relative standard deviation. The developed method was

applied to the analysis of drinking water (bottled and tap water), surface waters (seawater and river

water) and wastewaters (wastewater treatment plant (WWTP) influent and effluent). Due to the

selectivity and sensitivity of the optimized method, it was possible to detect pharmaceuticals in all the

aqueous environmental matrices considered, including in bottled water at concentrations up to 31 ng

L−1 (salicylic acid). In general, non-steroidal anti-inflammatory drugs/analgesics was the therapeutic

group most frequently detected, with the highest concentrations found in wastewaters (acetaminophen

and the metabolite carboxyibuprofen at levels up to 615 and 120 μg L−1, respectively).

47

Development of new efficient method for isolation of phenolics from sea algae prior to their

rapid resolution liquid chromatographic–tandem mass spectrometric determination

Bořivoj Klejdus, Merichel Plaza, Marie Ńnóblová, Lea Lojková

ABSTRACT

The extraction of phenolic compounds from 4 different sea algae samples, three brown algae

(Cystoseira abies-marina, C. abies-marina grinded under cryogenic conditions with liquid nitrogen,

Undaria pinnatifida and Sargassum muticum) and one red algae (Chondrus crispus) via solid phase

extraction using micro-elution solid-phase extraction (μ-SPE) plate method was studied. Prior to μ-

SPE, 50 mg of algae with 80% methanol mixture was extracted in hyphenated series by various

extraction techniques, such as pressurized liquid extraction and Ika Ultra-Turrax® Tube Drive, in

combination with ultrasound assisted extraction. The μ-SPE plate technique reduced the time of

sample pre-treatment thanks to higher sensitivity and pre-concentration effect. Selected groups of

benzoic acid derivatives (p-hydroxybenzoic, protocatechuic, gallic, vanillic, and syringic acids),

hydroxybenzaldehydes (4-hydroxybenzaldehyde, and 3,4-dihydroxybenzaldehyde), and cinnamic acid

derivatives (p-coumaric, caffeic, ferulic, sinapic, and chlorogenic acids) were determined using rapid

resolution liquid chromatography coupled to mass spectrometry detection with negative ion

electrospray ionization (RRLC-ESI–MS) using multiple reactions monitoring. LOQs of measured

samples varied in the range 0.23–1.68 ng/mL and LODs in the range 0.07–0.52 ng/mL. The applied

method allowed a simultaneous determination of phenolics (i.e. free, esters soluble in methanol,

glycosides, and esters insoluble in methanol) in less than 5 min (including alkaline or acidic hydrolysis

of raw extracts) from sea algae extracts.

Physico-chemical profiling of semisynthetic opioids

Károly Mazák, Sándor Hosztafi, Márta Kraszni, Béla Noszál

ABSTRACT

Species-specific acid–base and partition equilibrium constants were experimentally determined for the

therapeutically important semisynthetic opioid receptor agonist hydromorphone, dihydromorphine, and

mixed agonist-antagonist nalorphine and nalbuphine. The acid–base microequilibria were

characterized by combining pH-potentiometry and deductive methods using synthesized auxiliary

compounds. Independent of the pH, there are approximately 4.8 times as many zwitterionic

microspecies than non-charged ones in nalbuphine solutions, while for nalorphine it is the non-charged

form that predominates by the same ratio. The non-charged microspecies is the dominant one also in

the case of hydromorphone, although its concentration exceeds only 1.3 times that of its zwitterionic

protonation isomer. The pH-independent partition coefficients of the individual microspecies were

determined by a combination of experimentally measured, pH-dependent, conditional distribution

constants and a custom-tailored evaluation method, using highly similar auxiliary compounds. The pH-

independent contribution of the zwitterionic microspecies to the distribution constant is 1380, 1070,

3160 and 72,440 times smaller than that of the inherently more lipophilic non-charged one for

hydromorphone, dihydromorphine, nalbuphine and nalorphine, respectively.

48

Interaction of anticancer drug clofarabine with human serum albumin and human α-1 acid

glycoprotein Spectroscopic and molecular docking approach

Mohammad Rehan Ajmal, Saima Nusrat, Parvez Alam, Nida Zaidi, Rizwan Hasan Khan

ABSTRACT

The binding interaction between clofarabine, an important anticancer drug and two important carrier

proteins found abundantly in human plasma, Human Serum Albumin (HSA) and α-1 acid glycoprotein

(AAG) was investigated by spectroscopic and molecular modeling methods. The results obtained from

fluorescence quenching experiments demonstrated that the fluorescence intensity of HSA and AAG is

quenched by clofarabine and the static mode of fluorescence quenching is operative. UV–vis

spectroscopy deciphered the formation of ground state complex between anticancer drug and the two

studied proteins. Clofarabine was found to bind at 298 K with both AAG and HSA with the binding

constant of 8.128 × 103 and 4.120 × 103 for AAG and HSA, respectively. There is stronger interaction

of clofarabine with AAG as compared to HSA. The Gibbs free energy change was found to be

negative for the interaction of clofarabine with AAG and HSA indicating that the binding process is

spontaneous. Binding of clofarabine with HSA and AAG induced ordered structures in both proteins

and lead to molecular compaction. Clofarabine binds to HSA near to drug site II. Hydrogen bonding

and hydrophobic interactions were the main bonding forces between HSA-clofarabine and AAG-

clofarabine as revealed by docking results. This study suggests the importance of binding of anticancer

drug to AAG spatially in the diseases like cancers where the plasma concentration of AAG increases

many folds. Design of drug dosage can be adjusted accordingly to achieve optimal treatment outcome.

A novel method for the determination of chemical purity and assay of menaquinone-7

Comparison with the methods from the official USP monograph

Łukasz Jedynak, Maria Jedynak, Magdalena Kossykowska, Joanna Zagrodzka

ABSTRACT

An HPLC method with UV detection and separation with the use of a C30 reversed phase analytical

column for the determination of chemical purity and assay of menaquinone-7 (MK7) in one

chromatographic run was developed. The method is superior to the methods published in the USP

Monograph in terms of selectivity, sensitivity and accuracy, as well as time, solvent and sample

consumption. The developed methodology was applied to MK7 samples of active pharmaceutical

ingredient (API) purity, MK7 samples of lower quality and crude MK7 samples before purification.

The comparison of the results revealed that the use of USP methodology could lead to serious

overestimation (up to a few percent) of both purity and MK7 assay in menaquinone-7 samples.

49

Structure and pharmaceutical formulation development of a new long-acting recombinant

human insulin analog studied by NMR and MS

Elżbieta Bednarek, Jerzy Sitkowski, Wojciech Bocian, Piotr Borowicz, Lech Kozerski

ABSTRACT

A monomer structure of novel human insulin analog A22S-B3K-B31R (SK3R) has been characterized

by NMR in water/acetonitrile solution and compared with the structure of human insulin (HIS)

established in the same medium. The composition of the oligomer ensemble for neat insulins in water

was qualitatively assessed by monitoring, derived from NMR experiment, translational diffusion

coefficient Di x 10−10 m2 s−1, whose value is a population averaged of individual coefficients for

species in oligomeric ensemble. Nanospray ESI/MS experiment was used to establish the masses of

oligomers in pharmaceutical formulation of the SK3R insulin. The pharmacodynamic data were

established and compared to insulin glargine characterized by the same profile of action in diabetics.

The oligomerization process of insulin during development of pharmaceutical formulation with

routinely used excipients has been studied using translation diffusion coefficient Di x 10−10 m2 s−1

established in water solution. These properties were compared with those of human insulin (HIS)

which is a standard reference for novel recombinant insulins.

Simultaneous HPLC assay for pretomanid (PA-824), moxifloxacin and pyrazinamide in an

inhaler formulation for drug-resistant tuberculosis

Mohammad A.M. Momin, Sim J. Thien, Woravimol Krittaphol, Shyamal C. Das

ABSTRACT

A simple and sensitive reversed phase HPLC method has been developed for the simultaneous

quantitation of pretomanid (PA-824), moxifloxacin and pyrazinamide in a combination spray-dried

powder formulation for inhalation, without any use of an internal standard. Good resolution of the

analytes was achieved on a Luna C18 (2), 150 × 4.6 mm, 5 μm, 100 Å column using gradient elution

with a mobile phase containing methanol and triethylamine phosphate buffer (pH 2.5) at a flow rate of

1.0 mL/min in a total run time of 25 min. Pyrazinamide, moxifloxacin and pretomanid (PA-824) were

detected at wavelengths (retention times) of 269 nm (3.80 min), 296 nm (7.94 min) and 330 nm (17.46

min), respectively. The assay was linear for all analytes in the concentration range 2.5–100 μg/mL

(correlation coefficients >0.999) with LODs and LLOQs (μg/mL) of pretomanid (PA-824) 0.51 and

1.56, moxifloxacin 0.06 and 0.19 and pyrazinamide 0.35 and 1.06, respectively. Recoveries of the

three drugs were 99.6–106.8% with intra- and inter-day precisions (as relative standard deviation) of

<7%. The method was successfully applied to an evaluation of content uniformity and freedom from

interference by l-leucine of a spray-dried combination powder for inhalation.

50

Levothyroxine sodium revisited: A wholistic structural elucidation approach of new impurities

via HPLC-HRMS/MS, on-line H/D exchange, NMR spectroscopy and chemical synthesis

M. Ruggenthaler, J. Grass, W. Schuh, C.G Huber, R.J. Reischl

ABSTRACT

The structural elucidation of unknown pharmaceutical impurities plays an important role in the quality

control of newly developed and well-established active pharmaceutical ingredients (APIs). The United

States Pharmacopeia (USP) monograph for the API Levothyroxine Sodium, a synthetic thyroid

hormone, features two high pressure liquid chromatography (HPLC) methods using UV-VIS

absorption detection to determine organic impurities in the drug substance. The impurity profile of the

first USP method (―Procedure 1‖) has already been extensively studied, however for the second

method (―Procedure 2‖), which exhibits a significantly different impurity profile, no wholistic

structural elucidation of impurities has been performed yet. Applying minor modifications to the

chromatographic parameters of USP ―Procedure 2‖ and using various comprehensive structural

elucidation methods such as high resolution tandem mass spectrometry with on-line hydrogen-

deuterium (H/D) exchange or two-dimensional nuclear magnetic resonance spectroscopy (NMR) we

gained new insights about the complex impurity profile of the synthetic thyroid hormone. This resulted

in the characterization of 24 compounds previously unknown to literature and the introduction of two

new classes of Levothyroxine Sodium impurities. Five novel compounds were unambiguously

identified via isolation or synthesis of reference substances and subsequent NMR spectroscopic

investigation. Additionally, Collision-Induced Dissociation (CID)-type fragmentation of identified

major impurities as well as neutral loss fragmentation patterns of many characterized impurities were

discussed.

Verification of the authenticity of drugs by means of NMR relaxometry—Viagra® as an example

S. Wilczyńki, M. Petelenz, M. Florek-Wojciechowska, S. Kulesza, D. Kruk

ABSTRACT

1H spin-lattice Nuclear Magnetic Resonance (NMR) relaxometry, vibrational spectroscopy and

Atomic Force Microscopy (AFM) have been applied to differentiate between original and counterfeit

Viagra®. The relaxation studies have been performed in a frequency range covering four orders of

magnitude, from 4 kHz to 40 MHz. It has been shown that for the counterfeit product the relaxation is

bi-exponential in the whole frequency range, while for the original Viagra® the relaxation process is

always single exponential. Thus, even a qualitative analysis of the relaxation data makes it possible to

identify the falsified medicine. Moreover, it has been demonstrated that vibrational spectroscopy does

not allow for differentiating between the products, while AFM studies are likely to lead one to

deceptive conclusions regarding the originality of the medicine. Furthermore, a quantitative analysis of

the relaxation data has been performed to describe in detail the relaxation properties of the original and

falsified products.

51

Metabolomics study of Populus type propolis

Boban AnĎelković, Ljubodrag Vujisić, Ivan Vučković, Vele Teńević, Dejan GoĎevac

ABSTRACT

Herein, we propose rapid and simple spectroscopic methods to determine the chemical composition of

propolis derived from various Populus species using a metabolomics approach. In order to correlate

variability in Populus type propolis composition with the altitude of its collection, NMR, IR, and UV

spectroscopy followed by OPLS was conducted. The botanical origin of propolis was established by

comparing propolis spectral data to those of buds of various Populus species. An O2PLS method was

utilized to integrate two blocks of data. According to OPLS and O2PLS, the major compounds in

propolis samples, collected from temperate continental climate above 500 m, were phenolic glycerides

originating from P. tremula buds. Flavonoids were predominant in propolis samples collected below

400 m, originating from P. nigra and P. x euramericana buds. Samples collected at 400–500 m were of

mixed origin, with variable amounts of all detected metabolites.

Hydrophilic interaction liquid chromatography method development and validation for the

assay of HEPES zwitterionic buffer

Xiaolong Xu, Bert Gevaert, Nathalie Bracke, Han Yao, Bart De Spiegeleer

ABSTRACT

HEPES is a zwitterionic buffer component used as a raw material in the GMP-manufacturing of

advanced therapy medicinal products (ATMPs), hence requiring an adequate assay method with

sufficient selectivity toward related impurities. Therefore, a hydrophilic interaction chromatography

(HILIC) method was developed. Different factors were investigated towards the retention behavior of

HEPES, its analogue EPPS and its starting material isethionate: pH, ion concentration and organic

solvent ratio of the mobile phase, as well as column temperature. Moreover, stress testing resulted in

the N-oxide degradant, identified by high resolution MS. The final method consisted of an isocratic

system with an aqueous (pH 2.0 with H3PO4) acetonitrile (35:65, v/v) mobile phase on a zwitterionic

HILIC (Obelisc N) column with a flow rate of 0.5 mL/min and UV detection at 195 nm. The assay

method of HEPES was validated, obtaining adequate linearity (R2 = 0.999), precision (RSD of 0.5%)

and accuracy (recovery of 100.08%). Finally, the applicability of the validated method was

demonstrated by analysis of samples from different suppliers.

52

Simultaneous analysis of glucocorticosteroid fluticasone propionate and its metabolite

fluticasone propionate 17β-carboxylic acid in human plasma by UPLC–MS/MS at sub pg/mL

level

Sneha G. Nair, Daxesh P. Patel, Mallika Sanyal, Puran Singhal, Pranav S. Shrivastav

ABSTRACT

A highly sensitive and rapid ultra performance liquid chromatography-tandem mass spectrometry

method has been developed for the simultaneous determination of fluticasone propionate (FP) and its

major metabolite, fluticasone propionate-17beta-carboxylic acid (FP 17β-CA) in human plasma. The

analytes and their deuterated internal standards, FP-d3 and FP 17β-CA-d3 were extracted from 500 μL

plasma samples by solid phase extraction on Oasis MAX cartridges. The chromatographic analysis

was performed on ACQUITY UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) column using methanol-

acetonitrile (50:50, v/v) and 2.0 mM ammonium trifluroacetate (ATFA) (85:15, v/v) as the mobile

phase. Following separation of the analytes, protonated precursor → product ion transitions (FP: m/z

501.1 → 293.2, FP17β-CA: m/z 453.3 → 293.2, FP-d3: m/z 504.2 → 293.2, FP 17β-CA-d3: m/z 456.3

→ 293.2) were monitored on FP 17β-CA a triple quadrupole mass spectrometer, operating in multiple

reaction monitoring (MRM) and positive ionization mode. The calibration curves were established in

the range of 0.5–100 pg/mL with a correlation coefficient (r2) ≥ 0.9992 for both the analytes. The

intra-batch and inter-batch accuracy and precision varied from 95.5-103.4% and 0.74-5.06% across

quality controls for both the analytes. The mean assay recoveries for FP and FP 17β-CA were 84.2%

and 93.5% respectively. The validated method was successfully applied to support a bioequivalence

study of 200 μg FP, administered using nasal spray formulation in 18 healthy Indian subjects.

Reproducibility of the method was assessed by reanalysis of 98 incurred study samples.

Fast Screening of Tissue Samples for Glycogen

Raluca-Ioana Stefan-van Staden, Amalia Gabriela Diaconeasa, Camelia Stanciu-Gavan

ABSTRACT

Screening of tissue samples for glycogen is very important in assessing the ageing, but also the state of

health of the tissue. Therefore, two needle stochastic sensors based on maltodextrins presenting

different dextrose equivalence (DE) MD-m (DE 13.0-17.0), and MD-M (DE 16.5-19.5) immobilized

in diamond paste (obtained from synthetic diamond and paraffin oil) were designed and characterized.

These stochastic sensors were used reliable for both qualitative and quantitative analysis for the assay

of glycogen in tissue samples with limits of determination as low as 1 fmol L−1. select article Urinary

metabolic profiling of cisplatin nephrotoxicity and nephroprotective effects of <em>Orthosiphon

stamineus</em> leaves elucidated by <sup>1</sup>H NMR spectroscopy

53

Urinary metabolic profiling of cisplatin nephrotoxicity and nephroprotective effects of

Orthosiphon stamineus leaves elucidated by 1H NMR spectroscopy

Raghunath Pariyani, Intan Safinar Ismail, Amalina Azam, Alfi Khatib, Hazilawati Hamza

ABSTRACT

Orthosiphon stamineus (OS) is a popular medicinal herb used in traditional Chinese medicine as a

diuretic agent and for renal system disorders. This study employed 1H NMR based metabolomics

approach to investigate the possible protective activity of OS in cisplatin induced nephrotoxicity owing

to its diuretic and antioxidant activities. Aqueous (OSAE) and 50% aqueous ethanolic (OSFE) extracts

of OS leaves were orally administered at 400 mg/kg BW doses to rats which were then

intraperitoneally injected with cisplatin at 5 mg/kg BW dose. The 1H NMR profile of the urine

samples collected on day 5 after cisplatin administration were analyzed by multivariate pattern

recognition techniques, whereby 19 marker metabolites suggestive in the involvement of TCA cycle,

disturbed energy metabolism, altered gut microflora and BCAA metabolism pathways were identified.

It was observed that OSFE caused significant changes (p < 0.05) in the levels of 8 markers namely

leucine, acetate, hippurate, lysine, valine, 2-oxoglutarate, 3-HBT and acetoacetate resulting in a

moderate ameliorative effect, however, it did not completely protect from nephrotoxicity. OSAE did

not demonstrate significant down regulatory effects on any markers, albeit, it potentiated the cisplatin

nephrotoxicity by inducing significant increase in glucose, glycine, creatinine, citrate, TMAO, acetate

and creatine levels. A Principal Component Analysis (PCA) of the 1H NMR spectra of OS extracts

identified that OSFE had higher concentrations of the secondary metabolites such as caffeic acid,

chlorogenic acid, protocatechuic acid and orthosiphol, among others. Whereas, OSAE was

characterized by higher concentrations of acetate, lactate, succinic acid, valine and

phosphatidylcholine. This research denotes the first comprehensive analysis to identify the effects of

OS extracts on cisplatin nephrotoxicity.

54

Preparation, characterization and in vivo evaluation of a formulation of dantrolene sodium with

hydroxypropyl-β-cyclodextrin

Mengmeng Chen, Qijuan Wu, Juan Jiang, Xin Jin, Chunjie Zhao

ABSTRACT

Dantrolene sodium (Da) is an effective skeletal muscle relaxant. However, its pharmacological effects

are severely limited owing to its poor solubility and low oral bioavailability. In order to solve these

problems, an inclusion complex using hydroxypropyl-β-cyclodextrin (HP-β-CD) to improve the oral

bioavailability of Da was prepared successfully by freeze-drying. The prepared complex was

characterized by Powder X-ray diffractometry (PXRD), Fourier transform infrared spectroscopy

(FTIR) and evaluated by a dissolution test and a pharmacokinetic study. The results of PXRD and

FTIR proved the formation of a complex between Da and HP-β-CD. The dissolution rate of Da was

markedly improved from inclusion complex with more than 90% being released within 5 min. The in

vivo pharmacokinetics of Da and dantrolene sodium-hydroxypropyl-β-cyclodextrin (Da-HP-β-CD)

inclusion complex were investigated in rats using a UPLC/MS/MS method. The Cmax and AUC0-t of

the Da-HP-β-CD inclusion complex were 5- and 3-fold higher than that of the Da. These results

suggested that the Da-HP-β-CD inclusion complex markedly improved the dissolution rate and

bioavailability of Da. select article Promotion of classic neutral bile acids synthesis pathway is

responsible for cholesterol-lowing effect of Si-miao-yong-an decoction: Application of LC–MS/MS

method to determine 6 major bile acids in rat liver and plasma

Promotion of classic neutral bile acids synthesis pathway is responsible for cholesterol-lowing

effect of Si-miao-yong-an decoction: Application of LC–MS/MS method to determine 6 major

bile acids in rat liver and plasma

Ziying Liu, Yu Zhang, Ruowen Zhang, Liqiang Gu, Xiaohui Chen

ABSTRACT

Si-miao-yong-an decoction (SMYAD), a traditional Chinese medicine formula, significantly reduced

plasma TC, LDL-c levels and increased HDL-c level in hyperlipidemia rats. Liver function test and

tissue section examination indicated that SMYAD improved liver function and reduced fat

accumulation in hyperlipidemia rat liver. A LC–MS/MS method was established and well validated to

evaluate major bile acids derived from cholesterol metabolism through the classic neutral pathway and

the alternative acidic pathway (cholic acid, chenodeoxycholic acid and their taurine and glycine

conjugates) in liver and plasma. Increased total 6 bile acids concentrations in both liver and plasma

were observed after oral administration of 12 g/kg/d, 24 g/kg/d and 36 g/kg/d of SMYAD in a dose

dependent manner which contributed to eliminate of cholesterol. Cholic acid, taurocholic acid and

glycocholic acid act as the main products of bile acid classic neutral synthesis pathway and show sharp

increase (p < 0.01) after treatment of SMYAD at dosage of 24–36 g/kg/d. For liver samples,

taurocholic acid level act as the largest growth section, while in plasma samples, cholic acid act as the

largest growth section after SMYAD treatment, compared with Model group. By contrast, the main

products of alternative acidic pathway (chenodeoxycholic acid and its glycine and taurine conjugates)

show no significant increase after treatment of SMYAD. In conclusion, the cholesterol lowing effect of

SMYAD may be related with the accelerated transformation of cholesterol into bile acids through the

classic neutral pathway.

55

Analysis of amino acid and monoamine neurotransmitters and their metabolites in rat urine of

Alzheimer’s disease using in situ ultrasound-assisted derivatization dispersive liquid-liquid

microextraction with UHPLC–MS/MS

Xian-En Zhao, Yongrui He, Meng Li, Guang Chen, Jinmao You

ABSTRACT

Neurotransmitters (NTs) may play an important role in neurodegenerative disorders such as

Alzheimer‘s disease (AD). In order to investigate the potential links, a new simple, fast, accurate and

sensitive analytical method, based on in situ ultrasound-assisted derivatization dispersive liquid-liquid

microextraction (in situ UA-DDLLME) coupled with ultra high-performance liquid chromatography

tandem mass spectrometry (UHPLC–MS/MS), has been developed and validated. The quantitation of

amino acid neurotransmitters (AANTs) and monoamine neurotransmitters (MANTs) in urine of AD

rats were performed in this work. The in situ UA-DDLLME procedure involved the rapid injection of

the mixture of low toxic 4-bromoanisole (extractant) and acetonitrile (dispersant), which containing

the new designed and synthesized 4′-carbonyl chloride rosamine (CCR) as derivatization reagent, into

the aqueous phase of real sample and buffer. Under the selected conditions, the derivatization and

microextraction of analytes were simultaneously completed within 1 min. Good linearity for each

analyte (R > 0.992) was observed with low limit of detections (LODs, S/N > 3). Moreover, the

proposed method was compared with direct detection or other reported methods, and the results

showed that low matrix effects and good recoveries results were obtained in this work. Taken together,

in situ UA-DDLLME coupled with UHPLC–MS/MS analysis was demonstrated to be a good method

for sensitive, accurate and simultaneous monitoring of AANTs and MANTs. This method would be

expected to be highly useful in AD diseases‘ clinical diagnostics and may have potential value in

monitoring the efficacy of treatment.

Rapid determination of alkaloids in Macleaya cordata using ionic liquid extraction followed by

multiple reaction monitoring UPLC–MS/MS analysis

Linqiu Li, Mingyuan Huang, Junli Shao, Bokun Lin, Qing Shen

ABSTRACT

The ultrasonic-assisted extraction (UAE) and ionic liquid based dispersive liquid–liquid

microextraction (IL-DLLME) have been successfully applied in extracting of six alkaloids from M.

cordata. 1-hexyl-3-methylimidazolium tetrafluoroborate ([C6MIM][BF4]) aqueous solution was used

as extraction solvent. The target analytes in raw material were deposited into a single drop of 1-hexyl-

3-methylimidazolium hexafluorophosphate ([C6MIM][PF6]), which was in situ formed by mixing

[C6MIM][BF4] and potassium hexafluorophosphate ([K][PF6]. Afterwards, the extract was analyzed

by ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) in multiple-

reaction monitoring (MRM) mode. The proposed method was fully validated in terms of linearity

(0.9983–0.9992), LOD (0.080 ng mL−1), LOQ (0.25 ng mL−1), intra-day precision (<5.46%), inter-

day precision (<6.36%), and recovery (86.42–112.48%). The results indicate that the approach of

combining IL-DLLME with UPLC–MS/MS is powerful and practical for analyzing alkaloids in M.

cordata., and it also has great potential for comprehensive quality control of other herbal medicines.

56

Volumetric absorptive microsampling (VAMS) as an alternative to conventional dried blood

spots in the quantification of miltefosine in dried blood samples

A.E. Kip, K.C. Kiers, H. Rosing, J.H.M. Schellens, T.P.C. Dorlo

ABSTRACT

Miltefosine is an oral agent against the neglected tropical disease leishmaniasis, which is mostly

endemic in resource-poor areas. Dried blood spot (DBS) sampling is an attractive alternative to plasma

sampling for pharmacokinetic studies in these remote areas, but introduces additional variability in

analyte quantification due to possible blood spot inhomogeneity and variability in blood spot volume

and haematocrit values. Volumetric absorptive microsampling (VAMS) potentially overcomes a few

of these issues as the VAMS device absorbs a fixed volume that is processed as a whole. We

developed and validated an LC–MS/MS method for the quantification of miltefosine with this novel

sampling technique with good performance in terms of linearity, selectivity, accuracy (bias within

±10.8%), precision (CV% ≤ 11.9%), recovery, carry-over and matrix effect. VAMS samples were

stable for at least one month at room temperature and 37 °C. The impact of haematocrit on assay

accuracy was reduced compared to conventional DBS sampling, but indicated a declining recovery

with increased haematocrit due to haematocrit dependency in recovery from the sampling device. A

clinical validation will be required to investigate whether VAMS is an appropriate and cost-effective

alternative sampling method to conventional DBS sampling.

The integration of GC–MS and LC–MS to assay the metabolomics profiling in Panax ginseng

and Panax quinquefolius reveals a tissue- and species-specific connectivity of primary

metabolites and ginsenosides accumulation

Jia Liu, Yang Liu, Yu Wang, Ann Abozeid, Zhong-Hua Tang

ABSTRACT

The traditional medicine Ginseng mainly including Panax ginseng and Panax quinquefolius is the most

widely consumed herbal product in the world. Despite the extensive investigation of biosynthetic

pathway of the active compounds ginsenosides, our current understanding of the metabolic interlink

between ginsenosides synthesis and primary metabolism at the whole-plant level. In this study, the

tissue-specific profiling of primary and the secondary metabolites in two different species of ginseng

were investigated by gas chromatography- and liquid chromatography coupled to mass spectrometry.

A complex continuous coordination of primary- and secondary-metabolic network was modulated by

tissues and species factors during growth. The results showed that altogether 149 primary compounds

and 10 ginsenosides were identified from main roots, lateral roots, stems, petioles and leaves in P.

ginseng and P. quinquefolius. The partial least squares-discriminate analysis (PLS-DA) revealed

obvious compounds distinction among tissue-specific districts relative to species. To survey the

dedication of carbon and nitrogen metabolism in different tissues to the accumulation of ginsenosides,

we inspected the tissue-specific metabolic changes. Our study testified that the ginsenosides content

was dependent on main roots and lateral roots energy metabolism, whereas independent of leaves and

petiole photosynthesis during ginsenosides accumulation. When tow species were compared, the

results indicated that high rates of C assimilation to C accumulation are closely associated with

ginsenosides accumulation in P. ginseng main roots and P. quinquefolius lateral roots, respectively.

Taken together, our results suggest that tissue-specific metabolites profiling dynamically changed in

process of ginsenosides biosynthesis, which may offer a new train of thoughts to the mechanisms of

the ginsenosides biosynthesis at the metabolite level.

57

Hierarchical identification of bioactive components in a medicinal herb by preparative high-

performance liquid chromatography and selective knock-out strategy

Cheng Qu, Lin-Yan Wang, Hang Lin, Er-Xin Shang, Jin-Ao Duan

ABSTRACT

A novel and generally applicable approach was established to hierarchically identify the bioactive

components of a medicinal herb by preparative high-performance liquid chromatography (prep-HPLC)

and a selective knock-out strategy. In this study, the targeted components of an herbal medicine were

separated and knocked out using prep-HPLC. Subsequently, the contributions of the different target

components to the overall effect of the medicinal herb were comparatively evaluated and differentiated

by a heat map and a 3D score plot. This approach was successfully applied to investigate the bioactive

constituents of safflower. The contributions of 11 components to the overall effect of safflower were as

follows: anhydrosafflor yellow B (10) > 6-hydroxykaempferol 3,6-di-O-β-d-glucoside (8) >

hydroxysafflor yellow A (3) > kaempferol 3-O-β-rutinoside (11) > 6-hydroxykaempferol 3-O-β-

rutinoside (9) > 6-hydroxykaempferol 3,6-di-O-β-d-glucoside-7-O-β-d-glucuronide (4) > 6-

hydroxyapigenin 6-O-β-d-glucoside-7-O-β-d-glucuronide (6) > cytidine (1) > 6-hydroxykaempferol 3-

O-β-rutinoside-6-O-β-d-glucoside (7) > 6-hydroxykaempferol 3,6,7-tri-O-β-d-glucoside (5) >

adenosine (2). These results demonstrate that quinochalcone C-glycosides (3 and 10) and some

flavonoid glycosides containing C7-OH (such as 8, 9 and 11) made a greater contribution to the overall

effect of safflower than the other components that were knocked out. The results provided an

important reference for improving quality control and further development of safflower products. And

this approach should also be useful for investigating the bioactive constituents of other medicinal

herbs.

Volume 136, March 2017

Metabolomics: A potential way to know the role of vitamin D on multiple sclerosis

Diego Luque-Córdoba, María D. Luque de Castro

ABSTRACT

The literature about the influence of vitamin D on multiple sclerosis (MS) is very controversial,

possibly as a result of the way through which the research on the subject has been conducted. The

studies developed so far have been focused exclusively on gene expression: the effect of a given

vitamin D metabolite on target receptors. The influence of the vitamin D status (either natural or after

supplementation) on MS has been studied by measurement of the 25 monohydroxylated metabolite

(also known as circulating form), despite the 1,25 dihydroxylated metabolite is considered the active

form. In the light of the multiple metabolic pathways in which both forms of vitamin D (D2 and D3)

are involved, monitoring of the metabolites is crucial to know the activity of the target enzymes as a

function of both the state of the MS patient and the clinical treatment applied. The study of

metabolomics aspects is here proposed to clarify the present controversy. In ―omics‖ terms, our

proposal is to take profit from up-stream information—thus is, from metabolomics to genomics—with

a potential subsequent step to systems biology, if required.

58

Impact of space environment on stability of medicines: Challenges and prospects

Priti Mehta, Dhara Bhayani

ABSTRACT

To upkeep health of astronauts in a unique, isolated, and extreme environment of space is the primary

goal for a successful space mission, hence, safe and efficacious medications are essential for the

wellness of astronauts. Space medication has been challenged with problems related to efficacy. Along

with altered physiology, one of the possible reasons could be instability of space medications in the

presence of harsh spaceflight environmental conditions. Altered physical and chemical stability can

result in reduced potency which can result in reduced efficacy. Right now, medicines from the

International Space Station are replaced before their expiration. But, for longer duration missions to

Mars or any other asteroid, there will not be any chance of replacement of medicines. Hence, it is

desired that medicines maintain the shelf-life throughout the space mission. Stability of medicines used

for short term or long term space missions cannot be judged by drug stability guidelines based on

terrestrial environmental factors. Unique environmental conditions related to spaceflight include

microgravity, excessive vibration, hard vacuum, humidity variation, temperature differences and

excessive radiation, which may cause instability of medicines. This write-up provides a review of the

problem and countermeasure approaches for pharmaceuticals exposed to the space environment. The

first part of the article discusses thought processes behind outlining of International Conference on

Harmonization drug stability guidelines, Q1A (R2) and Q1B, and its acceptance limits for accelerated

stability study. The second part of the article describes the difference in the radiation environment of

deep space compared to radiation environment inside the space shuttle based on penetration power of

different types of radiation. In the third part of the article, various promising approaches are listed

which can be used for assurance of space medicine stability. One of the approaches is the use of

ground-based space simulation analogues and statistical treatment to data to calculate failure rate of

drugs and probabilistic risk assessment. Another approach is to innovate storage and packaging

technology using radiation hardens polymer or using cryogenic temperatures.

Evaluation of size-exclusion chromatography for the analysis of phosphorothioate

oligonucleotides

Atsuko Shimoyama, Aki Fujisaka, Satoshi Obika

ABSTRACT

We evaluated size exclusion chromatography (SEC) for the detection of high-order structure of

phosphorothioate oligonucleotides (PS-oligo). Because of strong interaction between PS-oligo and

column packing material, peaks were broader and elution time was longer than those of the

corresponding natural DNA oligonucleotides. However, single- and double-stranded structures of PS-

oligo were clearly separated and discriminated, while single-stranded with high-order structures such

as G-quadruplex and hairpin structure were not distinguished from each other. select article Molecular

insight into atypical instability behavior of fixed-dose combination containing amlodipine besylate and

losartan potassium.

59

Molecular insight into atypical instability behavior of fixed-dose combination containing

amlodipine besylate and losartan potassium

Tarun Handa, Shalu Jhajra, Shweta Bhagat, P.V. Bharatam, Saranjit Singh

ABSTRACT

Combination therapy with the use of fixed-dose combinations (FDCs) is evincing increasing interest of

prescribers, manufacturers and even regulators, evidently due to the primary benefit of improved

patient compliance. However, owing to potential of drug-drug interaction, FDCs require closer

scrutiny with respect to their physical and chemical stability. Accordingly, the purpose of the present

study was to explore stability behavior of a popular antihypertensive combination of amlodipine

besylate (AML) and losartan potassium (LST). Physical mixtures of the two drugs and multiple

marketed formulations were stored under accelerated conditions of temperature and humidity (40

°C/75% RH) in a stability chamber and samples were withdrawn after 1 and 3 months. The physical

changes were observed visibly, while chemical changes were monitored by HPLC employing a

method that could separate the two drugs and all other components present. The combination revealed

strong physical instability and also chemical degradation of AML in the presence of LST.

Interestingly, three isomeric interaction products of AML were formed in the combination, which

otherwise were reported in the literature to be generated on exposure of AML free base above its

melting point. The same unusual products were even formed when multiple marketed FDCs were

stored under accelerated conditions outside their storage packs. However, these were absent when

AML alone was stored in the same studied conditions. Therefore, reasons for physical and chemical

incompatibility and the mechanism of degradation of AML in the presence of LST were duly explored

at the molecular level. The outcomes of the study are expected to help in development of stable FDCs

of the two drugs.

Qualification of HSQC methods for quantitative composition of heparin and low molecular

weight heparins

Lucio Mauri, Giovanni Boccardi, Giangiacomo Torri, Michael Karfunkle, Marco Guerrini

ABSTRACT

An NMR HSQC method has recently been proposed for the quantitative determination of the mono-

and disaccharide subunits of heparin and low molecular weight heparins (LMWH). The focus of the

current study was the validation of this procedure to make the 2D-NMR method suitable for

pharmaceutical quality control applications. Pre-validation work investigated the effects of several

experimental parameters to assess robustness and to optimize critical factors. Important experimental

parameters were pulse sequence selection, equilibration interval between pulse trains and temperature.

These observations were needed so that the NMR method was sufficiently understood to enable

continuous improvement. A standard validation study on heparin then examined linearity,

repeatability, intermediate precision and limits of detection and quantitation; selected validation

parameters were also determined for LMWH.

60

Selecting optimal columns for clarithromycin impurity analysis according to the quantitative

relationship of hydrophobic subtraction model

Xia Zhang, Changqin Hu

ABSTRACT

Hydrophobic subtraction model (HSM) is widely applied to select columns of equivalent or different

selectivity compared with a reference column, but its application in identifying optimal columns for

specific separations of real samples is rare. In this work, a column selection method was proposed by

firstly directly correlating separation selectivity of different pairs of solutes to column parameters

based on the quantitative relationship of HSM and then selecting the optimal columns according to the

predicted selectivity in consideration of the total separation of all critical pairs of solutes. Three critical

pairs of solutes in clarithromycin impurity analysis were evaluated as examples. Starting with the

analysis of clarithromycin impurities on 15 columns with different selectivities, ten optimal columns

were finally identified for clarithromycin impurity analysis from the HSM column characterization

database containing nearly 600 columns and two of them were validated with satisfactory separations

for all critical peak pairs. The proposed methodology was also compared to the traditional column

selection procedure based on calculations of scalar measures of the Euclidean distance between

chromatographic columns. Results showed that our method provides an effective way to find the

desired columns that may be overlooked by the traditional column selection due to selection of an

inappropriate reference column or overestimation of column similarity, such as Fs introduced in HSM.

Efficacy of metformin in human single hair fibre by ATR-FTIR spectroscopy coupled with

statistical analysis

Kamatchi Sundaramoorthi, Gunasekaran Sethu, Sailatha Ethirajulu, Pavithra Raja Marthandam

ABSTRACT

Diabetes mellitus is chronic metabolic disorder, resulting from insulin deficiency, characterized by

hyperglycemia altered metabolism of carbohydrates, proteins and lipids and an increased risk of

vascular complications. There are different classes of anti-diabetic drugs in allopathic system of

medicine. Metformin (dimethyl biguanide) is a blood glucose lowering agent used in the treatment of

non-insulin dependent diabetes mellitus. Almost in all diseases the blood serves as the primary

metabolic transport system in the body. Its composition is the preferred indicator with respect to the

pathophysiological condition of the patient. Instead of analyzing blood to diagnose diabetes, hair could

be used to detect diabetes using FTIR-ATR technique. The most important components of hair are

fibrous proteins (keratins), melanins, glycogen, and lipids. Hair follicles are located 3–4 mm below the

surface of the skin and are surrounded by rich blood capillary system. In the present study, ten diabetic

subjects were considered to evaluate the efficacy of metformin hydrochloride for the treatment of

diabetes mellitus using FTIR-ATR spectroscopy. The spectra of diabetic hair fibre samples have been

recorded in the mid infrared region of 4000–450 cm−1. The hair samples of the diabetic subjects

before medication were taken as pre-treatment samples. The hair samples of diabetic subjects referred

to medication with metformin for a period of three month were taken as post-treatment sample. Some

remarkable spectral differences were elucidated between pre- and post-treatment hair fibre samples. A

comparative study on the FTIR-ATR hair spectra of patients (pre- and post-treatment) along with the

healthy subjects has been made.

61

Primer design for SNP genotyping based on allele-specific amplification—Application to organ

transplantation pharmacogenomics

Luis A. Tortajada-Genaro, Rosa Puchades, Ángel Maquieira

ABSTRACT

Diagnostic methods based on single nucleotide polymorphism (SNP) biomarkers are essential for the

real adoption of personalized medicine. Allele specific amplification in a homogeneous format or

combined to microarray hybridization are powerful approaches for SNP genotyping. However, primers

must be properly selected to minimize cross-reactivity, dimer formation and nonspecific hybridization.

This study presents a design workflow diagram for the selection of required oligonucleotides for

multiplex assays. Based on thermodynamic restrictions, the oligonucleotide sets are chosen for a

specific amplification of wild- and mutant-type templates. Design constraints include the structural

stability of primer-template duplexes, template-probe duplexes and self-annealing complexes or

hairpins for each targeted gene. The performance of the design algorithm was evaluated for the

simultaneous genotyping of three SNPs related to immunosuppressive drugs administered after solid

organ transplantation. The assayed polymorphisms were rs1045642 (ABCB1 gene), rs1801133

(MTHFR gene) and rs776746 (CYP3A5 gene). Candidates were confirmed by discriminating

homozygote and heterozygote populations using a fluorescence solution method and two colorimetric

microarray methods on polycarbonate chips. The analysis of patient samples provided excellent

genotyping results compared to those obtained by a reference method. The study demonstrates that the

development of the allele-specific methods as pharmacogenetic tools can be simplified.

Development and validation of a liquid chromatography–mass spectrometric assay for

simultaneous determination of tacrolimus and 13-O-desmethyl tacrolimus in rat kidney tissue

Tatian Kirresh, Sony Tuteja, D. Russo, P.D. Brophy, D.J. Murry

ABSTRACT

A sensitive and robust LC–MS/MS method has been developed and validated to determine the

concentrations of tacrolimus and its major metabolite 13-O-desmethyl tacrolimus (13-ODMT) in

kidney tissue from rats who received tacrolimus intra-peritoneally at doses of 0.5 mg/kg and 2 mg/kg.

The samples were prepared by a liquid-liquid extraction procedure using ethyl ether as the extraction

solvent and ascomycin as the internal standard. Chromatographic separation was achieved using

Phenomenex Kinetex column (2.6 μm C18 100 Å, 100 × 2.1 mm, Phenomenex, Torrance CA) and a

gradient mobile phase of water and methanol-acetonitrile (50:50, v/v) both containing 0.1% formic

acid. The limit of quantification was 0.25 ng/ml and the calibration curves covered a concentration

range from 0.25 to 50 ng/ml. Intra-and inter-assay precision and accuracy for both tacrolimus and 13-

ODMT were all within FDA guidelines for bioanalysis. Extraction efficiency for tacrolimus ranged

from 67.00 to 74.90% and from 66.70 to 78.40% for 13-ODMT. Several challenges interfering with

the performance of the method such as phospholipid build-up have also been addressed. Kidney tissue

samples from six rats receiving either 0.5 or 2 mg/kg dose were analyzed and resulted in a median

concentration of 11.54 and 0.72 ng/ml for tacrolimus and 13-ODMT, respectively, for the lower dose

level, and a median concentration of 8.89 ng/ml and 1.50 ng/ml for tacrolimus and 13-ODMT,

respectively, at the higher dose level.

62

Towards interference free HPLC-SERS for the trace analysis of drug metabolites in biological

fluids

Waleed A. Hassanain, Emad L. Izake, Arumugam Sivanesan, Godwin A. Ayoko

ABSTRACT

Sofosbuvir metabolite, 2′-deoxy-2′-fluoro-2′-C-methyluridine (PSI-6206) was studied for the first time

by surface enhanced Raman spectroscopy (SERS) using the paper-based SERS substrate. The

quantification limit of PSI-6206 by SERS was found to be 13 ng L−1 (R2 value = 0.959, RSD =

5.23%). For the structural and quantitative analysis of PSI-6206 in blood plasma, an interference-free

HPLC-SERS method was developed and compared to HPLC-DAD and HPLC–MS methods. The

SERS quantification of the drug by the paper substrate was 4 orders of magnitude more sensitive than

that by the diode array detector. In addition, the SERS detection provided unique structural

identification of the drug in blood plasma, similar to Mass spectroscopy detector. Due to the

disposable nature of the SERS substrate, the new method does not suffer from the known ―memory

effect‖ which is known to lead to false positive identification in traditional HPLC-SERS methods.

Therefore, the presented HPLC-paper SERS platform holds great potential for the sensitive and cost

effective determination of drugs and their metabolites in biological fluids.

1H NMR-based metabolomics study of liver damage induced by ginkgolic acid (15:1) in mice

Lei Jiang, Zhi-Hong Si, Ming-Hui Li, He Zhao, Jun-Song Wang

ABSTRACT

Ginkgolic acid (15:1) is a major toxic component in extracts obtained from Ginkgo biloba (EGb) that

has allergic and genotoxic effects. This study is the first to explore the hepatotoxicity of ginkgolic acid

(15:1) using a NMR (nuclear magnetic resonance)-based metabolomics approach in combination with

biochemistry assays. Mice were orally administered two doses of ginkgolic acid (15:1), and mouse

livers and serum were then collected for NMR recordings and biochemical assays. The levels of

activity of alanine aminotransferase (ALT) and glutamic aspartate transaminase (AST) observed in the

ginkgolic acid (15:1)-treated mice suggested that it had induced severe liver damage. An orthogonal

signal correction partial least-squares discriminant analysis (OSC-PLSDA) performed to determine the

metabolomic profile of mouse liver tissues indicated that many metabolic disturbances, especially

oxidative stress and purine metabolism, were induced by ginkgolic acid (15:1). A correlation network

analysis combined with information related to structural similarities further confirmed that purine

metabolism was disturbed by ginkgolic acid (15:1). This mechanism might represent the link between

the antitumour activity and the liver injury-inducing effect of ginkgolic acid (15:1). A SUS (Shared

and Unique Structure) plot suggested that a two-dose treatment of ginkgolic acid (15:1) had generally

the same effect on metabolic variations but that its effects were dose-dependent, revealing some of the

common features of ginkgolic acid (15:1) dosing. This integrated metabolomics approach helped us to

characterise ginkgolic acid (15:1)-induced liver damage in mice.

63

Dose-response characteristics of Clematis triterpenoid saponins and clematichinenoside AR in

rheumatoid arthritis rats by liquid chromatography/mass spectrometry-based serum and urine

metabolomics

Rui Li, Lin-Xiu Guo, Yi Li, Wen-Qi Chang, Gui-Zhong Xin

ABSTRACT

Clematidis Radix et Rhizoma is a traditional Chinese medicine widely used for treating arthritic

disease. Clematis triterpenoid saponins (TS) and clematichinenoside AR (C-AR) have been considered

to be responsible for its antiarthritic effects. However, the underling mechanism is still unclear because

of their low bioavailability. To address of this issue, metabolomics tools were performed to determine

metabolic variations associated with rheumatoid arthritis (RA) and responses to Clematis TS, C-AR

and positive drug (Triptolide, TP) treatments. This metabolomics investigation of RA was conducted

in collagen-induced arthritis (CIA) rats. Liquid chromatography/mass spectrometry and multivariate

statistical tools were used to identify the alteration of serum and urine metabolites associated with RA

and responses to drug treatment. As a result, 45 potential metabolites associated with RA were

identified. After treatment, a total of 24 biomarkers were regulated to normal like levels. Among these,

PC(18:0/20:4), 9,11-octadecadienoic acid, arachidonic acid, 1-methyladenosine, valine, hippuric acid

and pantothenic acid etc, were reversed in Clematis TS and C-AR groups. Tetrahydrocortisol was

regulated to normal levels in Clematis TS and TP groups, while 3,7,12-trihydroxycholan-24-oic acid

was regulated in C-AR and TP groups. Biomarkers like citric acid, p-cresol glucuronide, creatinine,

cortolone were reversed in TP group.

Enhancing analysis throughput, sensitivity and specificity in LC/ESI–MS/MS assay of plasma

25-hydroxyvitamin D3 by derivatization with triplex 4-(4-dimethylaminophenyl)-1,2,4-triazoline-

3,5-dione (DAPTAD) isotopologues

Shoujiro Ogawa, Hiroki Kittaka, Akiho Nakata, Kenji Komatsu, Tatsuya Higashi

ABSTRACT

The plasma/serum concentration of 25-hydroxyvitamin D3 [25(OH)D3] is a diagnostic index for

vitamin D deficiency/insufficiency, which is associated with a wide range of diseases, such as rickets,

cancer and diabetes. We have reported that the derivatization with 4-(4-dimethylaminophenyl)-1,2,4-

triazoline-3,5-dione (DAPTAD) works well in the liquid chromatography/electrospray ionization-

tandem mass spectrometry (LC/ESI–MS/MS) assay of the serum/plasma 25(OH)D3 for enhancing the

sensitivity and the separation from a potent interfering metabolite, 3-epi-25-hydroxyvitamin D3 [3-epi-

25(OH)D3]. However, enhancing the analysis throughput remains an issue in the LC/ESI–MS/MS

assay of 25(OH)D3. The most obvious restriction of the LC/MS/MS throughput is the

chromatographic run time. In this study, we developed an enhanced throughput method for the

determination of the plasma 25(OH)D3 by LC/ESI–MS/MS combined with the derivatization using the

triplex (2H0-, 2H3- and 2H6-) DAPTAD isotopologues. After separate derivatization with 1 of 3

different isotopologues, the 3 samples were combined and injected together into LC/ESI–MS/MS.

Based on the mass differences between the isotopologues, the derivatized 25(OH)D3 in the 3 different

samples were quantified within a single run. The developed method tripled the hourly analysis

throughput without sacrificing assay performance, i.e., ease of pretreatment of plasma sample (only

deproteinization), limit of quantification (1.0 ng/mL when a 5 μL-plasma was used), precision (intra-

assay RSD ≤ 5.9% and inter-assay RSD ≤ 5.5%), accuracy (98.7–102.2%), matrix effects, and

capability of separating from an interfering metabolite, 3-epi-25(OH)D3.

64

A highly sensitive quantum dots-DNA nanobiosensor based on fluorescence resonance energy

transfer for rapid detection of nanomolar amounts of human papillomavirus 18

Mojtaba Shamsipur, Vahid Nasirian, Kamran Mansouri, Ali Barati, Soheila Kashanian

ABSTRACT

A very sensitive and convenient nanobiosensor based on fluorescence resonance energy transfer

(FRET) was developed for the detection of a 22-mer oligonucleotides sequence in Human

Papillomavirus 18 virus (HPV18) gene. For this purpose, water-soluble CdTe quantum dots (QDs)

were synthesized and, subsequently, amino-modified 11-mer oligonucleotide as one of the two

necessary probes was attached to QDs surface to form functional QDs-DNA conjugates. Right after

addition of the QDs-DNA and a second Cyanine5 (Cy5)-labeled 11-mer oligonucleotide probe to the

DNA target solution, the sandwiched hybrids were formed. The resulting hybridization brings the Cy5

fluorophore as the acceptor to close proximity of the QDs as donor, so that an effective transfer of

energy from the excited QDs to the Cy5 probe would occur via FRET processing. The fluorescence

intensity of Cy5 found to linearly enhance by increasing the DNA target concentration from 1.0 to 50.0

nM, with a detection limit of 0.2 nM. This homogeneous DNA detection method does not require

excessive washing and separation steps of un-hybridized DNA, due to the fact that no FRET can be

observed when the probes are not ligated. Finally, feasibility and selectivity of the proposed one-spot

DNA detection nanobiosensor were investigated by analysis of derived nucleotides from HPV18 and

mismatched sequences.

Development and validation of stability indicating HPLC methods for related substances and

assay analyses of amoxicillin and potassium clavulanate mixtures

Esen Bellur Atici, Yücel Yazar, Çağan Ağtaş, Nurten Ridvanoğlu, Bekir Karlığa

ABSTRACT

Antibacterial combinations consisting of the semisynthetic antibiotic amoxicillin (amox) and the β-

lactamase inhibitor potassium clavulanate (clav) are commonly used and several chromatographic

methods were reported for their quantification in mixtures. In the present work, single HPLC method

for related substances analyses of amoxicillin and potassium clavulanate mixtures was developed and

validated according to international conference on harmonization (ICH) guidelines. Eighteen

amoxicillin and six potassium clavulanate impurities were successfully separated from each other by

using triple gradient elution using a Thermo Hypersil Zorbax BDS C18 (250 mm × 4.6 mm, 3 μm)

column with 50 μL injection volumes at a wavelength of 215 nm. Commercially unavailable impurities

were formed by degradation of amoxicillin and potassium clavulanate, identified by LC–MS studies

and used during analytical method development and validation studies. Also, process related

amoxicillin impurity-P was synthesized and characterized by using nuclear magnetic resonance

(NMR) and mass spectroscopy (MS) for the first time. As complementary of this work, an assay

method for amoxicillin and potassium clavulanate mixtures was developed and validated; stress-testing

and stability studies of amox/clav mixtures was carried out under specified conditions according to

ICH and analyzed by using validated stability-indicating assay and related substances methods.

65

Rapid analysis of drug dissolution by paper spray ionization mass spectrometry

Yang Liu, Ning Liu, Ya-nan Zhou, Lan Lin, Lan He

ABSTRACT

With a great quantity of solid dosage tested by dissolution technology, developing a rapid and sensitive

method to access the content of drug within dissolution media is highly desired by analysts and

scientists. Traditionally, dissolution media is not compatible with mass spectrometry since the

inorganic salts in the media might damage the mass spectrometer. Here, paper spray ionization mass

spectrometry (PSI-MS), one of the ambient mass spectrometry technologies, is developed to

characterize the content of drugs in dissolution media. The porous structure of paper can effectively

retain salts from entering mass spectrometer. This makes the measurement of drug content within

dissolution media by mass spectrometer possible. After the experimental parameters were optimized,

calibration curves of model drugs − enalapril, quinapril and benazepril were established by using

corresponding deuterated internal standards. PSI-MS was then deployed to characterize the content of

enalapril from the dissolution testing of enalapril tablets. The results from PSI-MS are comparable to

those from HPLC characterization. More importantly, the analysis time of 6 samples is shortened from

90 min to 6 min. Detection limit of enalapril maleate tablets by PSI-MS is 1/300 of LC. PSI-MS is

rapid, sensitive and accurate in analyzing drug content from dissolution tests.

Development and validation of a liquid chromatography-MS/MS method for simultaneous

quantification of tenofovir and efavirenz in biological tissues and fluids

Luisa Barreiros, Cassilda Cunha-Reis, Eduarda M.P. Silva, Joana R.B. Carvalho, Marcela A.

Segundo

ABSTRACT

Millions of people worldwide live with human immunodeficiency virus (HIV) infection thus justifying

the continuous search for new prevention and treatment strategies, including topical microbicide

products combining antiretroviral drugs (ARVs) such as tenofovir (TFV) and efavirenz (EFV).

Therefore, the aim of this work was to develop and validate a high performance liquid chromatography

method coupled to triple quadrupole-tandem mass spectrometry (HPLC–MS/MS) for the

quantification of TFV and EFV in biological matrices (mouse vaginal tissue, vaginal lavage and blood

plasma). Chromatographic separation was achieved using a reversed phase C18 column (3 μm, 100 ×

2.1 mm) at 45 °C and elution in gradient mode using a combination of 0.1% (v/v) formic acid in water

and 0.1% (v/v) formic acid in acetonitrile at 0.35 mL min−1. Total run time was 9 min, with retention

time of 2.8 and 4.1 min for TFV and EFV, respectively. The MS was operated in positive ionization

mode (ESI+) for TFV and in negative ionization mode (ESI-) for EFV detection. Data were acquired

in selected reaction monitoring (SRM) mode and deuterated ARVs were employed as internal

standards. Calibration curves were linear for ARV concentrations ranging from 4 to 500 ng mL−1 with

LOD and LOQ for both analytes ≤0.4 and ≤0.7 ng mL−1 in sample extracts, respectively. The method

was found to be specific, accurate (96.0–106.0% of nominal values) and precise (RSD < 2.4%) in all

matrices. Both TFV and EFV were found to be stable in all matrices after standing 24 h at room

temperature (20 °C) or in the autosampler, and after three freeze-thawing cycles. Mean recovery values

of ARVs spiked in mice tissues or fluids were ≥88.4%. Matrix effects were observed for EFV

determination in tissue and plasma extracts but compensated by the use of deuterated internal

standards. The proposed methodology was successfully applied to a pharmacokinetic study following

intravaginal administration of both ARVs.

66

Isolation, characterization using LC-ESI-QTOF, NMR and in vitro cytotoxicity assay of

niclosamide forced degradation products

Johnsirani P., Vishnuvardhan Ch., Lingesh A., Naidu V.G.M., Satheeshkumar N.

ABSTRACT

The present study describes the isolation, characterization and in vitro cytotoxic effect of all forced

degradation products of niclosamide (NCM) an anthelmintic class of drug used specifically to treat

tapeworms. NCM was subjected to forced degradation involving hydrolysis (acidic, alkaline and

neutral), oxidative, photolysis and thermal stress, as per ICH (Q1A (R2)) suggested conditions. The

drug under hydrolytic (acidic and basic) conditions showed extensive degradation, while it was stable

under neutral hydrolytic, oxidative, photolytic and thermal stress conditions. A total of four

degradation products (DPs) were observed and chromatographic separation of drug and its degradation

products were achieved on a reverse phase Fortis diphenyl column (150 × 4.6 mm, 5 μm) using 0.1%

formic acid and acetonitrile as mobile phase in gradient mode. All the four degradation products were

isolated by semi preparative LC and its structures were characterized and confirmed by high resolution

MS and 1H NMR spectroscopic techniques. In view of safety aspects, cytotoxicity assay were carried

out for NCM and its four degradation products on human mononuclear cells and cell lines depicting

the major organelle: neuronal (Neuro 2a), hepatic (HepG 2) and alveolar (A549). NCM was found to

be non toxic on human mononuclear cells and cell lines at tested concentrations. However DP-1, DP-2,

DP-3 and DP-4 showed significant increase in LDH release as compared to control at a concentration

of 100 μM. DP-1 and DP-3 exhibited toxicity on A549 cells with an IC50 of 92.18 ± 4.93 μM and

65.42 ± 6.29 μM respectively. DP-2, DP-3 and DP-4 were cytotoxic to Neuro 2a cells with an IC50 of

63.62 ± 3.85 μM, 86.09 ± 6.19 μM and 42.81 ± 8.10 μM respectively. The degradation products were

found to be nontoxic on HepG 2 cells.

Quantitative determinations using portable Raman spectroscopy

Chelliah V. Navin, Chaitanya Tondepu, Roxana Toth, Latevi S. Lawson, Jason D. Rodriguez

ABSTRACT

A portable Raman spectrometer was used to develop chemometric models to determine percent (%)

drug release and potency for 500 mg ciprofloxacin HCl tablets. Parallel dissolution and

chromatographic experiments were conducted alongside Raman experiments to assess and compare

the performance and capabilities of portable Raman instruments in determining critical drug attributes.

All batches tested passed the 30 min dissolution specification and the Raman model for drug release

was able to essentially reproduce the dissolution profiles obtained by ultraviolet spectroscopy at 276

nm for all five batches of the 500 mg ciprofloxacin tablets. The five batches of 500 mg ciprofloxacin

tablets also passed the potency (assay) specification and the % label claim for the entire set of tablets

run were nearly identical, 99.4 ± 5.1 for the portable Raman method and 99.2 ± 1.2 for the

chromatographic method. The results indicate that portable Raman spectrometers can be used to

perform quantitative analysis of critical product attributes of finished drug products. The findings of

this study indicate that portable Raman may have applications in the areas of process analytical

technology and rapid pharmaceutical surveillance.

67

Screening active compounds from Corydalis yanhusuo by combining high expression VEGF

receptor HEK293 cell membrane chromatography with HPLC - ESI - IT - TOF - MSn method

Fen Wei, Qi Hu, Jing Huang, Shengli Han, Sicen Wang

ABSTRACT

Corydalis Thizoma,or Yuanhu in China, is a common herbal drug used for thousands of years as

analgesic in Chinese medicine that has been reported to have potential anti-angiogenic effects. In this

study, a VEGFR/cell membrane chromatography (VEGFR/CMC) coupled with HPLC- ESI-IT-TOF-

MSn system was developed and successfully applied for identifying active components from YuanHu

extract acting on VEGFR. We identified tetrahydropalmatine and corydaline as bioactive components

with VEGFR activity, thus confirming their inhibitory activity on VEGFR engineered HEK293 cell

growth by MTT assay. The activity of tetrahydropalmatine and corydaline was compared with the

positive control sorafenib in a range of concentration from 6.25 to 50.0 μM, showing a dose-dependent

inhibitory trend. These results indicate that the VEGFR/CMC coupled with HPLC- ESI-IT-TOF-MSn

system can purify and identify specific bioactive components from complex systems, thus representing

a promising tool for screening molecules active towards VEGFR from natural herbs.

Volume 137, April 2017

Powerful combination of analytical and chemometric methods for the photodegradation of 5-

Fluorouracil

Cristian Gómez-Canela, Gabino Bolivar-Subirats, Romà Tauler, Silvia Lacorte

ABSTRACT

The photodegradation of the antineoplastic drug 5-fluorouracil (5-FU) under the action of UV light

was studied using UV–vis spectroscopy and High Performance Liquid Chromatography coupled to

Diode Array Detector and High Resolution Mass Spectrometry (HPLC-DAD-HRMS). To analyze,

integrate and interpret the degradation kinetics, the Multivariate Curve Resolution-Alternating Least

Squares (MCR-ALS) chemometric method has been applied. The high complexity of the

photodegradation process of this drug involving up to seven different photoproducts showed the

advantages of the proposed approach which provides simultaneously mechanistic and structural

information explaining the 5-FU photodegradability. Moreover, this study provides a new tool to be

used for elucidating the photodegradation kinetics at real time.

68

Separation of antibody drug conjugate species by RPLC: A generic method development

approach

Szabolcs Fekete, Imre Molnár, Davy Guillarme

ABSTRACT

This study reports the use of modelling software for the successful method development of IgG1

cysteine conjugated antibody drug conjugate (ADC) in RPLC. The goal of such a method is to be able

to calculate the average drug to antibody ratio (DAR) of and ADC product. A generic method

development strategy was proposed including the optimization of mobile phase temperature, gradient

profile and mobile phase ternary composition. For the first time, a 3D retention modelling was

presented for large therapeutic protein. Based on a limited number of preliminary experiments, a fast

and efficient separation of the DAR species of a commercial ADC sample, namely brentuximab

vedotin, was achieved. The prediction offered by the retention model was found to be highly reliable,

with an average error of retention time prediction always lower than 0.5% using a 2D or 3D retention

models. For routine purpose, four to six initial experiments were required to build the 2D retention

models, while 12 experiments were recommended to create the 3D model. At the end, RPLC can

therefore be considered as a good method for estimating the average DAR of an ADC, based on the

observed peak area ratios of RPLC chromatogram of the reduced ADC sample.

Time domain NMR as a new process monitoring method for characterization of pharmaceutical

hydrates

Stefanie Ulrike Schumacher, Benno Rothenhäusler, Alf Willmann, Jürgen Thun, Martin Kuentz

ABSTRACT

Hydrates are of great pharmaceutical relevance and even though they have been characterized

thoroughly by various analytical techniques, there is barely literature available on molecular mobility

of the hydrate water studied by NMR relaxation in the time domain. The aim of this work was to

examine the possibility of differentiating hydration states of drugs by 1H time domain NMR (TD-

NMR) regarding spin-spin and spin-lattice relaxation times (T2 and T1) using benchtop equipment.

Caffeine and theophylline were selected as model compounds and binary mixtures of hydrate to

anhydrate were analyzed for each drug using a spin echo and inversion recovery pulse sequence. It was

possible to extract a signal that was specific for the water in the hydrates so that differentiation from

anhydrous solid forms was enabled. Excellent calibrations were obtained for quantitative analysis of

hydrate/anhydrate mixtures and predicted water contents were in good agreement with water amounts

determined in desiccator sorption experiments. TD-NMR was therefore found to be a suitable new

technique to characterize pharmaceutical hydrates in a non-invasive and hence sample-sparing manner.

Quantification of the hydrate content in pharmaceutical mixtures appears highly attractive for product

development and process monitoring. TD-NMR provides here a valuable and complementary

technique to established process analytics, such as for example Raman spectroscopy.

69

LC-method development for the quantification of neuromedin-like peptides Emphasis on column

choice and mobile phase composition

Yannick Van Wanseele, Johan Viaene, Leslie Van den Borre, Kathleen Dewachter, Ann Van

Eeckhaut

ABSTRACT

In this study, the separation of four neuromedin-like peptides is investigated on four different core-

shell stationary phases. Moreover, the effect of the mobile phase composition, i.e. organic modifier

(acetonitrile and methanol) and additive (trifluoroacetic acid, formic acid, acetic acid, ammonium

formate and ammonium acetate) on the chromatographic performance is studied. An improvement in

chromatographic performance is observed when using the ammonium salt instead of its corresponding

acid as additive, except for the column containing a positively charged surface (C18+). In general, the

RP-Amide column provided the highest separation power with different mobile phases. However, for

the neuromedin-like peptides of interest, the C18+ column in combination with a mobile phase

containing methanol as organic modifier and acetic acid as additive provided narrower and higher

peaks. A three-factor, three-level design is applied to further optimize the method in terms of increased

peak height and reduced solvent consumption, without loss in resolution. The optimized method was

subsequently used to assess the in vitro microdialysis recovery of the peptides of interest. Recovery

values between 4 and 8% were obtained using a perfusion flow rate of 2 μL/min.

2D-LC as an on-line desalting tool allowing peptide identification directly from MS unfriendly

HPLC methods

Hao Luo, Wendy Zhong, Jiong Yang, Ping Zhuang, Christopher J. Welch

ABSTRACT

The increasing interest in peptides and proteins in pharmaceutical research and development has led to

many challenges for the researchers tasked with characterizing and analyzing these larger molecules.

Due to the more complicated impurity profile of peptides and proteins, multiple liquid chromatography

techniques are often needed to achieve comprehensive analysis. However, many of these separation

conditions require buffers, salts or additives that render them incompatible with mass spectrometry

(MS) detection. Previous researchers have demonstrated proof of concept for the use of two

dimensional liquid chromatography (2D-LC) to provide convenient second dimension online desalting

of components purified in the first chromatographic dimension. In this paper, we evaluated the Agilent

heart-cutting 2D-LC system connected with an Agilent Q-TOF mass spectrometer to address this

frequently encountered analytical challenge. On this 2D-LC/MS system, fractions containing the

compounds of interest are separated by the first dimension using an MS incompatible mobile phase,

then sent to a second dimension HPLC method where fast desalting using an MS compatible mobile

phase is performed prior to MS analysis. The system allows for fast and direct collection of MS

information for chromatographic peaks eluted in MS incompatible mobile phases, without requiring

difficult, time consuming and error-prone translation of chromatographic methods from MS

incompatible to MS compatible eluents, or off-line fraction collection and preparation. Several

examples showing the application of the approach to complex mixtures containing peptides with

impurities and positional isomers are presented.

70

Quantification of EC-18, a synthetic monoacetyldiglyceride (1-palmitoyl-2-linoleoyl-3-acetyl-rac-

glycerol), in rat and mouse plasma by liquid-chromatography/tandem mass spectrometry

Heon-Min Ryu, Yoo-Seong Jeong, Chang-Soon Yim, Jong-Hwa Lee, Suk-Jae Chung

ABSTRACT

EC-18 (i.e., 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol), an active ingredient in Rockpid®, has been

reported to be useful in controlling various types of inflammations, particularly those caused by

neutropenia. Although this product was originally approved as a functional food in Korea, it is

currently in phase II clinical trials for use in managing the severe chemotherapy-induced neutropenia

in patients with advanced breast cancer who are receiving intermediate febrile neutropenia risk

chemotherapy. The objective of this study was to develop a rapid, sensitive method for the

determination of EC-18 in rat and mouse plasma and to evaluate the applicability of the assay in

pharmacokinetic studies. EC-18 was extracted with MeOH from rat and mouse plasma samples, and

the extract directly introduced onto an LC–MS/MS system. The analyte and EC-18-d3, an internal

standard, were analyzed by multiple reaction monitoring (MRM) at m/z transitions of 635.4 → 355.4

for EC-18 and 638.4 → 338.4 for the internal standard, respectively. The lower limit of quantification

(LLOQ) was determined at 50 ng/mL, with an acceptable linearity in the range from 50 to 10,000

ng/mL (r > 0.999) for both matrices. Validation parameters such as accuracy, precision, dilution,

recovery, matrix effects and stability were found to be within the acceptance criteria of the assay

validation guidelines, indicating that the assay is applicable for estimating EC-18 in concentrations in

the range examined. EC-18 was readily determined in plasma samples for periods of up to 8 h

following an intravenous bolus injection of 1 mg/kg in rats and at 5 mg/kg in mice, respectively, and

up to 24 h following the oral administration of 2000 mg/kg in mice. The findings indicate that the

current analytical method is applicable for pharmacokinetic studies of EC-18 in small animals.

Compatibility study of a parenteral microdose polyethylene glycol formulation in medical

devices and identification of degradation impurity by 2D-LC/MS

Lulu Dai, Geoffrey K. Yeh, Yingqing Ran, Peter Yehl, Kelly Zhang

ABSTRACT

Polyethylene glycol (PEG) based formulation and polyvinylchloride (PVC) tubing are frequently used

for drug delivery and administration. The compatibility of a parenteral drug microdose formulation in

intravenous infusion (IV) devices was studied to support the clinical determination of absolute

bioavailability by the microdosing method. The investigational microdose formulation containing PEG

was found prone to significant loss of potency within hours of storage in the PVC IV tubing due to

degradation. Degradation occurred only when both PEG and PVC tubing were present. The

degradation product could not be detected by LC/MS due to the significant interference from the high

concentration of PEG (4%) matrix and the extremely low level of drug (0.6 ppm). To obtain structural

information of the degradation impurity and understand the cause of the degradation, a simple heart-

cutting 2D-LC/MS approach was utilized to effectively separate the impurity from the complex PEG

oligomers and overcome the matrix interference, enabling mass spectrometric analysis of the impurity.

An oxidation- dominated mechanism was proposed in which the combination of PEG auto-oxidation

and dehydrochlorination of the PVC tubing yielded an oxidative environment that enhanced radical

propagation and accelerated degradation of the investigational parent drug.

71

Use of mixture design in drug-excipient compatibility determinations: Thymol nanoparticles

case study

Felipe Q. Pires, Tamara Angelo, Joyce K.R. Silva, Lívia C.L. Sá-Barreto, Marcílio S.S. Cunha-Filho

ABSTRACT

The objective of this work was to access thymol-excipient compatibility using an alternative protocol

of mixture design subsidizing the development of nanostructures lipid carriers containing this drug.

Simultaneous DTA-TG analyses associated with infrared spectroscopy were performed according to

simplex centroid mixture designs with three components. Two designs were used: the design A

containing stearic acid (SA), soybean lecithin (LC), and sodium taurodeoxycholate (TAU) and the

design B, where TAU was replaced by polysorbate 80 (P80). Assays allowed for a quantitative

evaluation of thermal events involved with thymol (TML) – melting and evaporation –, as well as

events related to excipients decomposition and overall system stability. Although the anionic

surfactant TAU did not interact with TML in solid state, chemical and physical stability were

compromised after drug melting. Alternatively, nonionic surfactant P80 could be a good excipient

option, as TML formulation stability was not influenced by it. Fatty acid SA did not compromise TML

stability alone, but, when in combination with other formulation components, negative interaction

leading to a possible decomposition of the system was observed. Finally, phospholipid LC solubilizes

TML extending its evaporation to higher temperatures; hence, drug stability may be increased. In

conclusion, the use of mixture design in the evaluation of multicomponent systems is a valuable tool

for identification of synergistic effects of excipients, providing more complete information on

formulation development. In addition, the association of techniques employed allowed inferring with

certainty if thermal interactions could compromise formulation stability.

Rapid analysis of Aurantii Fructus Immaturus (Zhishi) using paper spray ionization mass

spectrometry

Xuemei Liu, Zhixin Gu, Yuan Guo, Jingjing Liu, Liping Wang

ABSTRACT

Paper spray-mass spectrometry (PS-MS) is a rapid, solvent-efficient, and high-throughput analytical

method for analyzing complex samples. In this study, a PS-MS method was developed to obtain MS

profiles of Aurantii Fructus Immaturus (aka Zhishi in Chinese) in positive and negative ion modes. In

combination with multivariate analyses, including principal component analysis and cluster analysis,

the PS-MS profiles of 25 batches of Zhishi were discriminated in 25 batches of Citri Reticulatae

Pericarpium Viride (aka Qingpi in Chinese; an adulterant of Zhishi). Moreover, a rapid quantitative

analysis of synephrine, a prescriptive quality control component of Zhishi listed in the Chinese

Pharmacopoeia, was conducted with PS-MS using synephrine-d2 as an internal standard (IS). The

linearity range was 1.68–16.8 μg/mL (R2 = 0.9985), the limit of quantitation was 0.5 μg/mL. Relative

standard deviations in the intra- and inter-day precision of the MS were 4.87 and 4.90%, respectively.

Compared with HPLC results, there was no significant difference in the quantitation of synephrine.

This study demonstrated that the PS-MS method is useful for the rapid discrimination and quality

control of Zhishi samples.

72

Rapid determination of 30 bioactive constituents in XueBiJing injection using ultra high

performance liquid chromatography-high resolution hybrid quadrupole-orbitrap mass

spectrometry coupled with principal component analysis

Lihua Zuo, Zhi Sun, Yurong Hu, Ya Sun, Xiaojian Zhang

ABSTRACT

Xuebijing injection (XBJ) is a traditional Chinese herbal prescription widely used in the treatment of

sepsis. Extensive chemical studies revealed that XBJ injection contains amino acids, phenolic acids,

flavonoid glycosides, terpeneglycosides and phthalides. In this study, the applicability of ultra high

performance liquid chromatography coupled with high resolution hybrid quadruple-orbitrap mass

spectrometry (UHPLC-Q-Orbitrap MS) for the simultaneous quantitative analysis of 30 bioactive

constituents in XueBiJing injection (XBJ) was investigated. The mass spectrometer was operated in

full MS scan mode. The use of 70,000 FWHM mass resolution and narrow mass windows (5 ppm)

could effectively improve the selectivity of the method. Separation was achieved on a Waters

ACQUITY UPLC® HSS C18 column (2.1 mm × 100 mm, 1.8 μm) with a gradient mobile phase

consisting of acetonitrile-water (containing 10 mM ammonium acetate) at a flow rate of 0.2 mL/min.

Satisfactory linearity was achieved within wide linear range and all correlation coefficients (r) of

analytes were more than 0.9996. The limits of detection (LODs) were in the range of 0.1180–27.82

ng/mL for different analytes. The relative standard deviations (RSDs) of inter- and intra-day precisions

were less than 3.0% and the recoveries of the assay were in the range of 98.5%–101.5%. The validated

method was successfully applied for simultaneous determination of 30 bioactive compounds in

XueBiJing injection from 10 batches samples by UHPLC-Q-Orbitrap MS within 10 min. Moreover,

the results were evaluated principal component analysis and two compounds might be the most

important chemical markers for chemical quality control of XBJ injection.

Generic DART-MS platform for monitoring the on-demand continuous-flow production of

pharmaceuticals: Advancing the quantitative protocol for caffeates in microfluidic biocatalysis

Yan Xu, Dong-Yang Zhang, Xiang-Yun Meng, Xi Liu, Fu-An Wu

ABSTRACT

Today, continuous processing is regarded as an effective on-demand production technique of

pharmaceuticals. Homemade microreactors packed with immobilized lipase under continuous-flow

conditions were first applied to tailor the production of high-value caffeic acid phenethyl ester (CAPE)

from methyl caffeate (MC) and 2-phenylethanol (PE) in cyclohexane via transesterification; however,

this method is challenging due to the lack of a rapid platform for monitoring caffeates in microfluidic

biocatalysis. The reactants were directly analyzed using Direct Analysis in Real Time Mass

Spectrometry (DART-MS), and the corresponding ionization parameters were investigated. Special

ions produced from MC (parent ion m/z 192.87 and product ion m/z 133.44) and CAPE (parent ion

m/z 282.93 and product ion m/z 178.87) were determined using DART-MS2 in the negative ion mode.

The peak areas of the select reaction monitoring (SRM) signals were calculated to develop the

standard curves for quantitative analyses of the concentration. Reasonable linear regression equations

of MC and CAPE were obtained in the range of 3.125–50.000 mg/L, with linear coefficients (R2) of

0.9515 and 0.9973, limits of detection (LOD) of 0.005 and 0.003 mg/L, limits of quantification (LOQ)

of 0.02 and 0.01 mg/L, and recovery ranges of 92.50–97.11% and 90.11–97.60%, respectively.

73

Potential impurities of anxiolytic drug, clobazam: Identification, synthesis and characterization

using HPLC, LC-ESI/MSn and NMR

Neeraj Kumar, Subba Rao Devineni, Shailendra Kumar Dubey, Pramod Kumar

ABSTRACT

During the optimization of process, eight impurities (CLB Imp-A to CLB Imp-H) were detected in few

of the laboratory batches of clobazam, used as anxiolytic agent, in the range of 0.02–0.12% using

gradient HPLC method with UV detection. On the basis of co-spiking analysis, six impurities (CLB

Imp-A to -F) enumerated by European Pharmacopoeia, however, not reported in the earlier literature,

have been harmonized and found to be two impurities are completely unknown (CLB Imp-G and -H).

These two new impurities structures were presumed based on LC-ESI/MSn study as 8-chloro-1-

methyl-5-phenyl-1,5-dihydro-3H-1,5-benzodiazepine-2,4-dione (CLB Imp-G) and 5-chloro-1-methyl-

3-phenyl-1H-benzo[d]imidazol-2(3H)-one (CLB Imp-H). The presumed impurities structures were

confirmed by their synthesis followed by the complete spectral analysis such as ESI–MS, 1D NMR

(1H, 13C and DEPT), 2D NMR (HSQC, HMBC and COSY) and IR, and chromatographic retention

time profile. Identification, synthesis, structural characterization, prospects to the formation and

controlling of these new impurities were described in detail and reported first in this paper.

Simultaneous quantification of twenty-one ginsenosides and their three aglycones in rat plasma

by a developed UFLC–MS/MS assay: Application to a pharmacokinetic study of red ginseng

Qi-Le Zhou, Di-Na Zhu, Yan-Fang Yang, Wei Xu, Xiu-Wei Yang

ABSTRACT

To track the pharmacokinetic features of red ginseng (RG), a rapid and sensitive ultra fast liquid

chromatographic coupled with electrospray ionization triple quadrupole tandem mass spectrometry

(UFLC–MS/MS) method was developed for simultaneous quantification of twenty-one ginsenosides

and their three aglycones, including 18 prototype compounds (ginsenosides Rb1, Rb2, Rc, Rd, Re,

Rg1, Rg5, Rh4, Rk1, Rk3, 20(S)-Rf, 20(S)-Rg2, 20(R)-Rg2, 20(S)-Rg3, 20(R)-Rg3, 20(S)-Rh1,

20(R)-Rh1, 20(S)-NG-R2), and 6 metabolites (ginsenosides 20(S)-Rh2 and Rh3, 20(S)-

protopanaxadiol (PPD), 20(S)-protopanaxatriol (PPT), 20(R)-PPT, ginseng saponin compound K) of

RG in rat plasma after oral administration of RG water extract at a single dose of 4 g/kg body weight

to rats. All analytes with internal standard (digoxin) were detected by multiple reaction monitoring in

negative ionization mode and separated on an ACQUITY UPLC® BEH RP-C18 column (1.7 μm, 100

× 2.1 mm). This established method was well validated in terms of linearity, sensitivity, intra- and

inter-day precisions, accuracy, recovery, matrix effect, stability, and had a lower limit of quantification

at the concentration range of 0.12–8.12 ng/mL for all of analytes. This UFLC–MS/MS approach was

successfully applied to the pharmacokinetic study for RG water extract in rats. We firstly proposed that

Rb1, Rb2, Rc, Rd, Rg1, Rg5, 20(S)-Rg3, 20(S)-Rh2, and 20(S)-PPD measured in rat plasma were

suitable pharmacokinetic markers of RG extract in rats due to their high systemic exposure levels.

Thus, this specific and reliable method will be useful for future applications to pharmacokinetic studies

for various sources of ginsenoside samples and Panax herbs in vivo.

74

Metabolic profiles of exudates from chronic leg ulcerations

Adam Junka, Wojciech Wojtowicz, Adam Ząbek, Grzegorz Krasowski, Marzenna Bartoszewicz

ABSTRACT

Chronic leg ulceration is a disease usually associated with other comorbidities, and significantly

reduces patient quality of life. Infected leg ulcers can lead to limb-threatening sequelae or mortality.

Leg ulcerations are colonized by a number of microbes that are able to cause life-threating infections

in susceptible patients. Wound exudate is a body fluid that collects metabolites from patient eukaryotic

cells and from prokaryotic bacterial communities inhabiting the wound. This study aimed at

identification of metabolites in exudates collected from chronic leg ulcers, and correlation of this

metabolome with patient comorbidities and microbiological status of the wound. By means of NMR

spectroscopy we detected 42 metabolites of microbial or patient origin. The metabolites that were in

abundance in exudates analyzed were lactate, lysine, and leucine. Metabolites were associated with the

presence of neutrophils in wounds and destruction of high quantities of microbes, but also with

hypoxia typical for venous insufficiency. The combination of nuclear magnetic resonance

spectroscopy technique and partial least squares discriminant analysis allowed us to further

discriminate groups of metabolites with regards to potential clinical meaning. For example, to

discriminate between S.aureus versus all other isolated microbial species, or between patients suffering

from type I or II diabetes versus patients without diabetes. Therefore, wound exudate seems to be

highly applicable material for discriminant analysis performed with the use of NMR technique to

provide for rapid metabolomics of chronic wound status.

Development and validation of a sensitive and fast UPLC–MS/MS method for simultaneous

determination of seven bioactive compounds in rat plasma after oral administration of Guizhi-

gancao decoction

Bin Ji, Limeng Zhuo, Bin Yang, Yang Wang, Zhiguo Yu

ABSTRACT

Rapid, sensitive, selective and accurate UPLC–MS/MS method was developed and fully validated for

simultaneous determination of cinnamaldehyde, cinnamic acid, 2-methoxy cinnamic acid, glycyrrhizic

acid, glycyrrhetinic acid, liquiritigenin and isoliquiritin in rat plasma after oral administration of

Guizhi-gancao decoction. Plasma samples were processed with a simple protein precipitation

technique using acetonitrile, followed by chromatographic separation using a Thermo Hypersil GOLD

C18 column. A 11.0 min linear gradient elution was used at a flow rate of 0.2 mL/min with a mobile

phase of 0.1% acetic acid containing 0.2 mM ammonium acetate in water and acetonitrile. The

analytes and internal standard, schisandrin, were detected using both positive and negative ion

electrospray ionization in multiple reaction monitoring mode. The developed method was validated for

intra-day and inter-day accuracy and precision whose values fell in the acceptable limits. Matrix effect

was found to be minimal. Recovery efficiency of all the analytes was found to be >60%. Stability

results showed that the analytes were stable at all the conditions. This validated method was

successfully used to study the pharmacokinetics of multiple compounds in rat plasma after oral

administration of Guizhi-gancao decoction.

75

Universal efavirenz determination in transport study, rat placenta perfusion and placenta lysate

by HPLC-UV

Lucie Zelena, Josef Reznicek, Martina Ceckova, Hana Sklenarova

ABSTRACT

Efavirenz is an antiretroviral drug used in the treatment of HIV-positive patients. A simple, fast and

sensitive high-performance liquid chromatography (HPLC) method was developed in order to

determine efavirenz in three types of samples provided from pharmacokinetic studies. The analysis

took 5 min and was performed using a C18 analytical column (Discovery HS C18, 150 × 4.6 mm,

particle size of 5 μm) in isocratic mode with a mobile phase containing acetonitrile and water (65:35,

v/v), a flow rate of 1.6 mL min−1, a sample volume of 10 μL and UV detection at 245 nm. Three

different sample matrices (Opti-MEM medium, Krebs perfusion liquid and tissue lysate) and their

treatment (dilution, SPE) were considered. The validated method was applied for the analysis of 805

real samples arising from in vitro transcellular transport assays and in vivo organ perfusion

experiments in order to evaluate the interaction of efavirenz with ATP-dependent drug efflux

transporters. The lack of interaction of efavirenz with ABCB1, ABCG2 and ABCC2 transporters as

well as technical aspects of this analysis, including the adhesion of efavirenz to the plastic materials

and the stability of the drug during different tissue lysis approaches are discussed.

Metabolite characterization of a novel sedative drug, remimazolam in human plasma and urine

using ultra high-performance liquid chromatography coupled with synapt high-definition mass

spectrometry

Ying Zhou, Pei Hu, Ji Jiang

ABSTRACT

Remimazolam is a new chemical entity belonging to the benzodiazepine class of sedative drugs, which

shows faster-acting onset and recovery than currently available short-acting sedatives. In the present

study, ultra high performance liquid chromatography with synapt high-definition mass spectrometry

method combined with MassLynx software was established to characterize metabolites of

remimazolam in human plasma and urine. In total, 5 human metabolites were detected, including 3

phase I and 2 phase II metabolites. There was no novel human metabolite detected compared to that in

rat. Hydrolysis, glucuronidation and oxidation were the major metabolic reactions. To our knowledge,

this is the first report of the human metabolic profile of remimazolam.

76

Chiral separation of new sulfonamide derivatives and evaluation of their enantioselective affinity

for human carbonic anhydrase II by microscale thermophoresis and surface plasmon resonance

Tiphaine Rogez-Florent, Catherine Foulon, Anne-Sophie Drucbert, Nadège Schifano, Jean-François

Goossens

ABSTRACT

The aim of this study was to develop a method combining chiral separation and biophysical techniques

to evaluate the enantioselective affinity of original sulfonamide derivatives towards their therapeutic

target, the human carbonic anhydrase II (hACII). The first step consisted in the preparation of the

enantiomers by chromatographic separation. The performances of HPLC and Supercritical Fluid

Chromatography (SFC) were studied at the analytical scale by optimization of various experimental

conditions using adsorbed polysaccharide chiral stationary phases (amylose AD-H and cellulose OD-

H). Since SFC allowed obtaining higher enantioresolutions per time unit, it was selected for the semi-

preparative scale and successfully used to isolate each enantiomer with a satisfactory enantiomeric

purity (>98%). Secondly, microscale thermophoresis (MST) method and surface plasmon resonance

(SPR) used as reference method were developed to measure potential enantioselective affinities of

these enantiomers towards the hACII. The optimizations of both methods were performed using a

reference compound, i.e. acetazolamide, which affinity for hCAII has previously been demonstrated.

For all compounds, KD values obtained using MST and SPR were in good agreement, leading to

similar affinity scales despite both approaches totally differ (labeling for MST versus immobilization

of the protein for SPR). The equilibrium dissociation constants of our original compounds for the

hCAII were in the range 100–1000 nM and an enantioselectivity was observed using the MST and

SPR methods for the diarylpyrazole 2.

Ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS)

for determination of GHB, precursors and metabolites in different specimens: Application to

clinical and forensic cases

Francesco Paolo Busardò, Chrystalla Kyriakou, Emilia Marchei, Roberta Pacifici, Simona Pichini

ABSTRACT

Gamma-hydroxybutyric acid (GHB) acts as a precursor and metabolite of the inhibitory central

nervous system (CNS) neurotransmitter gamma-aminobutyric acid (GABA). Sodium salt of GHB has

been used as a medication for narcolepsy and alcohol withdrawal. Moreover, GHB and its precursor

gamma-butyrolactone (GBL), are illegal recreational drugs of abuse. A procedure based on ultra-high-

performance liquid chromatography tandem mass spectrometry has been developed and validated in

plasma, urine, cerebrospinal fluid and hair for acute and chronic exposure to GHB and in seized

preparations coming from black market. In biological matrices, GHB was investigated together with its

glucuronide (GHB-Gluc) as a potential marker of exposure, GABA as endogenous precursor and

metabolite and GBL as eventual exogenous precursor. GBL was sought together with GHB in illegal

preparations. Chromatographic separation was achieved at ambient temperature using a reverse-phase

column and an isocratic elution with two solvents: 0.1% formic acid in water and pure methanol.

Multiple reaction monitoring (MRM) was used. The method was linear for all analytes under

investigation from limit of quantification (LOQ) to 500 μg mL−1 plasma, urine and cerebrospinal

fluid, from LOQ to 100 ng mg−1 hair and from LOQ to 10 mg mL−1 illicit preparations with good

correlation coefficients (r2 = 0.99) for all substances.

77

Impedimetric nanostructured genosensor for detection of schistosomiasis in cerebrospinal fluid

and serum samples

Giselle S. Santos, Cesar A.S. Andrade, Igor S. Bruscky, Leandro B. Wanderley, Maria D.L. Oliveira

ABSTRACT

Schistosomiasis is a neglected disease closely related to the low levels of social development and a

serious public health problem. In this work, we performed an electrochemical detection of

Schistosoma mansoni DNA with a self-assembled monolayer of mercaptobenzoic acid (MBA)

immobilizing nanostructures composed of gold nanoparticles (AuNPs) and magnetite nanoparticles

(Fe3O4_NPs). Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used

to monitor the hybridization process. MBA-Fe3O4_NPs-AuNPs-DNAprobe system reveals an

effective electrochemical response indicating the surface modification. The proposed biosystem was

capable to recognize specific nucleotide sequence of S. mansoni present in cerebrospinal fluid (CFS)

and serum samples at different genome DNA concentrations. The biorecognition resulted in an

increase in the electron transfer resistance and a decrease of the current peaks at higher DNA

concentrations during electrochemical measurements. The developed platform showed a DNA

detection limit of 0.781 and 0.685 pg μL−1 for serum and CFS, respectively. Therefore, the obtained

biosensor can be considered as a useful tool for specific detection of S. mansoni at low concentrations

in various biological fluids.

LC–MS/MS method for the simultaneous determination of Lys-MCC-DM1, MCC-DM1 and

DM1 as potential intracellular catabolite of the antibody-drug conjugate trastuzumab emtansine

(T-DM1)

Yazhong Liu, Fang Zhou, Hua Sang, Hui Ye, Jingwei Zhang

ABSTRACT

Lysine-MCC-DM1, MCC-DM1 and DM1 are potential catabolites of trastuzumab emtansine (T-

DM1). A convenient liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was

developed and validated to detect these catabolites simultaneously in in vitro investigations for the first

time. Protein precipitation was utilized to prepare the samples. Chromatographic separation was

achieved on a Phenomenex Kinetex C18 column (100 × 2.1 mm, 2.6 μm) with mobile-phase gradient

elution. The calibration curves of each analyte ranging from 1 to 100 nM showed good linearity (r2 >

0.995). The method was validated successfully and applied to the intracellular catabolism and

regulation of T-DM1.

78

Quantification of hydroxyurea in human plasma by HPLC–MS/MS and its application to

pharmacokinetics in patients with chronic myeloid leukaemia

Xin Hai, Meihua Guo, Chunlu Gao, Jin Zhou

ABSTRACT

Hydroxyurea (HU) has been used in the treatment of chronic myeloid leukaemia (CML) and other

myeloproliferative malignancies. Considering patient‘s wide variation in clinical response to HU, a

new and simple liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was

developed and validated to monitor patients‘ compliance to treatment and investigate the

pharmacokinetics of HU in patients with CML. Stable isotope labeled HU-13C1,15N2 was used as

internal standard. Plasma samples were treated with acetonitrile to precipitate protein. The supernatant

was injected directly without derivatization and separated on a hydrophilic interaction liquid

chromatography column. HU was quantitatively analyzed with a mobile phase of acetonitrile-1.5 mM

ammonium formate (90:10, V:V) within 3 min. The proposed method provided a linearity range of 1–

200 μg/mL. The coefficients of variation for intra- and inter-day precision were less than 2.07% and

4.28%, respectively, while the accuracy (bias) was in the range of −3.77 to 2.96%. This method was

satisfactorily applied to the determination of HU in two patients with CML. It is suitable for

supporting pharmacokinetic studies and clinical therapeutic monitoring.

Determination of rodenticides and related metabolites in rabbit liver and biological matrices by

liquid chromatography coupled to Orbitrap high resolution mass spectrometry

Marina López-García, Roberto Romero-González, Antonia Garrido Frenich

ABSTRACT

An analytical method based on ultra-high performance liquid chromatography (UHPLC) coupled to

Orbitrap high resolution mass spectrometry was developed for the determination of rodenticides

(bromadiolone, brodifacoum, difenacoum, chlorophacinone, diphacinone, coumachlor and warfarin) in

liver matrix. Different extraction conditions were tested, obtaining the best results when the ―dilute and

shoot‖ method (acidified acetonitrile as extraction solvent) and a clean-up step with primary secondary

amine (PSA) were used. The optimized method was validated, obtaining recoveries ranging from 60 to

120%. Repeatability and reproducibility were evaluated obtaining values lower than 20%, except for

brodifacoum at 10 μg/kg. Limits of quantification (LOQs) ranged from 0.1 to 0.5 μg/kg, except for

brodifacoum, which was 100 μg/kg. Six liver samples were analyzed and diphacinone and

chlorophacinone were detected in three samples at concentrations ranging from 4 μg/kg to 13 μg/kg.

Moreover a retrospective screening of rodenticide metabolites in those samples and in animal forensic

samples was developed based on Orbitrap capabilities. Brodifacoum was detected in three samples,

and warfarin alcohol, which is a metabolite of warfarin, was also detected in one sample.

79

Comparative pharmacokinetic profiles of selected irreversible tyrosine kinase inhibitors,

neratinib and pelitinib, with apigenin in rat plasma by UPLC–MS/MS

Hadir M. Maher, Nourah Z. Alzoman, Shereen M. Shehata, Ashwag O. Abahussain

ABSTRACT

Neratinib (NER) and pelitinib (PEL) are irreversible tyrosine kinase inhibitors (TKIs) that have been

recently employed in cancer treatment. Apigenin (API), among other flavonoids, is known to have

antioxidant, anti-proliferative, and carcinogenic effect. API can potentiate the antitumor effect of

chemotherapeutic agents and/or alleviate the side effects of many anticancer agents. Since TKIs are

mostly metabolized by CYP3A4 enzymes and that API could alter the enzymatic activity, potential

drug interactions could be expected following their co-aministration. In the present study, a

bioanalytical UPLC–MS/MS method has been developed and validated for the quantification of NER

and PEL in rat plasma, using domperidone (DOM) as an internal standard. Sample preparation was

carried out using solid phase extraction (SPE) with C18 cartridges with good extraction recovery of

not less than 92.42% (NER) and 89.73% (PEL). Chromatographic analysis was performed on a Waters

BEH C18 column with a mobile phase composed of acetonitrile and water, (70:30, v/v), each with

0.1% formic acid. Quantitation was performed using multiple reaction monitoring (MRM) of the

transitions from protonated precursor ions [M+H]+, at m/z 557.30 (NER), m/z 468.21 (PEL), and at

m/z 426.27 (DOM), to selected product ions at m/z 112.05 (NER), m/z 395.22 (PEL), and at m/z

175.18 (DOM). The method was fully validated as per the FDA guidelines over the concentration

range of 0.5–200 ng/mL with very low lower limit of quantification (LLOQ) of 0.5 ng/mL for both

NER and PEL. The intra- and inter-day assay precision and accuracy were evaluated for both drugs

and the calculated values of percentage relative standards deviations (%RSD) and relative errors (%Er)

were within the acceptable limits (<15%) for concentrations other than LLOQ and 20% for LLOQ.

The applicability of the method was extended to study the possibility of drug interactions following the

oral co-administration of NER/PEL with API. Thus, this study could be readily applied in therapeutic

drug monitoring (TDM) of cancerous patients receiving such drug combinations.

LC–MS/MS determination of d-mannose in human serum as a potential cancer biomarker

Lyndsey White, Jing Ma, Su Liang, Beatriz Sanchez-Espiridion, Dong Liang

ABSTRACT

Several metabolites in human serum have been identified as potential cancer biomarkers for early

detection. This study focuses on the LC–MS/MS method development and validation of d-mannose in

human serum. Surrogate blank serum, coupled with stable isotope d-mannose-13C6, as internal

standard, was used for generating standard curves ranging from 1 to 50 μg/mL. Separation was

achieved by an Agilent 1200 series HPLC equipped with a SUPELCOGELTM Pb, 6% Crosslinked

column with HPLC water as a mobile phase at flow rate of 0.5 mL/min at 80 °C. Mass detection was

performed under negative ionization electrospray. Inter- and intra-day accuracy and precision were

<2%. The extraction recovery and matrix effect were 104.1%–105.5% and 97.0%–100.0%,

respectively. This method was successfully applied for the quantification of d-mannose in the serum

samples of 320 esophageal cancer patients and 323 healthy volunteers. We report a simple, specific

and reproducible LC–MS/MS method for the quantification of d-mannose in human serum as a

potential cancer biomarker.

80

Assessment of in vitro cardiotoxicity of extract fractions and diterpene alkaloids from Aconitum

leucostomum Worosch: A short communication

Jihong Nie, Fang Wang, Tengfei Ji, Jun Zhao, Feicui Zhao

ABSTRACT

Aconitum leucostomum Worosch is a traditional Chinese medicine (TCM) and has a broad spectrum

of health effects, but with a narrow therapeutic window. It is important to identify both the therapeutic

ingredients and the toxic components to better utilize this TCM. The present study investigated the

cardiotoxicity of the selected compounds in Aconitum leucostomum Worosch. The effects of extract of

A. leucostomum Worosch and the isolated compounds on cardiocardiomyocytes were evaluated in

vitro. Five known compounds in this TCM, including three C18-diterpene alkaloids, lappaconitine (2),

N-deacetyllappaconitine (3), and ranaconitine (5), and two C19-diterpene alkaloids, delvestidine (1)

and anthranoyllycoctonine (4), were isolated from A. leucostomum Worosch. The cardiotoxicity of

these components and extract fractions, as measured by lactate dehydrogenase release and apoptosis,

was ranked as follows, in descending order: delvestidine > anthranoyllycoctonine > pH 4 fraction > pH

8 fraction > aconitine > N-deacetyllappaconitine > ranaconitine > lappaconitine. The cytotoxicity of

these compounds was shown to be dose-dependent, with delvestidine (1) and anthranoyllycoctonine

(4) being the two most toxic compounds to cardiomyocytes in our assays. These results provide a basis

for future rational use of this TCM, reducing side effects while retaining therapeutic effects.

Development and validation of a liquid chromatography–tandem mass spectrometry method for

the assay of tafamidis in rat plasma: Application to a pharmacokinetic study in rats

Hun-Chan Hyun, Jong-Woo Jeong, Hye-Rim Kim, Ji-Hoon Oh, Tae-Sung Koo

ABSTRACT

Tafamidis is a first-in-class inhibitor of transthyretin amyloid fibril formation. It has been available in

Argentina, Japan, and Mexico for the treatment of transthyretin amyloidosis in adult patients with

early-stage symptomatic polyneuropathy. In this study, a rapid and sensitive liquid chromatography-

tandem mass spectrometry method was developed and validated for the assay of tafamidis in rat

plasma. The method was also assessed for its applicability to pharmacokinetic studies in rats.

Tafamidis was extracted from rat plasma by the liquid-liquid extraction method using hydrochloric

acid and ethyl acetate. A reversed-phase C18 column and a mobile phase consisting of 10 mM

ammonium formate and acetonitrile were used to achieve chromatographic separation. The flow rate

for the mobile phase was set at 0.3 mL/min. Tafamidis and 2-CBC, which was used as the internal

standard (IS), were analyzed by multiple reaction monitoring in negative ESI mode at m/z transitions

of 305.4 → 261.4 for tafamidis and 271.7 → 227.8 for the IS. The lower limit of quantification of

tafamidis was obtained as 3 ng/mL, and the calibration curve was linear over a concentration range of

3–3000 ng/mL (R2 > 0.99). The validation parameters investigated, which were specificity, precision,

accuracy, matrix effect, recovery, and stability, were well within acceptable limits. The method was

successfully used for the evaluation of the pharmacokinetics of tafamidis in rats.

81

Isolation and characterization of a novel dithio-carbodenafil analogue from a health supplement

Chee-Leong Kee, Min-Yong Low, Xiaowei Ge

ABSTRACT

A novel dithio-carbodenafil compound was isolated and identified from a health supplement. The

structure of the unknown compound has been characterized using LC-UV, Fourier Transform Infrared

(FTIR), high-resolution mass spectrometry, 1D and 2D nuclear magnetic resonance (NMR)

spectroscopy. It has been revealed as 3,5-dimethylpiperazinyl dithio-desmethylcarbodenafil as a result

of two additional methyl groups on the piperazine ring.

Preparation and 68Ga-radiolabeling of porous zirconia nanoparticle platform for PET/CT-

imaging guided drug delivery

Andras Polyak, Lívia Naszalyi Nagy, Judith Mihaly, Sebastian Görres, Tobias L. Ross

ABSTRACT

This paper describes the preparation of gallium-68 (68Ga) isotope labeled porous zirconia (ZrO2)

nanoparticle (NP) platform of nearly 100 nm diameter and its first pharmacokinetic and biodistribution

evaluation accomplished with a microPET/CT (μPet/CT) imaging system. Objectives of the

investigations were to provide a nanoparticle platform which can be suitable for specific delivery of

various therapeutic drugs using surface attached specific molecules as triggering agents, and at the

same time, suitable for positron emission tomography (PET) tracing of the prospective drug delivery

process. Radiolabeling was accomplished using DOTA bifunctional chelator. DOTA was successfully

adsorbed onto the surface of nanoparticles, while the 68Ga-radiolabeling method proved to be simple

and effective. In the course of biodistribution studies, the 68Ga-labeled DOTA-ZrNPs showed proper

radiolabeling stability in their original suspension and in blood serum. μPet/CT imaging studies

confirmed a RES-biodistribution profile indicating stable nano-sized labeled particles in vivo. Results

proved that the new method offers the opportunity to examine further specifically targeted and drug

payload carrier variants of zirconia NP systems using PET/CT imaging.

82

Determination of panduratin A in rat plasma by HPLC–MS/MS and its application to a

pharmacokinetic study

Minsoo Kim, Seungmok Choi, Keumhan Noh, Changhee Kim, Wonku Kang

ABSTRACT

Although panduratin A, a chalcone derivative isolated from the rhizome of Kaempferia pandurata

Roxb, represents a variety of pharmacological activities, no validated method for determination of

panduratin a levels in biological samples is yet available. Thus, we developed a liquid

chromatographic method using a tandem mass spectrometry for the determination of panduratin A in

rat plasma. After simple protein precipitation with methanol including flufenamic acid (internal

standard, IS), the analytes were chromatographed on a reversed-phased column with a mobile phase of

distilled water and acetonitrile including 2% of formic acid (2:8, v/v). The ion transitions of the

precursor to the product ion were principally deprotonated ions [M−H] − at m/z 405.2 → 165.9 for

panduratin A and 280.1 → 236.0 for the IS. The accuracy and precision of the assay were in

accordance with FDA regulations for the validation of bioanalytical methods. This analytical method

was successfully applied to monitor the plasma concentrations of panduratin a following oral

administration in rats.

Simultaneous determination of creatinine and acetate by capillary electrophoresis with

contactless conductivity detector as a feasible approach for urinary tract infection diagnosis

Wojciech Grochocki, Michał J. Markuszewski, Joselito P. Quirino

ABSTRACT

Urinary tract infection (UTI) is one of the most common bacterial infections in human but its diagnosis

is difficult. Metabolomic studies with nuclear magnetic resonance of urine have shown that acetic

acid/creatinine ratio may be used for early UTI diagnosis. Here, a method for simultaneous

determination of acetate and creatinine by capillary zone electrophoresis with contactless conductivity

detector was developed for the first time. The separation was with 40 mM MES and 20 mM l-histidine

as a background solution. The total time of a single run, including capillary conditioning, was less than

12 min. The method was successfully demonstrated for analysis of actual and fortified human urine

samples after methanol dilution. Analytical figures of merit such as linearity, LOQ, and repeatability

(intraday and interday) were studied.

83

Analytical method development and validation for the analysis of verapamil hydrochloride and

its related substances by using ultra perfomance liquid chromatography

S. Vijayabaskar, V. Mahalingam, Kalaivani

ABSTRACT

A novel, economic, and time-efficient stability-indicating, reverse-phase ultra-performance liquid

chromatographic (RP-UPLC) method has been developed for the analysis of verapamil hydrochloride

in the presence of both impurities and degradation products generated by forced degradation. When

verapamil hydrochloride was subjected to acid hydrolysis, oxidative, base hydrolysis, photolytic, and

thermal stress, degradation was observed only in oxidative and base hydrolysis. The drug was found to

be stable to other stress conditions. Successful chromatographic separation of the drug from impurities

formed during synthesis and from degradation products formed under stress conditions was achieved

on a Shimpak XR ODS, 75 mm × 3.0 mm, 1.7 μ particle size column, UV detection at 278 nm and a

gradient elution of ammonium formate, orthophosphoric acid and acetonitrile as mobile phase. The

method was validated for specificity, precision, linearity, accuracy, robustness and can be used in

quality control during manufacture and for assessment of the stability samples of verapamil

hydrochloride. To the best of our knowledge, a validated UPLC method which separates all the sixteen

impurities disclosed in this investigation has not been published elsewhere. Total elution time was

about 18 min which allowed quantification of more than 100 samples per day. The analytical method

discussed in British Pharmacopeia was pH sensitive and not compatible to LC–MS analysis but the

method reported in this study is not involved any pH adjustment.

Evaluation of in silico pharmacokinetic properties and in vitro cytotoxic activity of selected

newly synthesized N-succinimide derivatives

Natasa P. Milosevic, Vesna Kojic, Jelena Curcic, Dimitar Jakimov, Roman Kaliszan

ABSTRACT

Design of a new drug entity is usually preceded by analysis of quantitative structure activity

(properties) relationships, QSA(P)R. Six newly synthesized succinimide derivatives have been

determined for (i) in silico physico-chemical descriptors, pharmacokinetic and toxicity predictors, (ii)

in vitro biological activity on four different carcinoma cell lines and on normal fetal lung cells and (iii)

lipophilicity on liquid chromatography. All compounds observed were predicted for good permeability

and solubility, good oral absorption rate and moderate volume of distribution as well as for modest

blood brain permeation, followed by acceptable observed toxicity. In silico determined lipophilicity,

permeability through jejunum and aqueous solubility were correlated with experimentally obtained

lipophilic constants (by use of high pressure liquid chromatography) and linear correlations were

obtained. Absorption rate and volume of distribution were predicted by chromatographic lipophilicity

measurements while permeation through blood bran barrier was predicted dominantly by molecular

size defined with molecular weight. Five compounds have demonstrated antiproliferative activity

toward cervix carcinoma HeLa cell lines; three were cytotoxic against breast carcinoma MCF-7 cells,

while one inhibited proliferation of colon carcinoma HT-29 cell lines. Only one compound was

cytotoxic toward normal cell lines, while other compounds were proven as safe. Antiproliferative

potential against HeLa cells was described as exponential function of lipophilicity. Based on obtained

results, lead compounds were selected.

84

Biopharmaceutical characterization of praziquantel cocrystals and cyclodextrin complexes

prepared by grinding

Martina Cugovčan, Jasna Jablan, Jasmina Lovrić, Dominik Cinčić, Mario Jug

ABSTRACT

Mechanochemical activation using several different co-grinding additives was applied as a green

chemistry approach to improve physiochemical and biopharmaceutical properties of praziquantel

(PZQ). Liquid assisted grinding with an equimolar amount of citric acid (CA), malic acid (MA),

salicylic acid (SA) and tartaric acid (TA) gained in cocrystal formation, which all showed pH-

dependent solubility and dissolution rate. However, the most soluble cocrystal of PZQ with MA was

chemically unstable, as seen during the stability testing. Equimolar cyclodextrin complexes prepared

by neat grinding with amorphous hydroxypropyl-β-cyclodextrin (HPβCD) and randomly methylated β-

cyclodextrin (MEβCD) showed the highest improvement in drug solubility and the dissolution rate, but

only PZQ/HPβCD product presented an acceptable chemical and photostability profile. A combined

approach, by co-grinding the drug with both MA and HPβCD in equimolar ratio, also gave highly

soluble amorphous product which again was chemical instable and therefore not suitable for the

pharmaceutical use. Studies on Caco-2 monolayer confirmed the biocompatibility of PZQ/HPβCD

complex and showed that complexation did not adversely affect the intrinsically high PZQ

permeability (Papp(PZQ) = (3.72 ± 0.33) × 10−5 cm s−1 and Papp(PZQ/HPβCD) = (3.65 ± 0.21) ×

10−5 cm s−1; p > 0.05). All this confirmed that the co-grinding with the proper additive is as a

promising strategy to improve biopharmaceutical properties of the drug.

Development of an enzyme-linked immunosorbent assay for the detection of isomiroestrol, an

identical marker, in White Kwao Krua using a monoclonal antibody

Tharita Kitisripanya, Kamonthip Jutathis, Chadathorn Inyai, Jukrapun Komaikul, Waraporn Putalun

ABSTRACT

Pueraria candollei var. mirifica or White Kwao Krua (WKK) is a phytoestrogen-rich plant widely used

among women to improve climacteric symptoms. Additionally, the tuberous roots of this plant have

been added as an active ingredient for skin rejuvenation and breast enlargement effects in various

functional foods. However, most of the products on the market containing WKK have not been

sufficiently standardized with respect to the active compound or identical marker. To control the

quality of these plant materials, an enzyme-linked immunosorbent assay (ELISA) using anti-

isomiroestrol antibodies was established for the determination of isomiroestrol, an identical marker in

WKK. Polyclonal and monoclonal antibodies against isomiroestrol were generated and their specificity

characterized in this study. Monoclonal antibody 12C1 showed higher specificity to isomiroestrol and

was thus selected to develop the ELISA. Based on the validation analysis and the tested performance

of the developed ELISA in variably sourced WKK samples, the assay can provide an alternative

approach that is reliable and highly sensitive for the quantitative analysis of isomiroestrol in plant.

85

Volume 138 May 2017

Fabrication of a novel hemin-based monolithic column and its application in separation of

protein from complex bio-matrix

Xiaoya Jiang, Doudou Zhang, Xueying Li, Xixi Wang, Hongyuan Yan

ABSTRACT

A novel polymer-based monolithic column was prepared via redox initiation system within the

confines of a stainless steel column with 4.6 mm i.d. In the processes, hemin and lauryl methacrylate

were used as co-monomers; ethylene dimethacrylate as crosslinking agent; n-butyl alcohol, ethanediol,

and N, N-dimethylformamide as tri-porogens; benzoyl peroxide and N, N-dimethyl aniline as redox

initiation system. The resulting polymer-based monolithic columns were characterized by scanning

electron microscopy, nitrogen adsorption-desorption instrument, and mercury intrusion porosimeter,

respectively. The results illustrated that the improved monolith had relative uniform porous structure,

good permeability, and low back pressure. Aromatic compounds were used to test the chromatographic

behavior of the monolith, resulting in highest column efficiency of 19 880 plates per meter with

reversed-phase mechanism. Furthermore, the homemade monolith was used as the stationary phase of

high performance liquid chromatography to separate proteins from complex bio-matrix, including

human plasma, egg white, and snailase. The results showed that the monolithic column occupied good

separation ability with these complex bio-samples.

High-throughput NIR-chemometric methods for chemical and pharmaceutical characterization

of sustained release tablets

Alina Porfire, Cristina Filip, Ioan Tomuta

ABSTRACT

The aim of this study was the development and validation of methods based on near-infrared

spectroscopy (NIRS) and chemometry, useful for characterization of sustained release (SR) tablets

with indapamide, in terms of tablet composition (API and two excipients), in vitro drug release

mechanism (k and n Peppas) and crushing strength. A calibration set consisting of 25 different tablets

formulations containing API, HPMC and lactose at five different content levels in the range 100 ±

20% relative to a targeted tablet composition, were manufactured by direct compression in order to

develop the methods for prediction of tablet composition, and in vitro drug release mechanism. On the

other hand, a 15 batches calibration set prepared at five different compression forces was used for

development of methods for prediction of crushing strength. Moreover, independent batches were

manufactured for validation of all methods Intact tablets were analyzed by transmission mode with

NIRS, the spectra were pre-processed, and partial least square (PLS) regression was used to build

prediction models. Cross-validation was carried out in order to select the optimal number of PLS

factors for all models, and the best model was chosen based on their RMSECV and bias. All developed

methods were validated in terms of trueness, precision and accuracy. Based on the validation results,

the methods proposed in this work can successfully be applied for routine determination of

indapamide, HPMC and lactose content of sustained release tablets, as well as for prediction of their in

vitro drug release mechanism (k and n Peppas) and crushing strength.

86

Optimized multi-step NMR-crystallography approach for structural characterization of a stable

quercetin solvate

Xenia Filip, Maria Miclaus, Flavia Martin, Claudiu Filip, Ioana Georgeta Grosu

ABSTRACT

Herein we report the preparation and solid state structural investigation of the 1,4-dioxane-quercetin

solvate. NMR crystallography methods were employed for crystal structure determination of the

solvate from microcrystalline powder. The stability of the compound relative to other reported

quercetin solvates is discussed and found to be in perfect agreement with the hydrogen bonding

networks/supra-molecular architectures formed in each case. It is also clearly shown that NMR

crystallography represents an ideal analytical tool in such cases when hydrogen-bonding networks are

required to be constrained at a high accuracy level.

Study on the forced degradation behaviour of ledipasvir: Identification of major degradation

products using LC–QTOF–MS/MS and NMR

Debasish Swain, Gananadhamu Samanthula

ABSTRACT

Ledipasvir, a novel NS5A inhibitor is used in the management of hepatitis C virus infections. The drug

was subjected to forced degradation studies as per the conditions prescribed in ICH Q1 (R2) guideline.

Ledipasvir degraded in hydrolytic (acid, alkaline and neutral) and oxidative stress conditions. The drug

was found to be stable in thermal and photolytic conditions. Eight novel degradation products were

obtained and were well separated using an HPLC C18 stationary phase (150 × 4.6 mm, 5 μm) and

mobile phase composed of formic acid/acetonitrile in gradient elution mode. All the degradation

products were characterised using tandem mass spectrometry with a time-of-flight analyser and the

major degradation products of hydrolytic and oxidative stress were isolated and their structural

confirmation was studied using 1H and 13C NMR. A well resolved chromatographic method proposed

in this study suggests that the proposed analytical method finds its application as a stability indicating

assay method for the drug and can be used in routine analysis.

87

Raman scattering-based multiconformational analysis for probing the structural differences

between acetylcholine and acetylthiocholine

Belén Hernández, Pascal Houzé, Fernando Pflüger, Sergei G. Kruglik, Mahmoud Ghomi

ABSTRACT

Acetylcholine is the first discovered neurotransmitter that has received a great attention regarding its

capability of binding to several cellular targets. The chemical composition of acetylcholine, including

a positively charged trimethylammonium and a carbonyl group, as well as its conformational

flexibility was pointed out as the key factors in the stabilization of its interactions. Here, the

possibilities offered by a Raman scattering-based multiconformatioal analysis to access the most stable

conformers of acetylcholine, is discussed. To control the validity of this protocol, acetylcholine and

one of its closely structured analogues, acetylthiocholine, were simultaneously analyzed. Solution

Raman spectra revealed distinct and well resolved strong markers for each molecule. Density

functional theory calculations were consistent with the fact that the energy order of the low energy

conformers is considerably affected by the acyloxy oxygen → sulfur atom substitution. Raman spectra

were calculated on the basis of the thermal average of the spectra arising from the low energy

conformers. It has been evidenced that the carbonyl and trimethylammonium groups are the most

favorable hydration sites in aqueous environment. Taking into account the large gap between the

carbonyl bond-stretch and aliphatic bending bands, Raman spectra also allowed separation of the HOH

bending vibrations arising from the bound and bulk water molecules.

Characterization of metabolites in different kiwifruit varieties by NMR and fluorescence

spectroscopy

Nur Ashikin Abdul Hamid, Ahmed Mediani, M. Maulidiani, Faridah Abas, S. Gorinstein

ABSTRACT

It is known from our previous studies that kiwifruits, which are used in common human diet, have

preventive properties of coronary artery disease. This study describes a combination of 1H NMR

spectroscopy, multivariate data analyses and fluorescence measurements in differentiating of some

kiwifruit varieties, their quenching and antioxidant properties. A total of 41 metabolites were identified

by comparing with literature data Chenomx database and 2D NMR. The binding properties of the

extracted polyphenols against HSA showed higher reactivity of studied two cultivars in comparison

with the common Hayward. The results showed that the fluorescence of HSA was quenched by Bidan

as much as twice than by other fruits. The correlation between the binding properties of polyphenols in

the investigated fruits, their relative quantification and suggested metabolic pathway was established.

These results can provide possible application of fruit extracts in pharmaceutical industry.

88

Comparison of bioactive components and pharmacological activities of ophiopogon japonicas

extracts from different geographical origins

Min Zhao, Wan-feng Xu, Han-yuan Shen, Pei-qiang Shen, Li-juan Cao

ABSTRACT

Ophiopogon japonicus (Linn. f.) Ker-Gawl (O. japonicas), mainly cultivated in Sichuan and Zhejiang

province in China, has different bioactive components and therefore their pharmacological activities.

To explain the different clinical efficacy of O. japonicas derived preparations, herein we report

differences of pharmacological activities between Sichuan and Zhejiang O. japonicas and behind them

the exact differences of bioactive components. Based on a LC/MS-IT-TOF method, the differences of

bioactive components between Sichuan and Zhejiang O. japonicas extracts were analyzed and

respective characteristic components were picked out. We determined 39 ophiopogonones and 71

ophiopogonins compounds in Sichuan and Zhejiang O. japonicas extracts and found the contents of

these compositions have several times difference. Evidenced by experimental data of pharmacological

activities in inhibiting cardiomyocyte damage induced by H2O2, mouse macrophage cell inflammation

induced by lipopolysaccharide and cytotoxicity in vitro, Zhejiang O. japonicas extract had a stronger

antioxidant and anti-inflammatory capacity than Sichuan O. japonicas extract, and the two O.

japonicas extracts exhibited selective cytotoxicity on different cancer cell lines in vitro. These data

shed light on the links between bioactive components and pharmacological activities of O. japonicas

derived preparations. Thus, geographical origin of O. japonicas should be considered to be a key factor

in efficacy studies and further clinical application.

Tiered analytics for purity assessment of macrocyclic peptides in drug discovery: Analytical

consideration and method development

Jingfang (Jenny) Qian Cutrone, Xiaohua (Stella) Huang, Edward S. Kozlowski, Ye Bao, Adrienne A.

Tymiak

ABSTRACT

Synthetic macrocyclic peptides with natural and unnatural amino acids have gained considerable

attention from a number of pharmaceutical/biopharmaceutical companies in recent years as a

promising approach to drug discovery, particularly for targets involving protein–protein or protein–

peptide interactions. Analytical scientists charged with characterizing these leads face multiple

challenges including dealing with a class of complex molecules with the potential for multiple isomers

and variable charge states and no established standards for acceptable analytical characterization of

materials used in drug discovery. In addition, due to the lack of intermediate purification during solid

phase peptide synthesis, the final products usually contain a complex profile of impurities. In this

paper, practical analytical strategies and methodologies were developed to address these challenges,

including a tiered approach to assessing the purity of macrocyclic peptides at different stages of drug

discovery. Our results also showed that successful progression and characterization of a new drug

discovery modality benefited from active analytical engagement, focusing on fit-for-purpose analyses

and leveraging a broad palette of analytical technologies and resources.

89

Enantiomeric separation of the antiuremic drug colchicine by electrokinetic chromatography

Method development and quantitative analysis

Nuria Menéndez-López, Jesús Valimaña-Traverso, María Castro-Puyana, Antonio Salgado, María

Luisa Marina

ABSTRACT

Two analytical methodologies were developed by CE enabling the enantiomeric separation of

colchicine, an antiuremic drug commercialized as pure enantiomer. Succinyl-γ-CD and Sulfated-γ-CD

were selected as chiral selectors after a screening with different anionic CDs. Under the optimized

conditions, chiral resolutions of 5.6 in 12 min and 3.2 in 8 min were obtained for colchicine with

Succinyl-γ-CD and Sulfated-γ-CD, respectively. An opposite enantiomeric migration order was

observed with these two CDs being S-colchicine the first-migrating enantiomer with Succinyl-γ-CD

and the second-migrating enantiomer with Sulfated-γ-CD. H NMR experiments showed a 1:1

stoichiometry for the enantiomer-CD complexes in both cases. However, the apparent and averaged

equilibrium constants for the enantiomer-CD complexes could be calculated only for Succinyl-γ-CD.

The developed methods were applied to the analysis of pharmaceutical formulations but only the use

of Succinyl-γ-CD enabled to detect a 0.1% of enantiomeric impurity in colchicine formulations.

Integrative hepatoprotective efficacy comparison of raw and vinegar-baked Radix Bupleuri

using nuclear magnetic resonance-based metabolomics

Jie Xing, Hui-Min Sun, Jin-Ping Jia, Xue-Mei Qin, Zhen-Yu Li

ABSTRACT

Radix Bupleuri (RB), with a Chinese name Chaihu, is one of the most popular Traditional Chinese

herbal drug. It can be baked with vinegar to afford vinegar-baked Radix Bupleuri (VBRB), which is

used in Traditional Chinese Medicine (TCM) for liver diseases treatment. In the present study, nuclear

magnetic resonance-based metabolomic approach was used to compare the liver protective effect of

RB and two types of VBRBs, which were prepared by two kinds of vinegar. The contents of 14

metabolites in the liver of carbon tetrachloride (CCl4) treated mice were significantly altered in

comparison with control group, and VBRB prepared by Shanxi vinegar showed best effect as revealed

by the amount and regulatory degree of the perturbed metabolites. The metabolism pathways analysis

showed that the liver protective effect was related with the energy metabolism, lipid metabolism,

ketone body metabolism, glutathione metabolism, amino acids metabolism and nucleotide synthesis.

The results presented here showed that metabolomic approach made it possible to disclose the subtle

biological difference between two types of VBRB, which highlight the potential of metabolomic

approach to quantitatively compare the pharmacological effect of the herbal drugs.

90

Systematic identification of flavonols, flavonol glycosides, triterpene and siraitic acid glycosides

from Siraitia grosvenorii using high-performance liquid chromatography/quadrupole-time-of-

flight mass spectrometry combined with a screening strategy

Zhi-Xing Qing, Huan Zhao, Qi Tang, Chang-ming Mo, Jian-Guo Zeng

ABSTRACT

The fruits of Siraitia grosvenorii are considered to be health-promoting because of the diversity of their

bioactive ingredients. In the present study, a screening method, using high-performance liquid

chromatography/quadrupole-time-of-flight mass spectrometry (HPLC-Q-TOF-MS) combined with a

screening strategy, has been established. The technology was used to systematically screening the

targeted metabolites, primarily from the complex matrix of S. grosvenorii. The compounds were then

identified by their exact masses and characteristic fragment ions, in comparison with the fragmentation

behaviors of 19 references. Finally, 122 compounds, including 53 flavonols and flavonol glycosides,

59 triterpene glycosides and 10 siraitic acid glycosides, were screened and identified in 10-, 50- and

80-day fruits, roots, stems and leaves of S. grosvenorii. 98 of them were reported for the first time.

Additionally, the distribution of all identified components in different parts of the plant was

determined and metabolic networks for flavonol and triterpene glycosides were proposed.

Rapid discrimination and determination of antibiotics drugs in plastic syringes using near

infrared spectroscopy with chemometric analysis: Application to amoxicillin and penicillin

Laetitia Minh Mai Lê, Luc Eveleigh, Ikram Hasnaoui, Patrice Prognon, Eric Caudron

ABSTRACT

The aim of this study was to investigate near infrared spectroscopy (NIRS) combined to chemometric

analysis to discriminate and quantify three antibiotics by direct measurement in plastic

syringes.Solutions of benzylpenicillin (PENI), amoxicillin (AMOX) and amoxicillin/clavulanic acid

(AMOX/CLAV) were analyzed at therapeutic concentrations in glass vials and plastic syringes with

NIR spectrometer by direct measurement. Chemometric analysis using partial least squares regression

and discriminative analysis was conducted to develop qualitative and quantitative calibration models.

Discrimination of the three antibiotics was optimal for concentrated solutions with 100% of accuracy.

For quantitative analysis, the three antibiotics furnished a linear response (R²>0.9994) for

concentrations ranging from 0.05 to 0.2 g/mL for AMOX, 0.1 to 1.0 MUI/mL for PENI and 0.005 to

0.05 g/mL for AMOX/CLAV with excellent repeatability (maximum 1.3%) and intermediate precision

(maximum of 3.2%). Based on proposed models, 94.4% of analyzed AMOX syringes, 80.0% of

AMOX/CLAV syringes and 85.7% of PENI syringes were compliant with a relative error including

the limit of ± 15%.NIRS as rapid, non-invasive and non-destructive analytical method represents a

potentially powerful tool to further develop for securing the drug administration circuit of healthcare

institutions to ensure that patients receive the correct product at the right dose.

91

Identification of UV-absorbing extractables from rubber closures used in containers of

injectable powder and safety assessment of leachables in the drug

Yulei Wei, Ying Wu, Tingli Zhu, Zhiyan Li, Yilan Zhang

ABSTRACT

Rubber closures have been of great concern to regulatory authorities on account of their potential

safety risks to patients. The aim of our work is to provide part of data about the compatibility of the

injectable powder and its packaging materials for the drug registration. In this report, methodologies

were established to study the system of the preparation. Firstly, three major extractables were isolated

by semi-preparative HPLC method combined with silica gel-based chromatographic methods. NMR

spectra including 1D NMR (1H, 13C, DEPT135) and 2D NMR (COSY, HSQC, HMBC) were

introduced to identify the extractables, besides HPLC, GC–MS, ESI–MS/MS and HRMS. The

extractables were determined to be N-(2-(2,2,4,4-tetramethylcyclohexyl)allyl) benzo[d]thiazol-2-

amine (1), 2,6-Di-tert-butyl-4-methylphenol (2) and sulfur (3) respectively. Then, to address safety

concerns, approaches including QSAR analysis, the TTC and comprehensive literature evaluation

methods to toxicological safety evaluation of the target compounds were established, where the safety

threshold such as TTC and PDE values were developed. Finally, the migration testing of the

extractables were performed to assess the leaching behavior of the rubber closures. An optimized

analysis method was proposed using SPE and HPLC with an ultraviolet detector, which demonstrated

good linearity, acceptable accuracy and precision.

Solid state characterization of azelnidipine–oxalic acid co-crystal and co-amorphous complexes:

The effect of different azelnidipine polymorphs

Yahui Pan, Wenzhe Pang, Jie Lv, Jing Wang, Wei Guo

ABSTRACT

In present study, based on the two polymorphs (α and β form) of azelnidipine (AZE), 12 complexes of

AZE and oxalic acid (OXA) were prepared by solvent-assisted grinding (SG) and neat powder

grinding (NG) methods at the AZE/OXA molar ratios of 2:1, 1:1, and 1:2. The effect of the different

polymorphs of AZE on the micro-structure of the complexes were investigated by powder X-ray

diffraction (PXRD), tempreture modulated differential scanning calorimetry and thermogravimetric

analysis, cryo-field emission scanning electron microscope system, fourier transform infrared (FTIR),

and solid-state nuclear magnetic resonance spectroscopy. β-AZE-OXA co-crystal was produced at β-

AZE/OXA molar ratio of 2:1 when SG method was used; while α-AZE was used to produce α-AZE-

OXA co-crystal at same condition. However, the other 10 combinations were in co-amorphous forms,

including the NG samples with α (or β)-AZE/OXA molar ratios of 2:1, 1:1 (SG and NG), and 1:2 (SG

and NG). Although the XRD pattern and IR spectra of the two co-crystals showed no difference, the

melting enthalpy and specific heat cp of the β-AZE-OXA co-crystal was higher than that of the α-

AZE-OXA co-crystal, indicating that the numbers of solvent molecules which entered the two co-

crystal lattices were different. Interestingly, obvious difference occurred in the IR spectra between the

α-AZE-OXA and β-AZE-OXA co-amorphous systems. 1745 cm−1 wave-numbers, which were

assigned to the free CO groups, appeared in the α-AZE-OXA co-amorphous systems even when just a

small amount of OXA was introduced, thereby indicating the presence of different intermolecular

forces in the two series of co-amorphous forms.

92

Biorelevant physicochemical profiling of (E) - and (Z)-resveratrol determined from isomeric

mixtures

Gábor Orgován, Imre Gonda, Béla Noszál

ABSTRACT

Biorelevant, isomer-specific physicochemical parameters of resveratrol, a multifunctional component

in red wines, with cardioprotective, anti-Alzheimer and several other pharmacologic activities were

determined. The parameters include site-specific basicities, lipophilicities, solubilities and diffusion

constants for the two geometric isomers. The protonation equilibria of (E)- and (Z)-resveratrol were

monitored by 1H NMR-pH titrations. Five closely related auxiliary compounds ((E)-pinostilbene, (Z)-

pinostilbene, (E)-pterostilbene, (Z)-pterostilbene and resorcinol) were also studied. Combining the

datasets, the group-specific protonation constants of resveratrol isomers were determined. The results

show that (Z)-resveratrol is more basic at every protonation site than the (E)-isomer. Lipophilicities are

quantified in terms of logP values and were determined by octanol/water partition experiments and

quantitative NMR spectroscopy: (E)-resveratrol was found to be more lipophilic. Since the molecular

geometries of the isomers differ, diffusion ordered NMR spectroscopy (DOSY) experiments were also

carried out to quantify the diffusion capabilities of the isomers: (Z)-resveratrol of bent shape has a

slightly higher diffusion coefficient than its extended (E) counterpart. A striking 10-fold difference of

water solubility was found in favor of the (Z) isomer, due obviously to the reduced water-repellent

character in the more compact molecule.

An improved size exclusion-HPLC method for molecular size distribution analysis of

immunoglobulin G using sodium perchlorate in the eluent

Hsiaoling Wang, Mark S. Levi, Alfred V. Del Grosso, William M. McCormick, Lokesh Bhattacharyya

ABSTRACT

Size exclusion (SE) high performance liquid chromatography (HPLC) is widely used for the molecular

size distribution (MSD) analyses of various therapeutic proteins. We report development and

validation of a SE-HPLC method for MSD analyses of immunoglobulin G (IgG) in products using a

TSKgel SuperSW3000 column and eluting it with 0.4 M NaClO4, a chaotropic salt, in 40 mM

phosphate buffer, pH 6.8. The chromatograms show distinct peaks of aggregates, tetramer, and two

dimers, as well as the monomer and fragment peaks. In addition, the method offers about half the run

time (12 min), better peak resolution, improved peak shape and more stable base-line compared to

HPLC methods reported in the literature, including that in the European Pharmacopeia (EP). A

comparison of MSD analysis results between our method and the EP method shows interactions

between the protein and the stationary phase and partial adsorption of aggregates and tetramer on the

stationary phase, when the latter method is used. Thus, the EP method shows lower percent of

aggregates and tetramer than are actually present in the products. In view of the fact that aggregates

have been attributed to playing a critical role in adverse reactions due to IgG products, our observation

raises a major concern regarding the actual aggregate content in these products since the EP method is

widely used for MSD analyses of IgG products. Our method eliminates (or substantially reduces) the

interactions between the proteins and stationary phase as well as the adsorption of proteins onto the

column. Our results also show that NaClO4 in the eluent is more effective in overcoming the

protein/column interactions compared to Arg-HCl, another chaotropic salt. NaClO4 is shown not to

affect the molecular size and relative distribution of different molecular forms of IgG.

93

Assessment of the structure of pegylated-recombinant protein therapeutics by the NMR

fingerprint assay

Derek J. Hodgson, Yves Aubin

ABSTRACT

A number of recombinant protein therapeutic products, such as filgrastim (methionyl granulocyte

colony stimulating factor [Met-GCSF] used to boost the immune system in chemotherapy treated

cancer patients), and interferon alpha-2 (used for the treatment of various viral infections), have been

chemically modified with the addition of a polyethylene glycol (PEG) chain. This modification

prolongs residency of the drug in the body and reduces metabolic degradation, which allows less

frequent administration of the products. Here we show how NMR spectroscopy methods can assess the

higher order structure (HOS) of pegylated-filgrastim (Neulasta®), pegylated interferon-α2a

(Pegasys®) pegylated interferon-α2b (PEG-Intron®) purchased from the marketplace. The addition of

the PEG moiety effectively doubles the molecular weight of the three products. This presents a

significant challenge for the application of NMR techniques. Nevertheless, the results showed that

high-resolution spectra could be recorded for two of the three products. Comparison of the spectra of

the pegylated protein and the non-pegylated protein shows that the chemical modification did not alter

the HOS of these proteins.

Prediction of the hydroxypropyl cellulose—poly (vinyl alcohol) ratio in aqueous solution

containing papaverine hydrochloride in terms of drug loaded electrospun fiber formation

Adrienn Kazsoki, Péter Szabó, Romána Zelkó

ABSTRACT

Papaverine hydrochloride loaded electrospun fibers were prepared for buccal drug delivery with the

aim of improving the oral bioavailability of the crystalline drug, which can be achieved by the

increased solubility and by the circumvention of the intensive first pass metabolism. The water soluble

hydroxypropyl cellulose (HPC) was chosen as a mucoadhesive polymer. In order to improve the

electrospinnability of HPC, the also mucoadhesive poly(vinyl alcohol) (PVA) was used. During the

experiments, the total polymer concentration was kept constantly at 15% (w/w), and only the ratio of

the two polymers was changed. Five different HPC:PVA ratios (5:5, 6:4, 7:3, 8:2, 9:1) were examined.

Combination of rheological measurements and molar reflectance characterization with scanning

electron microscopy was applied for the determination of the optimum composition of the gels for

fiber formation. The crystalline-amorphous transition of papaverine hydrochloride was also tracked by

Fourier transform infrared spectroscopy. A correlation was found between the macrostructural

properties of the polymer solutions and their electrospinnability and the consequent morphology of the

resultant samples. Along with the changes of the polymer ratio, the corresponding morphology of the

electrospun samples also varied. With decreasing HPC ratio of the system, a transition from the spray-

dried film-like structure through fibrous film to fibers was observed. Polymer solutions of the lowest

elasticity and smallest intermolecular interactions contributed to the best fiber characteristics of the

samples. The results enable the determination of the polymer ratio for the formation of applicable

quality of electrospun fibers. According to the results 5:5 and 6:4 polymer ratios enabled the best fiber

performance.

94

Optimization of dispersive liquid-phase microextraction based on solidified floating organic drop

combined with high-performance liquid chromatography for the analysis of glucocorticoid

residues in food

Yuan Huang, Zhiqun Zheng, Liying Huang, Hong Yao, Dandan Lin

ABSTRACT

A rapid, simple, cost-effective dispersive liquid-phase microextraction based on solidified floating

organic drop (SFOD-LPME) was developed in this study. Along with high-performance liquid

chromatography, we used the developed approach to determine and enrich trace amounts of four

glucocorticoids, namely, prednisone, betamethasone, dexamethasone, and cortisone acetate, in animal-

derived food. We also investigated and optimized several important parameters that influenced the

extraction efficiency of SFOD-LPME. These parameters include the extractant species, volumes of

extraction and dispersant solvents, sodium chloride addition, sample pH, extraction time and

temperature, and stirring rate. Under optimum experimental conditions, the calibration graph exhibited

linearity over the range of 1.2–200.0 ng/ml for the four analytes, with a reasonable linearity(r2:

0.9990–0.9999). The enrichment factor was 142–276, and the detection limits was 0.39–0.46 ng/ml

(0.078–0.23 μg/kg). This method was successfully applied to analyze actual food samples, and good

spiked recoveries of over 81.5%–114.3% were obtained.

A novel LCMSMS method for quantitative measurement of short-chain fatty acids in human

stool derivatized with 12C- and 13C-labelled aniline

James Chun Yip Chan, Dorinda Yan Qin Kioh, Gaik Chin Yap, Bee Wah Lee, Eric Chun Yong Chan

ABSTRACT

A novel liquid chromatography tandem mass spectrometry (LCMSMS) method for the quantitative

measurement of gut microbial-derived short-chain fatty acids (SCFAs) in human infant stool has been

developed and validated. Baseline chromatographic resolution was achieved for 12 SCFAs (acetic,

butyric, caproic, 2,2-dimethylbutyric, 2-ethylbutyric, isobutyric, isovaleric, 2-methylbutyric, 4-

methylvaleric, propionic, pivalic and valeric acids) within an analysis time of 15 min. A novel

sequential derivatization of endogenous and spiked SCFAs in stool via 12C- and 13C-aniline

respectively, facilitated the accurate quantitation of 12C-aniline derivatized endogenous SCFAs based

on calibration of exogenously 13C-derivatized SCFAs. Optimized quenching of derivatization agents

prior to LCMSMS analysis further reduced to negligible levels the confounding chromatographic peak

due to in-line derivatization of unquenched aniline with residual acetic acid present within the LCMS

system. The effect of residual acetic acid, a common LCMS modifier, in analysis of SCFAs has not

been addressed in previous SCFA assays. For the first time, a total of 9 SCFAs (acetic, butyric,

caproic, isobutyric, isovaleric, 2-methylbutyric, 4-methylvaleric, propionic and valeric acids) were

detected and quantitated in 107 healthy infant stool samples. The abundance and diversity of SCFAs in

infant stool vary temporally from 3 weeks onwards and stabilize towards the end of 12 months. This in

turn reflects the maturation of infant SCFA-producing gut microbiota community. In summary, this

novel method is applicable to future studies that investigate the biological roles of SCFAs in paediatric

health and diseases.

95

Mixed-mode reversed phase/positively charged repulsion chromatography for intact protein

separation

Ling Ding, Zhimou Guo, Zhuo Hu, Xinmiao Liang

ABSTRACT

A mixed-mode reversed phase/positively charged repulsion stationary phase C8PN composed of octyl

and amino group has been developed for separation of intact protein. Before the separation of proteins,

a set of probe compounds were employed to evaluate the chromatographic properties of C8PN,

demonstrating typical reversed phase/positively charged repulsion interaction on this stationary phase

as estimated. Then the new C8PN stationary phase was used to separate a standard protein mixture on

the reversed phase mode. Compared with a commercial C4 stationary phase, it showed different

selectivity for some proteins. In order to better understand the properties of C8PN, the effect of

acetonitrile content was investigated based on retention equation. Higher values of the equation

parameters on C8PN demonstrated that the protein retentions were more sensitive to the change of

acetonitrile content. Besides, the influences of buffer salt additives on the protein retentions were also

studied. The retention factors of the proteins got larger with the increase of buffer salt concentration,

which confirmed the positively charged repulsion interaction on the column. Finally, the C8PN was

further applied to separate oxidized- and reduced- forms of Recombinant Human Growth Hormone.

Our study indicated the advantages and application potential of mixed-mode reversed phase/positively

charged repulsion stationary phase for intact protein separation.

Quantitative determination of five metabolites of aspirin by UHPLC–MS/MS coupled with

enzymatic reaction and its application to evaluate the effects of aspirin dosage on the metabolic

profile

Jian-Ping Li, Jian-Ming Guo, Er-Xin Shang, Zhen-Hua Zhu, Jin-Ao Duan

ABSTRACT

Acetylsalicylic acid (Aspirin, ASA) is a famous drug for cardiovascular diseases in recent years.

Effects of ASA dosage on the metabolic profile have not been fully understood. The purpose of our

study is to establish a rapid and reliable method to quantify ASA metabolites in biological matrices,

especially for glucuronide metabolites whose standards are not commercially available. Then we

applied this method to evaluate the effects of ASA dosage on the metabolic and excretion profile of

ASA metabolites in rat urine. Salicylic acid (SA), gentisic acid (GA) and salicyluric acid (SUA) were

determined directly by UHPLC–MS/MS, while salicyl phenolic glucuronide (SAPG) and salicyluric

acid phenolic glucuronide (SUAPG) were quantified indirectly by measuring the released SA and SUA

from SAPG and SUAPG after β-glucuronidase digestion. SUA and SUAPG were the major

metabolites of ASA in rat urine 24 h after ASA administration, which accounted for 50% (SUA) and

26% (SUAPG). When ASA dosage was increased, the contributions dropped to 32% and 18%,

respectively. The excretion of other three metabolites (GA, SA and SAPG) however showed

remarkable increases by 16%, 6% and 4%, respectively. In addition, SUA and SUAPG were mainly

excreted in the time period of 12–24 h, while GA was excreted in the earlier time periods (0–4 h and

4–8 h). SA was mainly excreted in the time period of 0–4 h and 12–24 h. And the excretion of SAPG

was equally distributed in the four time periods. We went further to show that the excretion of five

metabolites in rat urine was delayed when ASA dosage was increased.

96

HPLC–MS/MS method for the simultaneous quantification of desmethylmebeverine acid,

mebeverine acid and mebeverine alcohol in human plasma along with its application to a

pharmacokinetics study

Natalia E. Moskaleva, Pavel A. Baranov, Natalia V. Mesonzhnik, Svetlana A. Appolonova

ABSTRACT

A new simple, rapid and sensitive high pressure liquid chromatography-tandem mass spectrometry

(HPLC–MS/MS) method was developed and validated for simultaneous analysis of mebeverine

metabolites as: mebeverine alcohol (MAL), mebeverine acid (MAC) and desmethylmebeverine acid

(DMAC) in human plasma. Sample preparation was performed by protein precipitation following the

separation of analytes using an Acquity UPLC BEN C8 column 1.7 mm 2.1 × 50 mm (Waters, USA).

2H5-desmethylmebeverine acid (2H5-DMAC) was used as the internal standard (IS). The proposed

method was validated with linear ranges of 0.1–10 ng/mL; 1–100 ng/mL and 5–1000 ng/mL for MAL,

MAC and DMAC, respectively. Accuracy for all analytes (%RE), given as deviation between nominal

and measured concentration and assay variability (CV) ranged from −4.04% to 4.60% and from 0.31%

to 6.43% respectively both for within- and between-run. The overall recoveries for all metabolites

were above 85%. The proposed method was used successfully for analysis of real samples from a

pharmacokinetics study.

The impact of ion-pairing reagents on the selectivity and sensitivity in the analysis of modified

oligonucleotides in serum samples by liquid chromatography coupled with tandem mass

spectrometry

Sylwia Studzińska, Rafał Rola, Bogusław Buszewski

ABSTRACT

Present study highlights the usage of various ion-pairing agents and their impact on the process of

separation and ionization of oligonucleotides in the fortified human serum samples. What is more,

retention studies involved different stationary phases, including: octadecyl, phenyl, pentafluorophenyl

groups and ligands with embedded polar groups. It was proved that retention of oligonucleotides

strongly depends on the alkyl chain branching in the structure of ion pairing reagent. Furthermore ion-

pairing agents build of straight alkyl chain are more easily adsorbed on the stationary phase modified

with octadecyl groups, while branching of alkyl chain caused more effective adsorption of studied

compounds at phenyl groups compared to octadecyl ones. The lowest limit of quantification values

were obtained for 5 mM N,N-dimethylbutylamine, while the highest values are characteristic for

hexylamine. Moreover it was shown that a 2-fold increase of ion-pairing agent concentration results in

higher LOQ. The greatest sensitivity was obtained for 2.5 mM N,N-dimethylbutylamine/150 mM

hexafluoroisopropanol. On the other hand Hypersil GOLD aQ column was the most appropriate in

terms of time and separation effectiveness. The developed method was successfully used for the

determination of studied oligonucleotides and their metabolites in human serum samples. The

compounds were separated in just 3.5 min with high sensitivity (0.09–0.16 ng).

97

A novel LC–MS/MS method for the simultaneous quantification of topiramate and its main

metabolites in human plasma

Daniela Milosheska, Robert Rońkar

ABSTRACT

The aim of the present report was to develop and validate simple, sensitive and reliable LC–MS/MS

method for quantification of topiramate (TPM) and its main metabolites: 2,3-desisopropylidene TPM,

4,5-desisopropylidene TPM, 10-OH TPM and 9-OH TPM in human plasma samples. The most

abundant metabolite 2,3-desisopropylidene TPM was isolated from patients urine, characterized and

afterwards used as an authentic standard for method development and validation. Sample preparation

method employs 100 μL of plasma sample and liquid–liquid extraction with a mixture of ethyl acetate

and diethyl ether as extraction solvent. Chromatographic separation was achieved on a 1290 Infinity

UHPLC coupled to 6460 Triple Quad Mass Spectrometer operated in negative MRM mode using

Kinetex C18 column (50 × 2.1 mm, 2.6 μm) by gradient elution using water and methanol as a mobile

phase and stable isotope labeled TPM as internal standard. The method showed to be selective,

accurate, precise and linear over the concentration ranges of 0.10–20 μg/mL for TPM, 0.01–2.0 μg/mL

for 2,3-desisopropylidene TPM, and 0.001–0.200 μg/mL for 4,5-desisopropylidene TPM, 10-OH TPM

and 9-OH TPM. The described method is the first fully validated method capable of simultaneous

determination of TPM and its main metabolites in plasma over the selected analytical range. The

suitability of the method was successfully demonstrated by the quantification of all analytes in plasma

samples of patients with epilepsy and can be considered as reliable analytical tool for future

investigations of the TPM metabolism.

UPLC–MS/MS method for the simultaneous quantification of three new antiretroviral drugs,

dolutegravir, elvitegravir and rilpivirine, and other thirteen antiretroviral agents plus cobicistat

and ritonavir boosters in human plasma

Marco Simiele, Alessandra Ariaudo, Amedeo De Nicolò, Fabio Favata, Antonio D‘Avolio

ABSTRACT

Rilpivirine (RPV), dolutegravir (DTG) and elvitegravir (EVG) are the latest antiretroviral drugs

approved for treatment of HIV infection. Currently, poor information is currently available concerning

their pharmacokinetic and pharmacodynamic properties, thus making the use of therapeutic drug

monitoring for these drugs not useful. This lack of information is partially due to the absence of a

high-throughput method for their simultaneous quantification together with other antiretroviral drugs.

In this work, we describe the development and validation of a new UPLC–MS/MS method to quantify

these drugs, together with other fourteen antiretroviral agents, in human plasma. One hundred

microliters of plasma samples were added with internal standard (6,7-Dimethyl- 2,3-di(2-pyridyl)

quinoxaline), underwent a simple protein precipitation with methanol:acetonitrile (50:50 v/v) followed

by sample dilution with water. Chromatographic separation was performed on a Acquity® UPLC HSS

T3 column (150 mm x 2.1 mm I.D) with a particle size of 1.8 μm and compounds were detected with a

tandem mass detector, monitoring two ion transitions for each drugs. The mean recovery of RPV, DTG

and EVG were 101%, 87% and 112.3% respectively.

98

Concentrations of antibodies against β-amyloid 40/42 monomer and oligomers in Chinese

intravenous immunoglobulins

Shengliang Ye, Renyong Zeng, Peng Jiang, Mingxia Hou, Changqing Li

ABSTRACT

Intravenous immunoglobulin (IVIg) preparations are being investigated as a potential agent for

treatment or prevention of Alzheimer‘s disease (AD). Antibodies towards soluble β-amyloid (Aβ)

contained in IVIg were considered to be the major component contributing to the beneficial effect of

the preparations in pilot studies. This study compared the antibody concentrations against Aβ in

Octagam® IVIg (Octapharma) and 9 IVIg preparations from different Chinese manufacturers by

ELISA, using Aβ40 monomer, Aβ40 soluble oligomers, Aβ42 monomer and Aβ42 soluble oligomers

as the antigens. The results showed that each preparation contained different antibody levels against

the four Aβ forms. The median values of the four antibody concentrations in Chinese IVIg

preparations were 16.53, 8.47, 24.36 and 33.25 μg/mL, which were remarkably higher than that in

Octagam® IVIg (1.66, 2.07, 4.61 and 4.64 μg/mL). Moreover, the anti-Aβ42 oligomer antibody levels

in almost all IVIg preparations were higher than the anti-Aβ42 monomer antibody, and the

concentrations of anti-Aβ42 antibodies in most of the IVIg preparations were significantly higher than

that of anti-Aβ40 antibodies. These findings will contribute to an increased understanding of the

uniqueness of Chinese IVIg preparations, and could provide support for a trial of a Chinese IVIg

product in AD patients.

Simultaneous quantification of estrogens, their precursors and conjugated metabolites in human

breast cancer cells by LC–HRMS without derivatization

Stefan Poschner, Martin Zehl, Alexandra Maier-Salamon, Walter Jäger

ABSTRACT

Liquid chromatography–mass spectrometry (LC–MS) is the state of the art technique for quantification

of steroid hormones. Currently used methods are typically limited by the need of pre-column

derivatization to increase ionization efficiency; however, this causes hydrolysis of conjugated

metabolites. Our newly established LC–HRMS method is able to simultaneously quantify conjugated

and unconjugated steroids without prior derivatization using deuterated internal standards and solid-

phase extraction. This assay was validated according to ICH Q2(R1) guidelines for the analysis of the

10 main steroids of the estrogenic pathway, namely 4-androstene-3,17-dione, dehydroepiandrosterone

(DHEA), DHEA-3-sulfate, estrone, 17β-estradiol, estriol (16α-OH-17β-estradiol), estrone-3-sulfate,

17β-estradiol-3-(β-d-glucuronide), 17β-estradiol-3-sulfate and testosterone. Assay performance

characteristics were excellent with results for accuracy (98.8–101.2%), precision (mean: 2.05%, all

≤2.80%), stability over five freeze–thaw-cycles (95.7–100.4%) and SPE accuracy (96.9–102.0%), as

well as suitable lower and upper limits of quantification for cell culture experiments (LLOQ 0.005–2

ng/ml, ULOQ 3–2000 ng/ml). Furthermore, we demonstrated the functionality of our method for the

monitoring of steroid levels in the human breast cancer cell line MCF-7. This sensitive assay allows

for the first time detailed investigations on estrogen metabolomics in breast cancer cells and may also

apply to other estrogen-dependent tumor entities.

99

Quantification and clinical application of carboplatin in plasma ultrafiltrate

Kim Downing, Berit Packert Jensen, Sue Grant, Matthew Strother, Peter George

ABSTRACT

Carboplatin is a chemotherapy drug used in a variety of cancers with the primary toxicity being

exposure-dependant myelosuppression. We present the development and validation of a simple, robust

inductively coupled plasma mass spectrometry (ICP-MS) method to measure carboplatin in plasma

ultrafiltrate. Plasma ultrafiltrates samples were prepared using Amicon Ultra 30,000 da cut-off filters

and then diluted with ammonia EDTA before ICP-MS analysis. The assay was validated in the range

0.19–47.5 mg/L carboplatin in ultrafiltrate. The assay was linear (r2 > 0.9999), accurate (<6% bias,

12% bias at LLOQ) and precise (intra- and inter-day precision of <3% coefficient of variation). No

matrix effects were observed between plasma ultrafiltrate and aqueous platinum calibrators and

recovery was complete. The assay was applied to 10 clinical samples from patients receiving

carboplatin. Incurred sample reanalysis showed reproducible values over 3 analysis days (<6% CV).

As plasma stability prior to ultrafiltration has been a major concern in previous clinical studies this was

studied extensively at room temperature (22 °C) over 24 h. Carboplatin was found to be stable in both

spiked plasma (n = 3) and real patient samples (n = 10) at room temperature for up to 8 h before

ultrafiltration. This makes routine measurement of carboplatin concentrations in clinical settings

feasible.

An LC–MS/MS method for the determination of digitoxigenin in skin samples and its

application to skin permeation and metabolic stability studies

Xinchi Feng, Joel Turley, Zijian Xie, Sandrine V. Pierre, Jinsong Hao

ABSTRACT

An LC–MS/MS method was developed for the determination of digitoxigenin in mice skin samples.

Chromatographic separation was achieved on an Agilent Poroshell 120 EC-C18 column. Mass

spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an ESI

interface operating in a positive ionization mode. Quantification was performed using selective

reaction monitoring of the precursor-product ion transitions of m/z 375.5 → 339 for digitoxigenin and

m/z 391.5 → 337 for internal standard (IS). The calibration curves were linear over the concentration

range of 1.00–500 ng/mL. The intra- and inter-batch precision was no more than 10.6% of the

coefficient of variation and the accuracy was within ±8.1% of the actual values. This validated method

has been successfully applied to skin permeation and skin metabolic stability studies of digitoxigenin

in mice. The steady-state flux and lag time of digitoxigenin permeated across the full-thickness mice

skin were 1.86 ± 0.45 μg/cm2/h and 0.46 ± 0.18 h, respectively. The metabolism of digitoxigenin in

the skin was not detected in our study.

100

Stress-induced changes of neurosteroid profiles in rat brain and plasma under immobilized

condition

Myeong Hyeon Park, Shaheed Ur Rehman, In Sook Kim, Min Sun Choi, Hye Hyun Yoo

ABSTRACT

In this study, various neurosteroids in brain and plasma were simultaneously determined using liquid

chromatography-tandem mass spectrometry and their profile changes in a stress-induced rat were

investigated. The investigated neurosteroids are as follows: progesterone (P4), 5α-dihydroprogesterone

(5α-DHP), 5β-dihydroprogesterone, estrone, androstenedione (AE), cortisol, cortisone, corticosterone

(CORT), dehydroepiandrosterone (DHEA), pregnanolone (3α,5β-THP), allopregnanolone (ALLO),

11-deoxycorticosterone (DOC), 11-deoxycortisol, pregnenolone (PREG), and 5α/5β-

tetrahydrodeoxycorticosterone (5α/5β-THDOC). Brain and plasma samples were processed using

solid-phase extraction with methanol and acetic acid (99:1), and derivatized with a hydroxylamine

reagent. Separation was achieved within 13 min at a flow rate of 0.4 mL/min with a C18 column (3.0 ×

50 mm, 2.7 μm). The triple quadrupole mass spectrometer was operated in the positive electrospray

ionization mode. Using this method, the neurosteroid level variation was quantitated and investigated

in the brain and plasma upon immobilization stress in rats. As a result, AE, CORT, DOC, P4, 5α-DHP,

5α/5β-THDOC, DHEA, 3α,5β-THP, ALLO, and PREG levels were significantly altered in both the

brain and plasma samples when stress was induced.

Identification, characterization and in silico ADMET prediction of Roflumilast degradation

products

Mariana S. Pinheiro, Gil M. Viana, Bárbara de A. Abrahim Vieira, Alessandra Mendonça Teles de

Souza, Valéria P. de Sousa

ABSTRACT

The present study reports the degradation behavior of roflumilast (RFL), a new drug developed for the

treatment of chronic obstructive pulmonary disease. The degradation of RFL was tested under various

stress conditions as per the guidelines of the International Conference on Harmonization. The

degradation products (DPs) of RFL were identified, characterized and in silico predictions were made

of their pharmacokinetic properties, absorption, distribution, metabolism, excretion and toxicity

(ADMET). RFL was subjected to various stress conditions including photodegradation, alkaline and

acidic hydrolysis, oxidative and metallic degradation. After analysis by HPLC-DAD, the DPs were

isolated by preparative TLC and characterized by high resolution mass spectrometry (HRMS), 1H

NMR, 13C NMR and infrared (IR) spectroscopy. RFL tablets were prepared by the addition of solid

stressing substances such as excipients and storage in an accelerated stability chamber (40 °C; 75%

r.h.) for sixteen months. Resulting DPs from the tablets were analyzed by UFLC-QTOF. The most

drastic degradation conditions for RFL were 5 M NaOH(aq), 6 M HCl(aq), 7.5% v/v peracetic acid,

which resulted in the isolation of four DPs. However, milder degradation conditions (1 M NaOH(aq)

and photolysis) generated six DPs (DP-1, 2, 3, 5, 7 and 8), and are more similar to the actual

conditions the drug will be exposed. For tablets containing RFL exposed to an alkaline reagent, two

DPs were formed: DP-1 and DP-11. Where as RFL-containing tablets exposed to acid and oxidizing

agents, formed one products DP-11. Forced degradation of RFL led to the formation of eleven DPs,

seven of which have never been previously reported. RFL is stable under metallic stress and it is

relatively stable during photodegradation testing.

101

A rapid and robust UHPLC-DAD method for the quantification of amphotericin B in human

plasma

Sebastiano Barco, Alessia Zunino, Antonio D‘Avolio, Laura Barbagallo, Giuliana Cangemi

ABSTRACT

Amphotericin B is an antifungal drug widely used in Intensive Care Units. Therapeutic drug

monitoring (TDM) of amphotericin B is recommended for the assessment of toxicity surveillance and

treatment optimization. In this paper we described the development and validation of a new Ultra High

Performance Liquid Chromatography coupled to Diode Array Detection (UHPLC-DAD) method for

the quantification of Amphotericin B in 200 μL human plasma over a wide range of concentrations

(0.125–10 mg/L). The new method has been validated following international guidelines on

bioanalytical method validation and showed high selectivity, high accuracy and precision and high

process efficiency. The new UHPLC-DAD method that we describe is robust, rapid, cost effective and

suitable for application to the routine TDM analyses.

Quantitative analysis of mycosporine-like amino acids in marine algae by capillary

electrophoresis with diode-array detection

Anja Hartmann, Adele Murauer, Markus Ganzera

ABSTRACT

Marine species have evolved a variety of physical or chemical strategies to diminish damage from

elevated environmental ultraviolet radiation. Mycosporine-like amino acids, a group of widely

distributed small water soluble compounds, are biologically relevant because of their photo-protective

potential. In addition, presumed antioxidant and skin protective strategies raise the interest for possible

medicinal and cosmetic applications. In this study the first CE method for the quantification of

mycosporine-like amino acids in marine species is presented. A borate buffer system consisting of 30

mM sodium tetraborate in water at a pH-value of 10.3 enabled the baseline separation of five MAAs,

namely palythine, mycosporine-serinol, asterina-330, shinorine and porphyra-334, in 27 min.

Separation voltage, temperature and detection wavelength were 25 kV, 25 °C and 320 nm,

respectively. The optimized method was fully validated and applied for the quantitative determination

of MAAs in the marine macroalgae Palmaria palmata, Porphyra umbilicalis, and Porphyra sp., as well

as the lichen Lichina pygmaea.

102

LC–MS/MS assay for the simultaneous quantitation of the ATM inhibitor AZ31 and the ATR

inhibitor AZD6738 in mouse plasma

Brian F. Kiesel, Jeffrey C. Shogan, Madani Rachid, Robert A. Parise, Jan H. Beumer

ABSTRACT

The ATM kinase inhibitor AZ31 and ATR kinase inhibitor AZD6738 are in various phases of

preclinical and clinical evaluation for their ability to potentiate chemoradiation. To support the

preclinical evaluation of their pharmacokinetics, we developed and validated an LC–MS/MS assay for

the simultaneous quantification of AZ31 and AZD6738 in mouse plasma. A ―dilute and shoot‖ method

was used to precipitate proteins from a sample volume of 50 μL. Chromatographic separation was

achieved using a Phenomenex Polar-RP column and a gradient mobile phase consisting of methanol–

water with 0.1% formic acid. Detection was accomplished using a Waters Quattro Micro mass

spectrometer in positive ionization mode. The assay utilizing 50 μL sample was linear from 10 to 5000

ng/mL and determined to be both accurate (−8.2 to 8.6%) and precise (<5.4% CV) and achieved the

criteria for U.S. FDA guidance for bioanalytical method validation. Quantification was achieved in

mouse tissue homogenate using a separate 200 μL sample preparation. This LC–MS/MS assay will be

essential for determining the tissue distribution and pharmacokinetics in future mouse studies.

The atypical excretion profile of meldonium: Comparison of urinary detection windows after

single- and multiple-dose application in healthy volunteers

Christian Görgens, Sven Guddat, Christina Bosse, Hans Geyer, Mario Thevis

ABSTRACT

Following a one-year monitoring program providing unequivocal analytical evidence for a high

prevalence in international elite sports, meldonium has been included in the World Anti-Doping

Agency‘s (WADA) list of prohibited substances that came into effect on 1 January 2016. Despite of

the polar and hydrophilic nature of the molecule, an unusual long detection window was observed in

pilot elimination studies. Consequently, in the present study, urinary excretion profiles after single-

dose (5 volunteers, 1 × 500 mg) and multiple-dose oral application (5 volunteers; 2 × 500 mg/day for 6

days) were determined in order to facilitate the result management concerning meldonium findings in

doping controls. Particularly the option to differentiate between recent use and tapering concentrations

was studied. Urinary meldonium concentrations were determined using an analytical approach based

on hydrophilic interaction liquid chromatography and high resolution tandem mass spectrometry. The

study corroborates the hypothesis of a non-linear, dose-depended and biphasic excretion profile after

oral application of meldonium and demonstrates that urinary detection windows are of considerable

extent with up to 65 and 117 days (concentrations > LOQ of 10 ng/mL) following single- and

multiple-dose applications, respectively.

103

Simultaneous quantitation of abiraterone, enzalutamide, N-desmethyl enzalutamide, and

bicalutamide in human plasma by LC–MS/MS

Kyu-pyo Kim, Robert A. Parise, Julianne L. Holleran, Lionel D. Lewis, Jan H. Beumer

ABSTRACT

Inhibiting the androgen receptor (AR) pathway is an important clinical strategy in metastatic prostate

cancer. Novel agents including abiraterone acetate and enzalutamide have been shown to prolong life

in men with metastatic, castration-resistant prostate cancer (mCRPC). To evaluate the

pharmacokinetics of AR-targeted agents, we developed and validated an LC–MS/MS assay for the

quantitation of enzalutamide, N-desmethyl enzalutamide, abiraterone and bicalutamide in 0.05 mL

human plasma. After protein precipitation, chromatographic separation was achieved with a

Phenomenex Synergi Polar-RP column and a linear gradient of 0.1% formic acid in methanol and

water. Detection with an ABI 4000Q mass spectrometer utilized electrospray ionization in positive

multiple reaction monitoring mode. The assay was linear over the ranges of 1–1000 ng/mL for

abiraterone and bicalutamide and 100–30,000 ng/mL for N-desmethyl enzalutamide and enzalutamide

and proved to be accurate (92.8–107.7%) and precise (largest was 15.3% CV at LLOQ for

bicalutamide), and fulfilled FDA criteria for bioanalytical method validation. We demonstrated the

suitability of this assay in plasma from patients who were administered enzalutamide 160 mg,

abiraterone 1000 mg and bicalutamide 50 mg once a day as monotherapy or in combination. The LC–

MS/MS assay that has been developed will be an essential tool that further defines the pharmacology

of the combinations of androgen synthesis or AR-receptor targeted agents.

New enantioselective LC method development and validation for the assay of modafinil

Marianna Harvanová, Taťána Gondová

ABSTRACT

For the first time, a new, fast and sensitive chromatographic method for the separation and

determination of modafinil enantiomers was developed on chiral stationary phase with macrocyclic

glycopeptide teicoplanin in the polar organic mode. The effect of the mobile phase composition and

column temperature on the retention and enantioseparation were studied. The separation was

performed using a Chirobiotic T column (250 × 4.6 mm, 5 μm) with methanol and triethylamine

(100/0.05, v/v) as the mobile phase at a flow rate of 1.0 mL/min, and UV detection at 225 nm. The

total analysis time is less than 6 min, which is faster than the previous chiral HPLC methods (total run

time of 12 min). Calibration curves were linear (R2 > 0.999) over a concentration range 5–150 μg/mL

for each modafinil enantiomer. Limit of detection (LOD) and limit of quantification (LOQ) for (R)-

modafinil were 15 and 45 ng/mL and for (S)-modafinil were 20 and 60 ng/mL, respectively. The

recoveries were in the range of 100.5-102.3% with the relative standard deviations ranging from 0.4%

to 1.0% for both enantiomers. It was demonstrated that the developed method was selective, precise

and robust. The validated method was successfully applied for the separation and determination of

modafinil enantiomers in the real samples.

104

Identification of a novel low-level impurity in fungicide pyraclostrobin by high-performance

liquid chromatography/tandem mass spectrometry

Kaimin Xia, ShanShan Shen, Qun Gao, Wei Shang, Jun Wu

ABSTRACT

Pyraclostrobin is one kind of new type methoxy acrylate fungicides that has been widely used in

agriculture at present, with a lot of advantages including broad spectrum, high efficiency and high

selectivity. In this work, a novel low-level impurity in the pyraclostrobin at about 0.2% was separated

and characterized by high-performance liquid chromatography/tandem mass spectrometry (HPLC–

MS). Firstly, the impurity was speculated to possess the same skeleton structure as the main product

pyraclostrobin while the methyl group on the methyl ester was substituted to be CH2CH2Cl on the

basis of the on-line multi-stage mass spectrometric behaviors compared with that of pyraclostrobin.

Then the accurate molecular weight and element composition of target impurity was verified to be

C20H19Cl2N3O4 by high resolution mass spectrometry. Finally, the proposed structure was further

confirmed by the 1H NMR data.

Paeonifiorin sulfonate as a characteristic marker for specifically inspecting Chinese patent

medicine Liu-Wei-Di-Huang-Wan contained sulfur-fumigated Moutan Cortex

Xiu-Yang Li, Fang Long, Jin-Di Xu, Hong Shen, Song-Lin Li

ABSTRACT

Sulfur fumigation can induce chemical transformation of bioactive components, consequently the

alteration of bioactivities or even toxicities of medicinal herbs. Inspecting Chinese patent medicines

(CPM) contained sulfur-fumigated constituent herbs is crucial for ensuring the safety and efficacy of

CPM. Paeonifiorin sulfonate is a sulfur-fumigation induced compound of Moutan Cortex (MC), one of

the main constituent herbs of a commonly used CPM Liu-Wei-Di-Huang-Wan (LWDHW). Herein, we

investigated the approach of paeonifiorin sulfonate as a characteristic marker for specifically

inspecting LWDHW potentially contained sulfur-fumigated MC (SFMC). First, mimic LWDHW

samples contained SFMC (SFMC-LWDHW) and non-fumigated MC (NFMC-LWDHW) were

prepared respectively. Second, an LC–MS method was developed and validated to qualitatively and

quantitatively determine paeonifiorin sulfonate in the mimic LWDHW samples. Third, the established

method was applied to analyze the commercial LWDHW samples. The results showed that

paeoniflorin sulfonate could only be detectable in SFMC-LWDHW, but not in NFMC-LWDHW

samples. The CPM matrix could enhance the response of paeoniflorin sulfonate in mass spectrometry

analysis. In addition, the LOQ, linearity, precision, accuracy and stability were also demonstrated to be

acceptable for quantifying paeoniflorin sulfonate in LWDHW. Commercial samples analysis indicated

that paeoniflorin sulfonate were detectable in 9 of 10 commercial LWDHW samples, with the content

varied between 105.53 μg/g and 438.61 μg/g. All the results suggested that paeoniflorin sulfonate

could be used as a characteristic and reliable chemical marker for specifically inspecting commercial

LWDHW contained SFMC. This study also provides a new strategy for the quality control of other

CPMs potentially produced from sulfur-fumigated constituent herbs.

105

Simple on-line pretreatment of column-switching coupled with ion chromatography for the

determination of lactic acid in lobaplatin

Zhendong Zhao, Jianzhong Zhu, Yanli Xie

ABSTRACT

In this study, a simple and sensitive on-line pretreatment of column-switching technique coupled with

ion chromatography method was achieved for the determination of key impurity of lactic acid in

lobaplatin. The first dimension of reverse phase liquid chromatography was used to reduce organic

matrices, whereas the second dimension of ion chromatography was applied for the separation and

detection of lactic acid. The results exhibited good linearity (R = 0.9994) ranging from 0.01 mg L−1 to

50 mg L−1. The limit of detection was below 0.0015 mg L−1. Also, a spiking study was performed

with satisfactory recoveries between 96% and 106%.

Discovery of discriminatory quality control markers for Chinese herbal medicines and related

processed products by combination of chromatographic analysis and chemometrics methods:

Radix Scutellariae as a case study

Fei Wang, Bo Wang, Long Wang, Zi-Yue Xiong, Hui-Jun Li

ABSTRACT

The processing procedure of traditional Chinese herbal medicines (CHMs) plays an essential role in

clinical applications. However, little progress has been made on the quality control of crude and

processed products. The present work, taking Radix Scutellariae (RS), wine-processed RS and

carbonized RS as a typical case, developed a comprehensive strategy integrating chromatographic

analysis and chemometric methods for quality evaluation and discrimination of crude RS and its

processed products. Chemical fingerprints were established by high-performance liquid

chromatography coupled with photodiode array detector and quadrupole time-of-flight mass

spectrometry, and similarity analyses were calculated based on eleven common characteristic peaks.

Subsequently, four chemical markers were discovered by back propagation-artificial neural network

(BP-ANN) modeling. The selected markers were quantified by the ‗single standard to determine multi-

components‘ (SSDMC) method, and then the quantitative data were subjected to principal component

analysis (PCA) and partial least-squares discriminant analysis (PLS-DA). Furthermore, support vector

machine (SVM) was employed to predict the different processed products of RS. Finally, a hotmap

visualization was conducted for clarifying the distribution of major flavonoids among different drugs.

Collectively, the proposed strategy might be well-acceptable for quality control of CHMs and their

related processed products from the processing mechanism-based perspective.

106

New and cost effective cell-based assay for Dialyzed Leukocyte Extract (DLE)-induced Jurkat

cells proliferation under azathioprine treatment

F.M. Cardoso, M. Tomkova, D. Petrovajova, M. Bubanova, T. Hornakova

ABSTRACT

The human Dialyzed Leukocyte Extract (DLE) is a heterogeneous mix of oligopeptides of <10 kDa,

extracted from leukocytes of healthy donors. There is significant clinical evidence of improvement

using DLE during treatment of allergies, cancer, immunodeficiencies, and in mycotic and viral

infections. Nevertheless, the DLE exact nature and mechanism of action have been elusive for more

than 50 years. DLE biological activity testing is necessary in DLE production and quality control. Both

in vitro and in vivo assays exist: E-rosette test, induction of delayed type hypersensitivity in mice,

leukocyte migration and IFN-γ secretion. The animal-origin materials and in vivo assays convey a

considerable logistic, ethic and economic burden, meanwhile the available in vitro assays have been

reported with limited reproducibility and sometimes contradictory results. Here we are reporting a new

DLE biological activity cell-based assay. The A20 and Jurkat cell lines were treated with (+Aza) or

without (−Aza) azathioprine, DLE (+DLE) or both (+Aza/+DLE). After 72 h, the cell proliferation was

analyzed by the MTT or BrdU incorporation assays. In +Aza/+DLE treated cells, we observed a

significant higher proliferation, when compared with +Aza/-DLE. In the absence of Aza, cells did not

present any proliferation difference between −DLE or +DLE treatments. Both assays, MTT and BrdU

showed similar results, being the MTT test more cost effective and we select it for validation as DLE

biological assay using Jurkat cells only. We tested three different lyophilized DLE batches and we

found consistent results with acceptable assay reproducibility and linearity. The DLE capacity for

rescuing Jurkat cell proliferation during +Aza treatment was consistent using different liquid and

lyophilized DLE batches, presenting also consistent chromatographic profiles. Finally, DLE treatment

in Jurkat cells did not result into significant IL-2 of IFN-γ secretion, and known lymphocyte

proliferative drugs failed to rescue Jurkat cells viability in presence of +Aza, as +DLE treatment did in

our MTT assay.

Spectroscopy analysis and molecular dynamics studies on the binding of penicillin V and

sulbactam to beta-lactamase II from Bacillus cereus

Penghui Shi, Pan Qiao, Yeli Zhang, Shuaihua Li, Liujiao Bian

ABSTRACT

The molecular recognition and interaction of beta-lactamase II from Bacillus cereus (Bc II) with

penicillin V (PV) and sulbactam (Sul) especially conformational changes of Bc II in the binding

process were studied through spectroscopy analysis in combination with molecular dynamics (MD)

simulation. The results show that in the binding process, a new coordination bond is observed between

the Zn2 of Bc II and the carboxyl-O of PV or Sul by replacing His204. Electrostatic interaction

between Zn2 and the ligand provide main driving force for the binding affinity. Compared with apo Bc

II, there are mainly four loops showing significant conformational changes in ligand-bound Bc II. A

weak conformational transformation from β-sheets to random coils is observed in the loop2 of ligand-

bound Bc II. The conformational transformation may depend on the functional group and binding pose

of the ligand, giving the binding pocket greater flexibility and accordingly allowing for an induced fit

of the enzyme-ligand binding site around the newly introduced ligand. The change in the loop2 of

ligand-bound Bc II may lead to the opening of the binding pocket of Bc II.

107

Variations in gut microbiota and fecal metabolic phenotype associated with depression by 16S

rRNA gene sequencing and LC/MS-based metabolomics

Meng Yu, Hongmei Jia, Chao Zhou, Yong Yang, Zhongmei Zou

ABSTRACT

As a prevalent, life-threatening and highly recurrent psychiatric illness, depression is characterized by

a wide range of pathological changes; however, its etiology remains incompletely understood.

Accumulating evidence supports that gut microbiota affects not only gastrointestinal physiology but

also central nervous system (CNS) function and behavior through the microbiota-gut-brain axis. To

assess the impact of gut microbiota on fecal metabolic phenotype in depressive conditions, an

integrated approach of 16S rRNA gene sequencing combined with ultra high-performance liquid

chromatography-mass spectrometry (UHPLC–MS) based metabolomics was performed in chronic

variable stress (CVS)-induced depression rat model. Interestingly, depression led to significant gut

microbiota changes, at the phylum and genus levels in rats treated with CVS compared to controls. The

relative abundances of the bacterial genera Marvinbryantia, Corynebacterium, Psychrobacter,

Christensenella, Lactobacillus, Peptostreptococcaceae incertae sedis, Anaerovorax, Clostridiales

incertae sedis and Coprococcus were significantly decreased, whereas Candidatus Arthromitus and

Oscillibacter were markedly increased in model rats compared with normal controls. Meanwhile,

distinct changes in fecal metabolic phenotype of depressive rats were also found, including lower

levels of amino acids, and fatty acids, and higher amounts of bile acids, hypoxanthine and stercobilins.

Moreover, there were substantial associations of perturbed gut microbiota genera with the altered fecal

metabolites, especially compounds involved in the metabolism of tryptophan and bile acids. These

results showed that the gut microbiota was altered in association with fecal metabolism in depressive

conditions. These findings suggest that the 16S rRNA gene sequencing and LC–MS based

metabolomics approach can be further applied to assess pathogenesis of depression.

Chemical profiling of Fufang-Xialian-Capsule by UHPLC-Q-TOF-MS and its antioxidant

activity evaluated by in vitro method

Shizhe Li, Shu Liu, Zifeng Pi, Fengrui Song, Zhiqiang Liu

ABSTRACT

Fufang Xialian (FXL) capsule, a traditional Chinese medicine (TCM) formula, has been used for a

long time to treat Chronic Atrophic Gastritis, but the pharmacodynamic material basis and mechanism

of action are unclear. In this research, ferric-reducing antioxidant power (FRAP) method and DPPH

scavenging method were utilized to evaluate the antioxidant activity of FXL in vitro. An ultra high-

performance liquid chromatography coupled with Linear Ion Trap Mass Spectrometer (UHPLC-IT-

MS) method and an ultra high-performance liquid chromatography coupled with quadrupole time-of-

flight mass spectrometry (UHPLC-Q-TOF-MS) method were utilized to acquire the constituents

information of FXL. Mass spectra were acquired in both positive and negative ion modes. As the

results, FXL showed a moderate DPPH scavenging activity and ferric-reducing antioxidant power. A

total of 73 components were identified or tentatively characterized in FXL, which included alkaloids,

flavones, flavone glycosides, ginsenoside, lignins, triterpene saponins, flavanones, chalcones and

coumarin. The results in this research could provide a basis for further study in bioactivity and

metabolites.

108

Volume 139 May 2017

Development of an UPLC–MS/MS method for quantification of Avitinib (AC0010) and its five

metabolites in human cerebrospinal fluid: Application to a study of the blood-brain barrier

penetration rate of non-small cell lung cancer patients

Weicong Wang, Xin Zheng, Hanping Wang, Lu Wang, Pei Hu

ABSTRACT

Avitinib (AC0010) is a mutant-selective epidermal growth factor receptor tyrosine kinase inhibitors

(EGFR-TKI), designed to be a targeted therapeutic agent for non-small cell lung cancer (NSCLC)

patients harboring EGFR active and T790M resistant mutations. A rapid and sensitive ultra-

performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method was

developed and validated for the determination of Avitinib and its five metabolites (M1, M2, M4, M7,

MII-6) in human cerebrospinal fluid (CSF). The samples were purified by protein precipitation and

separated on a BEH C18 column (2.1 × 50 mm, 1.7 μm). Electrospray ionization (ESI) in positive ion

mode and multiple reaction monitoring (MRM) were used to monitor the ion transitions at m/z

488/257, 474/403, 504/487, 434/377, 490/405, 476/391. The results indicated that the method had

excellent sensitivity and specificity. The linear range covered from 0.05 to 50 ng/mL for Avitinib, M1,

M4, M7, and MII-6, and from 0.01 to 10 ng/mL for M2. Intra-day and inter-day precisions (in terms

of% RSD) were all <15% and the accuracies (in terms of% RE) were within the range of ±15%. The

lower limit of quantification (LLOQ), matrix effect, extraction recovery, stability and dilution integrity

were also validated and satisfied with the criteria of validation. Finally, the method was successfully

applied to a blood-brain barrier (BBB) penetration rate research of NSCLC patients after an oral

administration of Avitinib.

The sodium salt of the enantiomers of ricobendazole: Preparation, solubility and chiroptical

properties

Roberto Cirilli, Paolo Guglielmi, Francesca Romana Formica, Adriano Casulli, Simone Carradori

ABSTRACT

Albendazole (ABZ) is a sulfanyl-benzimidazole anthelmintic drug used worldwide in the treatment

and prevention of parasitic diseases in animals and humans. Following oral administration, ABZ is

rapidly oxidized into the pharmacologically active chiral sulfoxide metabolite known as ricobendazole

(RBZ). As its achiral precursor, RBZ shows very low intestinal absorption due to its poor solubility in

water (0.06 mg mL−1). To the best of our knowledge, there is no known example in human medicine

of a water-soluble salt form of racemic or enantiomerically pure RBZ. In the present study, we

describe in detail the preparation of the sodium (Na) salt of the enantiomers of RBZ through a two-step

process: i) the multi-milligram resolution of RBZ by HPLC on the amylose-based Chiralpak IG chiral

stationary phase under polar organic mode; ii) the salification of the isolated enantiomers of RBZ by

reaction with sodium hydroxide solution. The spectroscopic and chiroptical properties of the RBZ-Na

enantiomers were determined. Due to their unique solubility in 0.01 M phosphate buffer at

physiological pH (14.49 mg mL−1) and the high sample throughput obtained on semipreparative

separation of the non-salified form, it is potentially possible to develop new anthelmintic enantiopure

formulations with improved pharmacokinetic properties and lower toxicity.

109

Application of design space optimization strategy to the development of LC methods for

simultaneous analysis of 18 antiretroviral medicines and 4 major excipients used in various

pharmaceutical formulations

Védaste Habyalimana, Jérémie Kindenge Mbinze, Achille Loconon Yemoa, Christelle Waffo, Roland

Djang'eing'a Marini

ABSTRACT

As one of the world‘s most significant public health challenges in low- and middle-income countries,

HIV/AIDS deserves to be treated with appropriate medicines, however which are not spared from

counterfeiting. For that, we developed screening and specific HPLC methods that can analyze 18

antiretroviral medicines (ARV) and 4 major excipients. Design of experiments and design space

methodology were initially applied for 15 ARV and the 4 excipients with prediction thanks to Monte

Carlo simulations and focusing on rapidity and affordability thus using short column and low cost

organic solvent (methanol) in gradient mode with 10 mM buffer solutions of ammonium hydrogen

carbonate. Two other specific methods dedicated to ARV in liquid and in solid dosage formulations

were also predicted and optimized. We checked the ability of one method for the analysis of a fixed-

dose combination composed by emtricitabine/tenofovir/efavirenz in tablet formulations. Satisfying

validation results were obtained by applying the total error approach taking into account the accuracy

profile as decision tool. Then, the validated method was applied to test two samples coded A and B,

and claimed to contain the tested ARV. Assay results were satisfying only for sample B.

Modeling and optimizing inhibitory activities of Nelumbinis folium extract on xanthine oxidase

using response surface methodology

Mangmang Sang, Guangyan Du, Jia Hao, Linlin Wang, Lifeng Han

ABSTRACT

Xanthine oxidase (XOD), which could oxidize hypoxanthine to xanthine and then to uric acid, is a key

enzyme in the pathogenesis of hyperuricemia and also a well-known target for the drug development

to treat gout. In our study, the total alkaloids of Nelumbinis folium markedly inhibited XOD activity,

with IC50 value being 3.313 μg/mL. UHPLC-Q-TOF-MS and 3D docking analysis indicated that

roemerine was a potential active ingredient. A response surface methodology combined with central

composite design experiment was further developed and validated for the optimization of the reaction

conditions between the total alkaloids of Nelumbinis folium and XOD, which could be considered as a

meaningful research for the development of XOD inhibitor rapidly and sensitively.

110

Comparative preclinical evaluation of 68Ga-NODAGA and 68Ga-HBED-CC conjugated

procainamide in melanoma imaging

György Trencsényi, Noémi Dénes, Gábor Nagy, Adrienn Kis, István Kertész

ABSTRACT

Malignant melanoma is the most aggressive form of skin cancer. The early detection of primary

melanoma tumors and metastases using non-invasive PET imaging determines the outcome of this

disease. Previous studies have shown that benzamide derivatives (e.g. procainamide) conjugated with

PET radionuclides specifically bind to melanin pigment of melanoma tumors. 68Ga chelating agents

can have high influence on physiological properties of 68Ga labeled bioactive molecules, as was

experienced during the application of HBED-CC on PSMA ligand. The aim of this study was to assess

this concept in the case of the melanin specific procaindamide (PCA) and to compare the melanin

specificity of 68Ga-labeled PCA using HBED-CC and NODAGA chelators under in vitro and in vivo

conditions. Procainamide (PCA) was conjugated with HBED-CC and NODAGA chelators and was

labeled with Ga-68. The melanin specificity of 68Ga-HBED-CC-PCA and 68Ga-NODAGA-PCA was

investigated in vitro and in vivo using amelanotic (MELUR and A375) and melanin containing (B16-

F10) melanoma cell lines. Tumor-bearing mice were prepared by subcutaneous injection of B16-F10,

MELUR and A375 melanoma cells into C57BL/6 and SCID mice. 21 ± 2 days after tumor cell

inoculation and 90 min after intravenous injection of the 68Ga-labelledlabeled radiopharmacons whole

body PET/MRI scans were performed. 68Ga-NODAGA-PCA and 68Ga-HBED-CC-PCA were

produced with excellent radiochemical purity (98%). In vitro experiments demonstrated that after 30

and 90 min incubation time 68Ga-NODAGA-PCA uptake of B16-F10 cells was significantly (p ≤

0.01) higher than the 68Ga-HBED-CC-conjugated PCA accumulation in the same cell line.

Furthermore, significant difference (p ≤ 0.01 and 0.05) was found between the uptake of melanin

negative and positive cell lines using 68Ga-NODAGA-PCA and 68Ga-HBED-CC-PCA. In vivo

PET/MRI studies using tumor models revealed significantly (p ≤ 0.01) higher 68Ga-NODAGA-PCA

uptake (SUVmean: 0.46 ± 0.05, SUVmax: 1.96 ± 0.25, T/M ratio: 40.7 ± 4.23) in B16-F10 tumors in

contrast to 68Ga-HBED-CC-PCA where the SUVmean, SUVmax and T/M ratio were 0.13 ± 0.01,

0.56 ± 0.11 and 11.43 ± 1.24, respectively.

Development and validation of a HILIC-ELSD method for simultaneous analysis of non-

substituted and acetylated xylo-oligosaccharides

Jianghua Pu, Xia Zhao, Lin Xiao, Haitao Zhao

ABSTRACT

A new HILIC-ELSD method was developed for compositional analysis of both xylo-oligosaccharides

(XOS) with degree of polymerization (DP) from 2 to 8 and acetylated XOS with DP from 3 to 8. The

method was carried out on a zwitterionic HILIC column using ELSD as a detector. The influences of

mobile phase composition, column temperature and flow rate on the retention time and resolution of

XOS were investigated. An excellent separation result was achieved with a linear gradient elution of

75%−50% acetonitrile in 30 min, at a flow rate of 1 mL/min and the column temperature at 35 °C. In

addition, LC–ESI–MS was employed to determine the structural information of X7, X8 and acetylated

XOS. The proposed method was simple, reliable, and no derivatization procedure was needed. It is

suitable for compositional analysis and quality control of XOS.

111

Enantioselective recognition of radezolid by cyclodextrin modified capillary electrokinetic

chromatography and electronic circular dichroism

Katarzyna Michalska, Ewa Gruba, Wojciech Bocian, Judyta Cielecka-Piontek

ABSTRACT

A method for the enantioseparation of radezolid (RAD), an analogue of a truly new class of

antibacterial agents, oxazolidinones, was developed based on capillary electrokinetic chromatography

using a cyclodextrin as a chiral pseudophase (CD-cEKC). The mechanism of RAD separation, together

with its precursor, were investigated to directly define the relationship between the oxazolidinone

structure and the complexation process. During the development of the method, anionic single isomer

cyclodextrins were tested. They were ranked in order from hydrophilic to hydrophobic as follows:

heptakis-(2,3-dihydroxy-6-sulfo)-β-cyclodextrin (HS-β-CD), heptakis-(2,3-diacetyl-6-sulfo)-β-

cyclodextrin (HDAS-β-CD) and heptakis-(2,3-dimethyl-6-sulfo)-β-cyclodextrin (HDMS-β-CD).

Experiments were performed at pH values of 2.5, 6.6, 8.2 and 9.6. The cyclodextrins that had an acetyl

or methyl group at the C2 and C3 positions, referred to as HDAS-β-CD and HDMS-β-CD,

respectively, exhibited partial and baseline separation of enantiomers in a low pH buffer. However,

higher temperatures were required for HDAS-β-CD and acetonitrile addition was required for HDMS-

β-CD. During the experiments, different organic solvents, varying in their amphiprotic or aprotic

nature, were tested. The best results for the separation of enantiomers using the CD-cEKC method

were obtained with 40 mM HDMS-β-CD dissolved in a 50 mM phosphate buffer (pH 2.5) with the

addition of acetonitrile (65:35, v/v) at 27 °C, reversed polarity and a voltage equal to 28 kV. The

apparent binding constants for each enantiomer to HDAS-β-CD or HDMS-β-CD were calculated.

Finally, the stereochemistry of (S) and (R)-RAD and the behaviour of selected complex formations

were established using electronic circular dichroism.

Identification of leachables observed in the size exclusion chromatograms of a low concentration

product stored in prefilled syringes

Joseph J. Valente, Michael B. Peddicord, Frank A. Rinaldi, Kathleen A. Kelly, Mark S. Bolgar

ABSTRACT

Requisite leachables testing of pharmaceutical products is commonly conducted with pre-defined

analytical methods on a subset of materials intended to be representative of the marketed product.

Throughout product development, leachables may occasionally be detected in other methods not

specifically intended for monitoring such impurities. We have identified two leachables, ethyl 4-

ethoxybenzoate (E4E) and 2,6-di(t-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadien-1-one (BHT-OH) in

a low concentration product stored in prefilled syringes (PFS). The leachables were initially detected

by size exclusion chromatography (SEC) as late-eluting impurity peaks. Syringe component extraction

studies indicated that the impurities were related to the syringe stoppers. Positive identification of E4E

was accomplished by reversed phase liquid chromatography- tandem mass spectrometry (RPLC–

MS/MS). Positive identification of BHT-OH required RPLC-solid phase extraction-cryoflow NMR

(RPLC-SPE-NMR), as initial RPLC–MS/MS investigations were unsuccessful in elucidating the

structure. We focus specifically on the efforts required to identify the leachables, and the fortuitous

mixed mode separation mechanism and low concentration nature of the product, which were the main

factors contributing to the unlikely detection of the leachables by SEC.

112

Isolation and characterization of bioactive polyacetylenes Panax ginseng Meyer roots

Chia-Rou Yeo, Jin-Jie Yong, David G. Popovich

ABSTRACT

Panax ginseng has been studied for its chemo-preventive properties and pharmaceutical potential.

Polyacetylenic compounds isolated from Panax ginseng root typically comprised of non-polar C17

compound have been reported to exhibit bioactive properties. The objective of this project is to extract,

isolate, and characterize bioactive polyacetylenes from Panax ginseng root using various extraction

and separation methods Ginseng was extracted by reflux using methanol, ethanol, hexane, ethyl

acetate, methanolic ultrasonication. The extracts were partitioned with hexane to obtain water-soluble

portion and hexane-soluble portion. Hexane was subsequently removed under vacuum, and formed a

crude polyacetylenes extract (crude PA). Silica gel chromatography and semi-preparative HPLC were

utilized to prepare 5 fractions and the polyacetylenes were measure by HPLC and molecular weights

confirm my APCI–MS and MNR. The bioactive effect was measured by MTT viability assay using

murine 3T3-L1 cells. Extraction with methanol under reflux produced significantly larger amount of

polyacetylenes (p < 0.05). Liquid-liquid extraction and column chromatography were used to separate

polyacetylenic compounds into five different fractions. Major polyacetylenes, panaxynol and

panaxydol were found in fraction 1 and 2 respectively. Dose-response relationships were observed in

3T3-L1 cells and LC50 were 13.52 ± 3.05 μg/mL (fraction 1), 3.69 ± 1.09 μg/mL (fraction 2), 52.88 ±

11.16 μg/mL (fraction 3), 85.91 ± 27.37 μg/mL (fraction 4) and 135.52 ± 32.91 μg/mL (fraction 5).

Fraction 2 containing panaxydol was found to have exhibited the greatest anti-proliferative effects on

3T3-L1 preadipocytes.

Purity assessment of ginsenoside Rg1 using quantitative 1H nuclear magnetic resonance

Bao-Ming Huang, Sheng-Yuan Xiao, Ting-Bo Chen, Ying Xie, Hua Zhou

ABSTRACT

Ginseng herbs comprise a group of the most popular herbs, including Panax ginseng, P. notoginseng

and P. quinquefolius (Family Araliaceae), which are used as traditional Chinese medicine (TCM) and

are some of the best-selling natural products in the world. The accurate quantification of ginsenoside

Rg1 is one of the major aspects of its quality control. However, the purity of the commercial Rg1

chemical reference substance (CRS) is often measured with high-performance chromatography

coupled with an ultraviolet detector (HPLC–UV), which is a selective detector with unequal responses

to different compounds; thus, this detector introduces probable error to purity assessments. In the

present study, quantitative nuclear magnetic resonance (qNMR), due to its absolute quantification

ability, was applied to accurately assess the purity of Rg1 CRS. Phenylmethyl phthalate was used as

the internal standard (IS) to calibrate the purity of Rg1 CRS. The proton signal of Rg1 CRS in

methanol-d4 at 4.37 ppm was selected to avoid interfering signals, enabling accurate quantitative

analysis. The relaxation delay, number of scans, and NMR windowing were optimized for data

acquisition. For post-processing, the Lorentz/Gauss deconvolution method was employed to increase

the signal accuracy by separating the impurities and noise in the integrated region of the quantitative

proton. The method validation showed that the developed method has acceptable sensitivity, linearity,

precision, and accuracy. The purity of the commercial Rg1 CRS examined with the method developed

in this research was 90.34 ± 0.21%, which was obviously lower than that reported by the manufacturer

(>98.0%, HPLC–UV).

113

Identification of forced degradation products of tedizolid phosphate by liquid

chromatography/electrospray ionization tandem mass spectrometry

Yinping Lei, Bo Jin, Chen Ma, Tingting Zhang, Tong Li

ABSTRACT

In this study, stress degradation behavior of tedizolid phosphate was investigated and structural

characterization of its degradation products were performed with the use of the UPLC–MSn and LC-

HRMS. The toxicity prediction of the degradation products was performed with web-based prediction

system. Tedizolid phosphate was subjected to forced degradation under hydrolytic (acid, base and

neutral), oxidative, photolytic and thermal conditions in accordance with ICH guidelines Q1A(R2).

The drug was degraded significantly under acid, base and oxidative conditions, while it was relatively

stable to neutral, thermal and photolytic conditions. A total of four degradation products were formed.

All of them have been identified and characterized based on QTRAP MSn and accurate mass

measurements. To the best of our knowledge, three of these impurities were identified for the first time

and two of them further synthesized and characterized by NMR spectroscopy.

Evaluation of automated Wes system as an analytical and characterization tool to support

monoclonal antibody drug product development

Jinyu Wang, Anulfo Valdez, Yingchen Chen

ABSTRACT

Monitoring and evaluation of critical quality attributes (cQA) in monoclonal antibodies (mAb) are a

regulatory requirement in pharmaceutical industry. High molecular weight (HMW) species are of

critical importance due to the potential risk associated with immunogenicity. HMW species are

typically monitored by size exclusion chromatography (SEC). Although low molecular weight (LMW)

species are also detected by SEC, low-resolution separation of LMW limits its capability to monitor

mAb fragmentation. Recently, we have developed new methods for LMW characterization and

evaluation based on the Wes instrument from ProteinSimple. The capillary western blot is based upon

size-based separation in a capillary system, and detection by specific immunoprobing, following the

separation. The capability of this method for characterization of mAb fragments were demonstrated.

The characterization was achieved by probing two antibodies targeted to specific regions (Fc region or

Fab region) of IgG1 protein. The specificity of these two antibodies was evaluated against F (ab‘) 2

and Fc/2 fragments generated from Ides enzyme treated IgG1 protein. The results showed the selected

antibodies provide high specificity to F (ab‘) 2 and Fc/2 fragments. Fractions collected from SEC were

used to evaluate this method. The detected fragments from SEC fractions were identified based on

their estimated molecular weight and antibody detection. The result proved the capability of the

capillary western blot as a characterization method for IgG1 fragments. In addition, with the specific

detection to IgG1 and IgG4, the power of the capillary western blot to specifically characterize and

evaluate individual IgG fragmentations in an IgG1 and IgG4 mixture was also demonstrated. When

heat stressed samples were used, results showed method capability as stability indicating in IgG1 and

IgG4 mixture samples. The stressed mixture samples were also evaluated by the total protein assay in

which protein samples were biotinylated after separation and were labeled with HRP linked

streptavidin to provide chemiluminescence detection. The results indicated total protein assay can be a

useful complementary method to capillary western blot immunoassay.

114

Pharmacokinetics and tissue distribution of 4, 5-dimethoxycanthin-6-one and its major

metabolites in rats

Xiaolei Miao, Junjun Wang, Yong Chen

ABSTRACT

4,5-Dimethoxycanthin-6-one and 5-hydroxy-4-methoxycanthin-6-one are the active ingredients of P.

quassiodes. In the present work, a LC–MS/MS method was developed for the determination of 4,5-

dimethoxycanthin-6-one and its major metabolites 5-hydroxy-4-methoxycanthin-6-one (M1) and 4-

hydroxy-5-methoxycanthin-6-one (M2) in rat plasma and tissues, and applied to study their

pharmacokinetics and tissue distribution after intramuscular administration of 4,5-dimethoxycanthin-6-

one to rats. By protein precipitation with methanol for plasma samples and liquid–liquid extraction

with ethyl acetate for tissue samples, the analytes were separated on an ODS C18 column with a

mobile phase consisted of methanol and water (0.1% formic acid), and quantified by a MS detector in

positive multiple reaction monitoring (MRM) mode. MS transitions were m/z 281.0 → 167.1 for 4,5-

dimethoxycanthin-6-one, m/z 267.0 → 168.1 for M1 and M2, m/z 251.0 → 195.1 for 3-methylcanthin-

2,6-dione (IS). The pharmacokinetic results indicate that 4,5-dimethoxycanthin-6-one is absorbed

rapidly (Tmax = 5.4–6.4 min), distributed rapidly and widely in the order of liver > kidney ≈ lung ≈

large intestine ≈ small intestine, and eliminated quickly (t1/2z = 64.9–77.7 min) following the

intramuscular administration. Furthermore, M1 and M2 were detected only in rat plasma and liver at

the indicated times after the intramuscular administration.

Development and validation of an ELISA method for the quantification of nivolumab in plasma

from non-small-cell lung cancer patients

Alicja Puszkiel, Gaëlle Noé, Pascaline Boudou-Rouquette, Chloé Le- Cossec, Benoit Blanchet

ABSTRACT

Nivolumab, an anti PD-1 monoclonal antibody, has been approved for the treatment of previously

treated advanced or metastatic non-small-cell lung cancer (NSCLC). The aim of this study was to

develop and validate an ELISA method for the quantification of nivolumab in plasma from patients

with NSCLC in order to perform future pharmacokinetic/pharmacodynamic (PK/PD) studies. A home-

made ELISA was developed and validated according to the general recommendations for the

immunoassays. Then, the ELISA method was applied to quantify plasma trough levels (Cmin) of

nivolumab (3 mg/kg every two weeks) in 27 NSCLC patients at days 14, 28 and 42 after start of

treatment. Blood samples were collected just before the infusion on days 0 (baseline), 14, 28 and 42

after start of treatment. The dynamic calibration range for nivolumab assay was 5–100 μg/mL. Within-

and between-day imprecision for quality controls (5, 20 and 75 μg/mL) were less than 5 and 12%,

respectively. The mean (± standard deviation) nivolumab Cmin was 17.3 ± 4.8 μg/mL (coefficient of

variation, CV = 27.8%), 25.0 ± 9.7 μg/mL (CV = 38.8%) and 33.0 ± 12.9 μg/mL (CV = 39.1%) on

days 14, 28 and 42, respectively. IgG (p = 0.002) and ALT (p = 0.041) were independently associated

with plasma nivolumab Cmin at day 42.

115

Bioanalysis of Pseudomonas aeruginosa alkyl quinolone signalling molecules in infected mouse

tissue using LC–MS/MS; and its application to a pharmacodynamic evaluation of MvfR

inhibition

Paul Turnpenny, Anthony Padfield, Patrick Barton, Joanne Teague, Aileen Rubio

ABSTRACT

Alkyl quinolone molecules 2-heptyl-4-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone

(PQS) are important quorum sensing signals, which play a mediatory role in the pathogenesis of acute

and chronic Pseudomonas aeruginosa infection. A targeted approach inhibiting the bacterial ‗multiple

virulence factor regulon‘ (MvfR) protein complex, offers the possibility to block the synthesis of

MvfR-dependant signal molecules. Here, a high throughput bioanalytical method was developed using

LC–MS/MS detection for the selective determination of HHQ and PQS in mouse tissue homogenate,

over a sensitive range of 1–5000 and 10–5000 pg/mL, respectively. Chromatographic peak distortion

of the iron chelator PQS was overcome with the applied use of a bidentate chelator mobile phase

additive 2-Picolinic acid at 0.2 mM concentration, giving an improved separation and response for the

analyte, whilst maintaining overall MS system robustness. Following thigh infection with P.

aeruginosa strain 2-PA14 in mice, the concentration and time course of HHQ and PQS (4-hydroxy-2-

alkyl-quinolone (HAQ) biomarkers) residing in the biophase were evaluated, and exhibited a low level

combined with a substantial inter-individual variability. Quantifiable levels could be obtained from

approximately 15 h post infection, to the study termination at 21–22 h. A dose dependant reduction in

HAQ tissue concentrations at selected time points were obtained following MvfR inhibitor

administration versus drug vehicle (p < 0.01, Kruskal–Wallis—one way ANOVA) and meta -analyses

of several studies enabled an inhibitory concentration (IC50) of 80 nM free drug to be determined.

Metabolites identification of berberine in rats using ultra-high performance liquid

chromatography/quadrupole time-of-flight mass spectrometry

Kun Wang, Liwei Chai, Xinchi Feng, Zhongbo Liu, Feng Qiu

ABSTRACT

Berberine (BBR), the principle component for many medicinal plants such as Coptis chinensis Franch,

Phellodendron chinense Schneid, and Mahonia bealei (Fort.) Carr, possesses diverse pharmacological

activities, including anti-bacterial, anti-inflammatory, antitumor, hypolipidemic and antidiabetic

activities. In this study, a rapid and reliable method using a five-step strategy based on the ultra-

performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

(UPLC/Q-TOF-MS), and metabolynx™ software with mass defect filter (MDF) technique was

developed to investigate the metabolism of BBR. Plasma, bile, urine and feces samples were collected

from rats after oral administration of BBR with a dose of 100 mg/kg/day for three consecutive days

and analyzed to characterize the metabolic profile of BBR. By comparing the molecular weights and

MS fragmentations of the metabolites with those of the parent drug and reference standards, a total of

97 metabolites were identified, including 68 metabolites in urine, 45 metabolites in plasma, 44

metabolites in bile and 41 metabolites in feces. Demethylation, demethylenation, reduction,

hydroxylation, and subsequent glucuronidation, sulfation and methylation were the major metabolic

pathways of BBR in vivo.

116

Quantitative chiral and achiral determination of ketamine and its metabolites by LC–MS/MS in

human serum, urine and fecal samples

Mahmoud Hasan, Robert Hofstetter, Georg M. Fassauer, Andreas Link, Stefan Oswald

ABSTRACT

Ketamine (KET) is a widely used anesthetic drug which is metabolized by CYP450 enzymes to

norketamine (n-KET), dehydronorketamine (DHNK), hydroxynorketamine (HNK) and

hydroxyketamine (HK). Ketamine is a chiral compound and S-ketamine is known to be the more

potent enantiomer. Here, we present the development and validation of three LC–MS/MS assays; the

first for the quantification of racemic KET, n-KET and DHNK in human serum, urine and feces; the

second for the separation and quantification of the S- and R-enantiomers of KET, n-KET and DHNK,

and the third for separation and quantification of 2S,6S-hydroxynorketamine (2S,6S-HNK) and 2R,6R-

hydroxynorketamine (2R,6R-HNK) in serum and urine with the ability to separate and detect 10

additional hydroxylated norketamine metabolites of racemic ketamine. Sample preparation was done

by liquid-liquid extraction using methyl tert-butyl ether. For achiral determination of KET and its

metabolites, an isocratic elution with ammonium acetate (pH 3.8; 5 mM) and acetonitrile on a C18

column was performed. For the separation of S- and R-enantiomers of KET, n-KET and DHNK, a

gradient elution was applied using a mobile phase of ammonium acetate (pH 7.5; 10 mM) and

isopropanol on the CHIRAL-AGP® column. The enantioselective separation of the HNK metabolites

was done on the chiral column Lux®-Amylose-2 with a gradient method using ammonium acetate (pH

9; 5 mM) and a mixture of isopropanol and acetonitrile (4:1). The mass spectrometric detection

monitored for each analyte 2–3 mass/charge transitions. D4-ketamine and D4-n-KET were used as

internal standards. The assays were successfully validated according to current bioanalytical guidelines

and applied to a pilot study in one healthy volunteer. Compared to previously published methods, our

assays have superior analytical features such as a lower amount of required matrix, faster sample

preparation, shorter analytical run time and higher sensitivity (LLOQ up to 0.1 ng/ml).

Irinotecan binds to the internal cavity of beta-lactoglobulin: A multi-spectroscopic and

computational investigation

N. Bijari, S. Ghobadi, K. Derakhshandeh

ABSTRACT

The binding of irinotecan, a potent anti cancer drug, to bovine beta-lactoglobulin (β-LG) was

investigated by various spectroscopic techniques including fluorimetry, circular dichroism (CD), UV–

vis, and Fourier transform infrared (FT-IR), in 10 mM phosphate buffer, pH 7.75, in combination with

a molecular docking study. Analysis of the fluorescence quenching data showed that combined static

and dynamic quenching occurs, with the predominant contribution of the static mode. Molecular

docking results were in full agreement with the results obtained from thermodynamic analysis of the

fluorescence data indicating the existence of one binding site for irinotecan in β-LG structure and

revealed the hydrophobic nature of the interaction between irinotecan and the protein. The binding

distance between β-LG and irinotecan, r, was estimated to be 5.74 nm based on the Förster‘s theory of

non-radiative energy transfer. The obtained results of near-UV CD and FT-IR experiments suggested

the occurrence of partial compactness of the protein structure upon irinotecan binding. Based on the

experimental data and the possible binding mode revealed by molecular docking study, we concluded

that irinotecan binds to the hydrophobic calyx of β-LG with induction of some alterations in the

secondary and tertiary structure of the protein.

117

Determination of drugs in plasma samples by disposable pipette extraction with C18-BSA phase

and liquid chromatography–tandem mass spectrometry

Mônia Ap. Lemos Pinto, Israel D. de Souza, Maria Eugênia C. Queiroz

ABSTRACT

This work describes restricted access material (RAM) constituted of porous octadecylsilane particles

with the outer surface covered with bovine serum albumin (C18-BSA) as a stationary phase to extract

drugs from plasma samples by disposable pipette extraction (DPX) for further analysis by liquid

chromatography–tandem mass spectrometry (LC–MS/MS). The C18-BSA phase simultaneously

excluded macromolecules by chemical diffusion barrier (BSA network) and enrichment of the interior

phase (C18) with drug traces by sorption. The hydrophilic barrier of the C18-BSA allows small

molecules (drugs) to permeate through the hydrophobic part (C18), while at the same time it excludes

the macromolecules by chemical diffusion barrier (BSA network). Optimization of the DPX variables

(sorption equilibration time, exclusion of endogenous compounds, and elution step) improved the

sensitivity and selectivity of the method, which presented a linear range from the lower limit of

quantification (0.5–20.0 ng mL−1) to the upper limit of quantification (32.5–10,500 ng mL−1), inter-

and intra-assay precision with coefficients of variation (CV) lower than 15%, and relative standard

error (RSE) of the accuracy ranging from −12% to 11%. The developed method was successfully used

to determine five antipsychotics (olanzapine, quetiapine, clozapine, haloperidol, and chlorpromazine)

in combination with seven antidepressants (mirtazapine, paroxetine, citalopram, sertraline, imipramine,

clomipramine, and fluoxetine), two anticonvulsants (carbamazepine and lamotrigine), and two

anxiolytics (diazepam and clonazepam) in plasma samples from schizophrenic patients for therapeutic

drug monitoring.

Differentiation of protein secondary structure in clear and opaque human lenses: AFM – IR

studies

C. Paluszkiewicz, N. Piergies, P. Chaniecki, M. Rękas, W.M. Kwiatek

ABSTRACT

Here, we present the first approach to human lenses investigations with and without cataract

development changes in nanoscale resolution using AFM – IR spectroscopy. We proved that the

application of this technique allowed us to better understand of structural changes connected with

advancing disease process in studied lenses. The obtained results show the impact of the disease

development on the secondary structure of proteins in these human tissues. The domination of the β–

turn protein secondary structure is observed in the clear (non-affected by cataract) lens. While, in the

case of the opaque (cataractous) samples the different degree of the degradation due to development of

cataract, was recognized. Briefly, this process is associated with the protein secondary changes from

β–turn/β–sheet parallel for less altered part of the lens to stable anti-parallel β–sheet for the more

degraded part. Interestingly, the AFM – IR technique provided estimation of the protein secondary

structure without the need for using deconvolution procedure.

118

Introduction of a carbon paste electrode based on nickel carbide for investigation of interaction

between warfarin and vitamin K1

Maryam Torkashvand, Mohammad Bagher Gholivand, Avat (Arman) Taherpour, Arash Boochani,

Arsalan Akhtar

ABSTRACT

In this paper a novel electrochemical sensor based on nickel carbide (Ni3C) nanoparticles as a new

modifier was constructed. Ni3C nanoparticle was synthesized and characterized by scanning electron

microscopy, X-ray diffraction and first-principles study. Electrochemical impedance spectroscopy

(EIS) and cyclic voltammetry (CV) studies confirmed the electrode modification. Afterwards, the new

electrode for the first time was used for interaction study between vitamin K1 and warfarin as an

anticoagulant drug by differential pulse voltammetry. The adduct formation between the drug and

vitamin K1 was improved by decreasing in anodic peak current of warfarin in the presence of different

amounts of vitamin K1. The binding constant between warfarin and vitamin K1 was obtained by

voltammetric and UV–vis and fluorescence spectroscopic methods. The molecular modeling method

was also performed to explore the structural features and binding mechanism of warfarin to vitamin

K1. The different aspects of modeling of vitamin K1 and warfarin and their adduct structures

confirmed the adduct formation by hydrogen bonding.

An integrated strategy using UPLC–QTOF-MSE and UPLC–QTOF-MRM (enhanced target)

for pharmacokinetics study of wine processed Schisandra Chinensis fructus in rats

Kuangyi Liu, Yonggui Song, Yali Liu, Mi Peng, Dan Su

ABSTRACT

Currently the pharmacokinetic (PK) research of herbal medicines is still limited and facing critical

technical challenges on quantitative analysis of multi-components from biological matrices which

often accompanied by lacking of authentic standards and low concentration. This present work

contributes to the development of an integrated strategy for extensive pharmacokinetics assessments,

and a selective and sensitive method independent of authentic standards for multi-components analysis

based on the use of ultra-performance liquid chromatography/quadrupole-time-of-flight/MSE (UPLC-

TOF-MSE) and UPLC-TOF-MRM (rnhanced target). Initially, phytochemicals were identified by

UPLC-TOF-MSE analysis, subsequently the identified components were matched with authentic

standards and pre-classified, and UPLC–QTOF-MRM method optimized and developed. To guarantee

reliable results, three rules are necessary: (1) detection with a mass error of less than 5 ppm; (2) same

class chemical compositions with structural high similarity between analytes with and without

authentic reference substance; (3) a matching retention time between TOF-MRM mode and TOF-MSE

within 0.2 min. The developed and validated method was applied for the simultaneous determination

of 12 lignans in rat plasma after administered with wine processed Schisandra Chinensis fructus

(WPSCF) extract. Such an approach was found capable of providing extensive pharmacokinetic

profiles of multi-components absorbed into blood after oral administrated with WPSCF extract. The

results also indicated that significant difference in pharmacokinetics parameters of

dibenzocyclooctadiene lignans was observed between schizandrin and gomisin compounds. For

lignans, the absorption via gastrointestinal tract were all rapid and maintained relatively long retention

time, especially for schisantherin A and schisantherin B with higher plasma exposure.

119

Simultaneous determination of glipizide and its four hydroxylated metabolites in human urine

using LC–MS/MS and its application in urinary phenotype study

Bo Tan, Aidong Yang, Weian Yuan, Yue Li, Furong Qiu

ABSTRACT

Cytochrome P450 (CYP) 2C9 and CYP2C19 genetic mutant could influence the plasma concentration

of glipizide in human subjects, which refers to glipizide safety and adverse effects in clinic practice. A

further study to investigate the relationship of the concentrations between glipizide and its metabolites

in human with different CYP mutants was valuable. We firstly develop a validated liquid

chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous quantification of

glipizide and its hydroxylated metabolites in human urine. After simple protein precipitation with

methanol including 4′-OH-tolbutamide and gliclazide (both are internal standards), the analytes were

chromatographed on a reversed-phased column with a mobile phase of 0.1% formic acid in acetonitrile

and 0.1% formic acid in water by a gradient elution. The ion transitions of the precursor to the product

ion were principally protonated ions [M + H]+ at m/z 446.4 → m/z 321.1 for glipizide, m/z 462.2 →

m/z 321.1 for the four hydroxylated forms of glipizide, m/z 287.2 → m/z 188.0 for 4′-OH-tolbutamide,

and m/z 324.1 → m/z 127.1 for gliclazide. The method was linear over a concentration range of 0.02–

20.0 ng/mL. The intraday and inter-day variances were less than 9.9%, and accuracy was within

±6.8%. The method was successfully applied to the urinary phenotyping study in volunteers after a

single oral administration of 5-mg glipizide tablet, and two new hydroxycyclohexyl metabolites of

glipizide (OH-gp), 4-cis-OH-gp and 3-trans-OH-gp, were found in this study.

Plasma pharmacokinetics and bioavailability of verticillin A following different routes of

administration in mice using liquid chromatography tandem mass spectrometry

Jiang Wang, Xiaohua Zhu, Shamala Kolli, Hongyan Wang, Mitch A. Phelps

ABSTRACT

Verticillin A is a natural product isolated from fungal cultures and has displayed potent antibiotic,

antiviral, nematocidal, and anticancer properties in vitro. While in vivo studies have been limited due

to sparse supply, the in vivo efficacy data that does exist demonstrates potent anti-tumor activity in

murine cancer models. The current study aims to investigate the pharmacokinetics and bioavailability

of verticillin A in mice to provide guidance for further efficacy assessment in mouse models. A

sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and

validated for the quantification of verticillin A in mouse plasma. Sample preparation was

accomplished through protein precipitation, and chromatographic separation was achieved on an

Agilent Zorbax Extend C18 column with a security guard cartridge C8 using a binary gradient with

mobile phase A (water/0.1% formic acid) and B (ACN/0.1% formic acid) at a flow rate of 400 μl/min.

Elution of verticillin A and internal standard, hesperetin, occurred at 4.87 and 2.06 min, respectively.

The total chromatographic run time was 8 min, and the assay was linear in the concentration range of

1–1000 nM. The within- and between day precisions and accuracy were in the range of 2.58–8.71 and

90–105%, respectively. The assay was applied to determine plasma drug concentration in a mouse

pharmacokinetic study. It was found that intraperitoneal dosing of 3 mg/kg resulted in high systemic

exposure and achieved Cmax of 110 nM with plasma concentrations sustained above 10 nM for the

24-h duration of the study.

120

Robust quantitation of basic-protein higher-order aggregates using size-exclusion

chromatography

David Gervais, Andrew Downer, Darryl King, Patrick Kanda, Stuart Smith

ABSTRACT

Detection of higher-order aggregates (HOA) using size-exclusion chromatography (SEC) was found to

be variable for a basic protein, using exposed-silanol or diol-silica-based SEC columns. Preparations

of the tetrameric biopharmaceutical enzyme Erwinia chrysanthemi l-asparaginase (ErA), which has an

isoelectric point of 8.6, were analysed using a diol-silica SEC column. Although the proportions of

ErA main peak and octamer species were unaffected, HOA recovery and detection were extremely

variable and had poor agreement with an orthogonal measurement technique, analytical

ultracentrifugation (AUC). The observation that only HOA was selectively affected by non-specific

silanol interactions was unexpected, so alternatives were sought. Coated-silica SEC columns improved

the resolution and reproducibility of HOA detection for this alkaline-pI protein, and improved the

agreement of HOA with the AUC method. Basic proteins, such as ErA, should be thoroughly

evaluated in SEC method development, to ensure that resolution of larger aggregate species is not

compromised.

A metabolomic approach shows sphingosine 1-phosphate and lysophospholipids as mediators of

the therapeutic effect of liver growth factor in emphysema

A. Navarrete, F.J. Rupérez, T.O. Mendes, S. Pérez-Rial, A. García

ABSTRACT

Tobacco smoke exposure is the principal cause of lung tissue destruction, which in turn results in

emphysema that leads into shortness of breath. Liver growth factor (LGF, a cell and tissue

regenerating factor with therapeutic activity in several organs) has antifibrotic and antioxidant

properties that could be useful to promote lung tissue regenerating capacity in damaged lungs. The

current study has examined differences in metabolite profiles (fingerprints) of plasma from mice

(strain C57BL/6J, susceptible to develop emphysema) exposed to tobacco smoke during six months.

One group of mice received a treatment with Liver Growth Factor (LGF) after emphysema was

established, whereas the other group did not receive the treatment. Age and sex-matched mice not

exposed to smoke were also maintained with or without treatment as controls. Metabolic fingerprints

(untargeted analysis) of plasma after protein precipitation were obtained by LC-QTOF-MS. The

signals were processed and a large number of possible metabolites were found (23944). Multivariate

data analysis provided models that highlighted the differences between control and smoke exposed

mice in both conditions. Accurate masses of features (possible compounds) representing significant

differences were searched using online public databases. Lipid mediators, related to intracellular

signaling in inflammation, were found among the metabolites putatively identified as markers of the

different conditions and among them, sphingosine, sphingosine 1-phosphate and lysophospholipids

point at the relevance of such metabolites in the regulation of the processes related to tissue

regeneration mediated by LGF. These results also suggest that metabolomic fingerprinting could

potentially guide the characterization of relevant metabolites leading the regeneration of lungs in

emphysema disease.

121

Hepatic and renal metabolism of genistein: An individual-based model to predict

glucuronidation behavior of genistein in different organs

Junjin Liu, Xiaoming Yu, Shilong Zhong, Weichao Han, Lan Tang

ABSTRACT

The present study aims to develop an individual-based model to predict the glucuronidation

characteristics of genistein in 45 human livers. The model was validated in 18 human kidneys. Ten

UDP-glucuronosyltransferases (UGTs) were expressed in liver abundantly. Among them, UGT1A1,

UGT1A3, UGT1A6, UGT1A9 and UGT2B7 contributed 95.6% to genistein glucuronidation in human

liver, with significant correlation between gene expression of the five UGTs and the glucuronidation of

genistein. The genistein glucuronidation differences between individuals were varied enormously;

meanwhile the expression variations can explain 24.7% total metabolic variation in livers. In kidney,

UGT1A6, UGT1A9 and UGT2B7 were amply expressed. Their gene expressions and glucuronidation

rates of genistein had high correlations. Expression variations of UGT1A9/2B7 can explain up to

40.9% of the total variation of genistein glucuronidation in kidney. The model was therefore

established based on UGT expression between individuals taking into account an important assessment

which is the ratio of metabolic rate of each recombinant UGT isoform. Excellent linear correlations

between predicted and observed glucuronidation rate revealed that the model could predict metabolic

activity of genistein using quite a small size of tissue. In conclusion, the individual-based model can be

employed for predicting individual glucuronidation behavior of genistein in different organs.

Determination of free polysaccharide in Vi glycoconjugate vaccine against typhoid fever

C. Giannelli, E. Cappelletti, R. Di Benedetto, F. Pippi, F. Micoli

ABSTRACT

Glycoconjugate vaccines based on the Vi capsular polysaccharide directed against Salmonella enterica

serovar Typhi are licensed or in development against typhoid fever, an important cause of morbidity

and mortality in developing countries. Quantification of free polysaccharide in conjugate vaccines is

an important quality control for release, to monitor vaccine stability and to ensure appropriate immune

response. However, we found that existing separation methods based on size are not appropriate as

free Vi non-specifically binds to unconjugated and conjugated protein. We developed a method based

on free Vi separation by Capto Adhere resin and quantification by HPAEC-PAD. The method has been

tested for conjugates of Vi derived from Citrobacter freundii with different carrier proteins such as

CRM197, Tetanus Toxoid and Diphtheria Toxoid.

122

HPLC–MS/MS method for quantification of paclitaxel from keratin containing samples

Emily A. Turner, Alexandra C. Stenson, Saami K. Yazdani

ABSTRACT

Local drug delivery of paclitaxel is becoming ever more prevalent. As complex drug/excipient

combinations are being developed and tested, new high performance liquid chromatography-mass

spectrometry (HPLC–MS) techniques capable of quantifying paclitaxel from such formulations are

needed. Here a method for quantifying paclitaxel from aqueous, protein and oil containing samples

was developed and validated. Keratin, derived from human hair, is the protein component/paclitaxel

excipient in the development and validation of said method. The novelty of this method is described by

its ability to overcome water solubility issues and address clean-up of residual solvents in clinical

grade paclitaxel injection composition. The method evaluates tert-butyl methyl ether and ethanol as

extraction solvents with an extraction efficiency of 31.9 ± 2.3% and 86.4 ± 4.5% respectively. Upon

evaporation and rehydration, samples were evaluated by HPLC–MS and a method was developed for

paclitaxel quantification. The method developed had an inter-day precision of 9.1% relative standard

deviation and an intra-day precision of 4.3% relative standard deviation normalized to a docetaxel

internal standard. The described method is applicable to any aqueous paclitaxel sample containing

protein and/or oils.

Development of an enzyme-linked immunosorbent assay for detection of CDCP1 shed from the

cell surface and present in colorectal cancer serum specimens

Yang Chen, Brittney S. Harrington, Kevin C.N. Lau, Lez J. Burke, John D. Hooper

ABSTRACT

CUB domain containing protein 1 (CDCP1) is a transmembrane protein involved in progression of

several cancers. When located on the plasma membrane, full-length 135 kDa CDCP1 can undergo

proteolysis mediated by serine proteases that cleave after two adjacent amino acids (arginine 368 and

lysine 369). This releases from the cell surface two 65 kDa fragments, collectively termed ShE-

CDCP1, that differ by one carboxyl terminal residue. To evaluate the function of CDCP1 and its

potential utility as a cancer biomarker, in this study we developed an enzyme-linked immunosorbent

assay (ELISA) to reliably and easily measure the concentration of ShE-CDCP1 in biological samples.

Using a reference standard we demonstrate that the developed ELISA has a working range of 0.68–

26.5 ng/ml, and the limit of detection is 0.25 ng/ml. It displays high intra-assay (repeatability) and high

inter-assay (reproducibility) precision with all coefficients of variation ≤7%. The ELISA also displays

high accuracy detecting ShE-CDCP1 levels at ≥94.8% of actual concentration using quality control

samples. We employed the ELISA to measure the concentration of ShE-CDCP1 in human serum

samples with our results suggesting that levels are significantly higher in serum of colorectal cancer

patients compared with serum from individuals with benign conditions (p < 0.05). Our data also

suggest that colorectal cancer patients with stage II–IV disease have at least 50% higher serum levels

of ShE-CDCP1 compared with stage I cases (p < 0.05). We conclude that the developed ELISA is a

suitable method to quantify ShE-CDCP1 concentration in human serum.

123

Volume 140 June 2017

Capillary blood collected on volumetric absorptive microsampling (VAMS) device for

monitoring hydroxychloroquine in rheumatoid arthritis patients

Ying Qu, Kelley Brady, Robert Apilado, Tyler O‘Malley, Thierry Dervieux

ABSTRACT

A novel technique for collection of capillary blood, termed volumetric absorptive microsampling

(VAMS), has been recently cleared by the FDA for collection of human blood. VAMS absorbs a fixed

volume of blood (10 μl) and overcomes area bias and homogeneity issues associated with dried blood

spot (DBS). This study is the application of VAMS for therapeutic drug monitoring (TDM) in human

capillary blood. A liquid chromatography–tandem mass spectrometry (LC–MS/MS) workflow for

analysis of VAMS samples was developed and validated. Concentrations of hydroxychloroquine

(HCQ) and its metabolites, desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and

bisdesethylchloroquine (BDCQ), in capillary blood on VAMS samplers were compared to those in

venous blood in rheumatoid arthritis patients. Feasibility of capillary blood collected on both VAMS

and DBS cards were evaluated on patients. Stability of dried capillary blood on VAMS was also

examined. Our results established that VAMS is a simple and accurate sampling technique that

delivers the benefits of DBS sampling while overcoming the issues associated with hematocrit and

homogeneity. It requires a small blood volume, simplifies sample logistics management, and may

allow sample collection in the patient‘s home setting.

Isolation and characterization of novel degradation products of Doxofylline using HPLC, FTIR,

LCMS and NMR

Ch. Krishnam Raju, Avadhesh K. Pandey, Gururaj S., Kaushik Ghosh, Sameer G. Navalgund

ABSTRACT

Forced degradation of Doxofylline (DFL) in different stress (base and peroxide) conditions gave rise to

two potential unknown impurities. These unknown degradation products DFL DEG-I and DFL DEG-II

were evaluated using a new-reverse-phase high performance liquid chromatography (HPLC), where it

was eluted at 0.44 and 1.09 relative retention times to DFL peak. DFL DEG-I and DFL DEG-II were

isolated using preparative HPLC from degradation mixtures. The structure of DFL DEG-I and DFL

DEG-II were elucidated using high resolution MS, multi-dimensional NMR and FTIR spectroscopic

techniques, and characterized. The stereochemistry of the enantiomers in DFL DEG-II has further been

investigated using computational techniques. To the best of our knowledge, DFL DEG-I and DFL

DEG-II are novel impurities and not reported elsewhere.

124

Enantiomers of triclabendazole sulfoxide: Analytical and semipreparative HPLC separation,

absolute configuration assignment, and transformation into sodium salt

Rosella Ferretti, Simone Carradori, Paolo Guglielmi, Marco Pierini, Roberto Cirilli

ABSTRACT

Direct HPLC separation of the enantiomers of triclabendazole sulfoxide (TCBZ-SO), which is the

main metabolite of the anthelmintic drug triclabendazole, was carried out using the polysaccharide-

based Chiralpak AS-H and Chiralpak IF-3 chiral stationary phases (CSPs). The chromatographic

behaviour of both CSPs was evaluated and compared using normal-phase and reversed-phase eluents

at different column temperatures. The eluent mixture of n-hexane-2-propanol-trifluoroacetic acid

70:30:0.1 (v/v/v) and a column temperature of 40 °C were identified as the best operational conditions

to carry out semipreparative enantioseparations on a 1-cm I.D. AS-H column. Under these conditions,

12.5 mg of racemic sample were resolved in a single chromatographic run within 15 min. Comparison

of calculated and experimental chiroptical properties provided the absolute configuration assignment at

the sulfur atom. The salification of the isolated enantiomers of TCBZ-SO by reaction with sodium

hydroxide solution produced water-soluble Na salts which are potentially useful in the development of

new anthelmintic enantiomerically pure formulations.

Separation and characterization of unknown impurities and isomers in flomoxef sodium by LC-

IT-TOF MS and study of their negative-ion fragmentation regularities

Xu Yu, Fan Wang, Jiani Li, Weiguang Shan, Jian Wang

ABSTRACT

Thirteen unknown impurities in flomoxef sodium were separated and characterized by liquid

chromatography coupled with high resolution ion trap/time-of-flight mass spectrometry (LC-IT-TOF

MS)with positive and negative modes of electrospray ionization method for further improvement of

official monographs in pharmacopoeias. The fragmentation patterns of impurities in flomoxef in the

negative ion mode were studied in detail, and new negative-ion fragmentation regularities were

discovered. Chromatographic separation was performed on a Kromasil C18 column (250 mm × 4.6

mm, 5 μm). The mobile phase consisted of (A) ammonium formate aqueous solution (10 mM)–

methanol (84:16, v/v) and (B) ammonium formate aqueous solution (10 mM)–methanol (47:53, v/v).

In order to determine the m/z values of the molecular ions and formulas of all detected impurities, full

scan LC–MS in both positive and negative ion modes was firstly executed to obtain the m/z value of

the molecules. Then LC–MS2 and LC–MS3 were carried out on target compounds to obtain as much

structural information as possible. Complete fragmentation patterns of impurities were studied and

used to obtain information about the structures of these impurities. Structures of thirteen unknown

degradation products in flomoxef sodium were deduced based on the high resolution MSn data with

both positive and negative modes. The forming mechanisms of degradation products in flomoxef

sodium were also studied.

125

Heparin and homogeneous model heparin oligosaccharides form distinct complexes with

protamine: Light scattering and zeta potential analysis

Cynthia D. Sommers, Hongping Ye, Jian Liu, Robert J. Linhardt, David A. Keire

ABSTRACT

Large multimolecular complexes of heparin with positively charged proteins such as platelet factor 4

(PF4) or protamine can initiate immune responses associated with heparin use in patients, including

the most significant adverse event, heparin-induced thrombocytopenia (HIT). Current evidence

suggests that platelet-activating antibodies that recognize large multi-molecular complexes (300–700

kDa) of PF4 bound to heparin cause HIT [1] and in very rare cases anti-protamine-heparin antibodies

can induce thrombocytopenia [2]. Heparin is administered as a mixture of sulfated

glycosaminoglycans of variable lengths and sulfation levels. To date the potential impact of chain

length, sulfation level and impurities on the formation, size and immunogenicity of heparin-protamine

complexes has not been addressed due to the lack of purified, homogenous heparin chains for testing

purposes. Here, a set of well-characterized model heparin oligosaccharides was used with protamine

sulfate to evaluate the physicochemical properties of the resulting complexes. Hydrodynamic radii and

zeta potential profiles of heparin-protamine complexes were observed to be dependent upon the

sulfation location, size and concentration of the model heparin oligosaccharides. The well-

characterized oligosaccharide-protamine complexes analyzed in this work will be useful for

establishing links between heparin-protamine complex physiochemical attributes to their potential to

illicit cellular immunogenicity.

Quantitative analysis of binary polymorphs mixtures of fusidic acid by diffuse reflectance FTIR

spectroscopy, diffuse reflectance FT-NIR spectroscopy, Raman spectroscopy and multivariate

calibration

Canyong Guo, Xuefang Luo, Xiaohua Zhou, Beijia Shi, Xiaoxia Zhang

ABSTRACT

Vibrational spectroscopic techniques such as infrared, near-infrared and Raman spectroscopy have

become popular in detecting and quantifying polymorphism of pharmaceutics since they are fast and

non-destructive. This study assessed the ability of three vibrational spectroscopy combined with

multivariate analysis to quantify a low-content undesired polymorph within a binary polymorphic

mixture. Partial least squares (PLS) regression and support vector machine (SVM) regression were

employed to build quantitative models. Fusidic acid, a steroidal antibiotic, was used as the model

compound. It was found that PLS regression performed slightly better than SVM regression in all the

three spectroscopic techniques. Root mean square errors of prediction (RMSEP) were ranging from

0.48% to 1.17% for diffuse reflectance FTIR spectroscopy and 1.60–1.93% for diffuse reflectance FT-

NIR spectroscopy and 1.62–2.31% for Raman spectroscopy. The results indicate that diffuse

reflectance FTIR spectroscopy offers significant advantages in providing accurate measurement of

polymorphic content in the fusidic acid binary mixtures, while Raman spectroscopy is the least

accurate technique for quantitative analysis of polymorphs.

126

Mesoporous silica nanoparticles incorporated hybrid monolithic stationary phase immobilized

with pepsin for enantioseparation by capillary electrochromatography

Shujuan Xu, Rongzhen Mo, Can Jin, Xiaoqin Cui, Yibing Ji

ABSTRACT

In this study, a novel mesoporous silica nanoparticles incorporated chiral hybrid monolithic stationary

phase was developed. The stationary phase was firstly prepared by an in situ copolymerization of

amino-modified mesoporous silica nanoparticles (NH2-MSN), glycidyl methacrylate (GMA), and

ethylene dimethacrylate (EDMA) and then functionalized with pepsin as chiral selector. The

incorporated mesoporous silica nanoparticles provided additional interactions sites, and in turn yielded

different enantioselectivity thus enhancing the overall separation. The column was successfully

employed for enantioseparation of fifteen basic chiral drugs in capillary electrochromatography.

Effects of nanoparticles percentage, pepsin concentration, the pH of running buffer and the applied

voltage were investigated. All the analytes could be eluted in less than ten minutes and nine of them

could achieve baseline separation. Satisfactory repeatabilities with relative standard deviations less

than 4.2% were achieved through intraday, interday, column-to-column and batch-to-batch

investigations. These results indicated that the simultaneous utilization of the unique properties of

mesoporous silica nanoparticles and versatile features of monoliths could be a promising strategy for

enantioseparation.

Host-guest kinetic interactions between HP-β-cyclodextrin and drugs for prediction of bitter

taste masking

Zhen Guo, Fei Wu, Vikramjeet Singh, Tao Guo, Jiwen Zhang

ABSTRACT

Cyclodextrins (CD) are widely used bitter taste masking agents, for which the binding equilibrium

constant (K) for the drug-CD complex is a conventional parameter for quantitating the taste masking

effects. However, some exceptions have been reported to the expected relationship between K and

bitterness reduction and the relationship between kinetic parameters of a drug-CD interaction,

including association rate constant (Ka) and disassociation rate constant (Kd), and taste masking

remains unexplored. In this study, based upon a database of kinetic parameters of drugs-HP-β-CD

generated by Surface Plasmon Resonance Imaging for 485 drugs, the host-guest kinetic interactions

between drugs and HP-β-CD for prediction of taste masking effects have been investigated. The taste

masking effects of HP-β-CD for 13 bitter drugs were quantitatively determined using an electronic

gustatory system (α-Astree e-Tongue). Statistical software was used to establish a model based on

Euclidean distance measurements, Ka and Kd of the bitter drugs/HP-β-CD-complexes (R2 = 0.96 and

P < 0.05). Optimized parameters, Ka3, Kd, KaKd, Kd3, Ka2 and Ka/Kd with notable influence, were

obtained by stepwise regression from 12 parameters derived from Ka, Kd and K (Ka/Kd). 10-fold

cross-validation was used to verify the reliability of the model (correlation coefficient of 0.84, P <

0.05). The established model indicated a relationship between Ka, Kd, K and taste masking by HP-β-

CD and was successful in predicting the extent of taste masking by HP-β-CD of 44 bitter drugs, which

was in accordance with the literature reported. In conclusion, the relationship between kinetics of drug-

CD interactions and taste masking was established and providing a new strategy for predicting the

cyclodextrin mediated bitter taste masking.

127

Crystal structures and physicochemical properties of amisulpride polymorphs

Wen-Peng Zhang, Dong-Ying Chen

ABSTRACT

The purpose of this work was to investigate the crystal structures and physicochemical properties of

amisulpride polymorphs. Except of the previously reported polymorph (named as Form I), a new

polymorphic form (named as Form II) was discovered through comprehensive solid-state screening

experiments. Both polymorphic forms were characterized by single crystal X-ray structure analysis

(SXRD), powder X-ray diffraction (PXRD), dynamic vapor sorption (DVS) and thermal analysis

(TGA and DSC) as well. It has been found that the Forms I and II are of conformational polymorph

with the main conformational difference around ethylsulfonyl group. Form II possesses lower

hygroscopicity and better solubility compared with that of Form I, indicating Form II could be an

alternate solid form for formulation development.

A UHPLC method for the rapid separation and quantification of phytosterols using tandem

UV/Charged aerosol detection – A comparison of both detection techniques


ABSTRACT

The presented work describes the development and validation of a rapid UHPLC-UV/CAD method

using a core–shell particle column for the separation and quantitative analysis of seven plant sterols

and stanols. The phytosterols (ergosterol, brassicasterol, campesterol, fucosterol, stigmasterol, and β-

sitosterol) and the phytostanol stigmastanol were separated and analyzed in 8.5 min. The sample pre-

treatment procedure was optimized to be less time-consuming than any other published method,

especially due to no need of derivatization, evaporation and even reconstitution step. The

chromatographic separation was performed on the Kinetex 1.7 μ Phenyl-hexyl column (100 × 2.1 mm)

with a mobile phase acetonitrile/water according to the gradient program at a flow rate of 0.9 mL

min−1 and a temperature of 60 °C. A tandem connection of PDA and CAD (Corona Charged Aerosol

Detector) was used and both detection techniques were compared. The method was validated using

saponification as a first step in sample pre-treatment and an universal CAD as the detector. Recoveries

for all analyzed compounds were between 95.4% and 103.4% and relative standard deviation ranged

from 1.0% to 5.8% for within-day and from 1.4% to 6.7% for between-day repeatability. The limits of

detection were in the range of 0.4–0.6 μg mL−1 for standard solutions and 0.3–1.2 μg mL−1 for

phytosterols in real samples. Although several gradient programs and different stationary phases were

tested, two compounds, campesterol and campestanol, were not separated. Their peak was quantified

as a sum of both analytes.

128

Analysis of chemical constituents in an herbal formula Jitong Ning Tablet

Di Gao, Baojun Wang, Zhipeng Huo, Yi He, Lian-Wen Qi

ABSTRACT

Jitong Ning Tablet (JTNT), a traditional Chinese herbal formula, consists of Eucommia ulmodies oliv,

Angelicae pubescentis radix, Aconiti radix cocta, Corydalis yanhusuo w.t. wang, Glycyrrhizae radix et

rhizoma, Paeoniae radix rubra and Radix puerariae. It has been demonstrated to show protective

effects on ankylosing spondylitis and anti-inflammatory effects. The chemical compositions of JTNT,

playing a key role in quality control, remain unknown. In this study, an ultra-performance liquid

chromatography combined with quadrupole time of flight mass spectrometry (UPLC-Q–TOF–MS)

method in both positive and negative ion mode was established to investigate the chemical constituents

of JTNT formula. In total, 162 compounds including flavonoids, triterpenoids, coumarins, alkaloids,

phenylpropionic acids, lignans, terpenoids, and organic acids were detected, 152 of which were

unambiguously or tentatively identified by comparing their retention times and accurate mass

measurement with reference compounds and data in literatures. Our results would benefit quality

control and chemical basis for JTNT.

Molecular recognition of pseudodistamine isomeric precursor trans-3(4)-aminopiperidin-4(3)-ols

by EI mass spectrometry

D.M. Mazur, G.V. Grishina, A.T. Lebedev

ABSTRACT

Synthesis of drugs, biologically active compounds or their derivatives always requires precise and

reliable method of their identification, including differentiation of the possible isomers.

Pseudodistamines and their precursors became a matter of elevated attention due to their different

enzymatic inhibition. This paper deals with one of the groups of the pseudodistamine precursors −

trans-3(4)-aminopiperidin-4(3)-ols. Their synthesis brings to a mixture of 2 regioisomers, resulting in

the necessity of their reliable recognition. NMR spectroscopy commonly used by organic chemists

requires advance knowledge and experience to analyse the spectra of these regioisomers. Therefore,

we herein proposed a simpler way to recognize trans-3(4)-aminopiperidin-4(3)-ols using mass

spectrometry with electron ionization. Fragmentation of 4 pairs of aminopiperidinol regioisomers with

variation of amine moiety was studied. The obtained results allowed defining a group of 3 ions ([M-

18]+., [M-19]+, [M-43]+) related only to the structure of trans-4-aminopiperidin-3-ols and 1 ion (m/z

100) related to the structure of trans-3-aminopiperidin-4-ols. Besides, interrogation of intensity of ions

common for spectra of both regioisomers allows making differentiation as well.

129

Development and validation of a general derivatization HPLC method for the trace analysis of

acyl chlorides in lipophilic drug substances

Xiangyuan Zheng, Lan Luo, Jie Zhou, Xiaoling Ruan, Feng Zheng

ABSTRACTs

Acyl chlorides are important acylating agents in the synthesis of active pharmaceutical ingredients.

Determining the residual acyl chlorides in drug substances is a challenge due to their high reactivity

and the matrix interferences from drug substances and their related impurities. This paper describes a

general derivatization HPLC method for the determination of aromatic and aliphatic acyl chlorides in

lipophilic drug substances. Since most drug substances have weak absorptions in the visible range

(above 380 nm), the nitro-substituted anilines and nitro-substituted phenylhydrazines were selected as

the derivatization reagents due to their weak basicity and red-shift of UV absorption spectra. The

maximum wavelength and absorption intensity of nitro-substituted anilines decreased after

derivatization with acyl chlorides, whereas the derivatization products of nitro-substituted

phenylhydrazines showed the slight increases of maximum wavelength and absorbance intensity.

Hence, 2-nitrophenylhydrazine was selected as the suitable derivatization reagent because the

derivatives have the maximum UV wavelength absorbance at 395 nm, which could largely minimize

the matrix interferences. The optimization of the concentration of 2-nitrophenylhydrazine is important

for the sensitivity and stability of derivatives. Other reaction conditions including reaction temperature,

time and the influence of three competitive solvents (water, methanol and ethanol) on the reaction

efficiency were also studied. After derivatization with 100 μg mL−1 2-nitrophenylhydrazine at room

temperature for 30 min, the method was validated for high specificity and sensitivity with the detection

limits in the range of 0.01–0.03 μg mL−1. The proposed method was applied as a generic method to

determine the residual acyl chlorides in lipophilic drug substances.

An isocratic hydrophilic interaction liquid chromatographic method for simultaneous

determination of iodixanol and its related impurities in drug substance

Bruno D. Rondon, Neila M. Cassiano, Quezia B. Cass

ABSTRACT

This work reports a simple isocratic hydrophilic interaction liquid chromatographic (HILIC) method

for the simultaneous quantification of iodixanol and of its related impurities C, D and E in drug

substance. The chromatographic separation was carried out with a Kinetex™ HILIC column, using

acetonitrile and formic acid aqueous solution (1.0 mmol/L, pH 3.2) (92:08, v/v) as eluent at a flow rate

of 0.8 mL/min. The autosampler and column temperature were maintained at 20 °C and UV detection

was set at 243 nm. The method was validated in accordance to the ICH guideline and employed for the

analysis of two different lots of iodixanol drug substance. The developed method is presented as a

valuable alternative to the current methods described in the USP monograph.

130

Electrochemical and optical study of metallothionein interactions with prion proteins

Alzbeta Cardova, Pavlina Adam, Stefano Mariani, Lukas Richtera, Vojtech Adam

ABSTRACT

The prion protein (PrPC) can be structurally shifted to its PrPSc isoform causing a wide range of

neurodegenerative diseases, which are currently incurable. There is an evidence that metallothioneins

(MTs), and especially MT-3, are associated with neurodegenerative diseases. PrPC and MTs play

pivotal roles in maintaining metal homeostasis; therefore, it is conceivable that each of them has its

own significance in prion diseases. In this paper, we study the nature of interactions between PrPC,

MT, and copper ions, Cu(II), using the method of differential pulse voltammetry (DPV) coupled with

adsorptive transfer stripping technique (AdTS). Electrochemical properties of PrP itself and its

interactions with both the Cu(II) ions and MTs have been found. Based on the results obtained, we

hypothesised the formation of the complex in molar ratio 2:1 (PrPC:MT). Surface plasmon resonance

imaging (SPRi) was used as a control reference assay to further confirm results obtained by the

electrochemical approach, such as the specific interactions between PrPC and MT-3.

Development of matrix effect-free MISPE-UHPLC–MS/MS method for determination of

lovastatin in Pu-erh tea, oyster mushroom, and red yeast rice

Pavel Svoboda, Daniel Sander, Kateřina Plachká, Lucie Nováková

ABSTRACT

Matrix effect-free UHPLC–MS/MS method was developed and validated for the determination of

cholesterol-lowering lovastatin in food samples represented by Pu-erh tea, oyster mushroom, and red

yeast rice. The resulting method was fully validated in terms of intra-day and inter-day precision,

accuracy, linearity, range, LOD, LOQ, and matrix effects. The matrix effect phenomenon evaluated by

comparison of slopes of calibration curves was completely eliminated by solid-phase extraction based

on the technique of molecularly imprinted polymers (MIPs). Comparison of elution profiles obtained

on the MIP and corresponding control non-imprinted polymer (NIP) showed selectivity of the

extraction procedure. In addition, selectivity of the MIP material and the molecularly imprinted solid-

phase extraction (MISPE) was also proved by experiments evaluating retention of analytes physico-

chemically similar to the target molecule. Extraction recoveries of these analytes represented by

estrogen derivatives (estrone, estriol, 17α‐ethinylestradiol, and β-estradiol) were very low or even null.

Synthesis and preparation of the resulting MIP sorbent was characterized by excellent repeatability

expressed as RSD 7.7% (n = 9) of extraction recoveries. The determined capacity of the MIP material

reaching 375 ng/mg is sufficient for analysis of the evaluated statin in its natural sources. Suitability of

the resulting MISPE-UHPLC–MS/MS procedure for real sample analysis was verified by the

determination of lovastatin in one dietary supplement based on the red yeast rice with a given amount

of the target analyte. Finally, three mushroom and fifteen tea samples obtained in Czech food stores

and tearooms were subjected to analysis. Low or null amount of lovastatin was found in these samples.

131

Facile preparation of fibrin coated open tubular column for characterization of monoclonal

antibody variants by capillary electrochromatography

Xue Xiao, Wentao Wang, Yamin Zhang, Li Jia

ABSTRACT

Monoclonal antibodies (mAbs) are one of the most promising classes of therapeutic protein

biopharmaceuticals. However, the complexity of mAbs poses a daunting analytical challenge for

heterogeneity characterization of mAbs. In this study, inspired by blood coagulation, we adopted a

fibrin coating as a novel stationary phase in open tubular (OT) column for the separation of the mAbs

variants by capillary electrochromatography. The fibrin coating was prepared by in situ polymerization

of fibrin in the presence of thrombin as a catalyst inside a fused-silica capillary. Scanning electron

microscopy and electroosmotic flow measurement were carried out to characterize the fibrin coated

OT columns. The average thickness of the fibrin coating was about 1.13 μm. And the EOF of the

column was pH-dependent. The electrochromatographic performance of the prepared columns was

evaluated by characterization of the variants of three mAbs (cetuximab, trastuzumab and rituximab).

The columns demonstrated good repeatability with the run-to-run, day-to-day and column-to-column

relative standard deviations of migration times less than 2.42%. The study highlighted the potential of

adsorbed proteins as stationary phases for the separation of mAbs variants. Furthermore, the study

provided a new platform for characterization of heterogeneity of mAbs in pharmaceutical industry.

Development and validation of a fast SFC method for the analysis of flavonoids in plant extracts

Yang Huang, Ying Feng, Guangyun Tang, Minyi Li, Zhengjin Jiang

ABSTRACT

Flavonoids from plants always show a wide range of biological activities. In the present study, a rapid

and highly efficient supercritical fluid chromatography (SFC) method was developed for the separation

of 12 flavonoids. After careful optimization, the 12 flavonoids were baseline separated on a ZORBAX

RX-SIL column using gradient elution. A 0.1% phosphoric acid solution in methanol was found to be

the most suitable polar mobile phase component for the separation of flavonoids. From the viewpoint

of retention and resolution, a backpressure of 200 bar and a temperature of 40 °C were shown to give

the best results. Compared with a previously developed reverse phase liquid chromatography method,

the SFC method could provide flavonoid separations that were about three times faster, while

maintaining good peak shape and comparable peak efficiency. This SFC method was validated and

applied to the analysis of five flavonoids (kaempferol, luteolin, quercetin, luteoloside, buddleoside)

present in Chrysanthemum morifolium Ramat. from different cultivars (Chuju, Gongju, Hangju, Boju).

The results indicated a good repeatability and sensitivity for the quantification of the five analytes with

RSDs for overall precision lower than 3%. The limits of detection ranged from 0.73 to 2.34 μg/mL,

while the limits of quantification were between 2.19 and 5.86 μg/mL. The method showed that SFC

could be employed as a useful tool for the quality assessment of Traditional Chinese medicines

(TCMs) containing flavonoids as active components.

132

A multi-matrix HILIC-MS/MS method for the quantitation of endogenous small molecule

neurological biomarker N-acetyl aspartic acid (NAA)

Dewakar Sangaraju, Sheerin K. Shahidi-Latham, Braydon L. Burgess, Brian Dean, Xiao Ding

ABSTRACT

A multi-matrix hydrophilic interaction liquid chromatography tandem mass spectrometric method

(HILIC-MS/MS) was developed for the quantitation of N-Acetyl Aspartic acid (NAA) using stable

isotope labeled internal standard, D3-NAA in various biological matrices such as human plasma,

human CSF, mouse plasma, brain and spinal cord. A high throughput 96-well plate format supported

liquid extraction (SLE) procedure was developed and used for sample preparation. Mass spectrometric

analysis of NAA was performed using selected reaction monitoring transitions in positive electrospray

ionization mode. As NAA is endogenously present, a surrogate matrix approach was used for

quantitation of NAA and the method was qualified over linear calibration curve range of 0.01–10

μg/mL. Intra and inter assay precision indicated by percent relative standard deviation (%RSD) was

less than 7.1% for low, medium, medium high and high QCs. The accuracy of the method ranged from

92.6-107.0% of nominal concentration for within-run and between-run for the same QCs. Extraction

recovery of NAA and D3-NAA was greater than 76%. Stability of NAA was established in the above

biological matrices under bench top (RT, 5 h), freeze thaw (–20 ± 10 °C, 3 cycles) and moues/human

plasma sample collection (Wet ice, RT) conditions. HILIC-MS/MS method was then used to quantify

and compare the NAA levels in human plasma and CSF of ALS patients versus control human

subjects. NAA CSF levels in control human subjects (73.3 ± 31.0 ng/mL, N = 10) were found to be

slightly higher than ALS patients (46.1 ± 22.6 ng/mL, N = 10) (P = 0.04). No differences were

observed in NAA plasma levels in human control subjects (49.7 ± 13.8 ng/mL, N = 9) as compared to

ALS patients (49.6 ± 8.1 ng/mL, N = 10) (P = 0.983). NAA endogenous concentrations in mouse

plasma, brain and spinal cord were found to be 243.8 ± 56.8 ng/mL (N = 6), 1029.8 ± 115.2 μg/g tissue

weight (N = 5) and 487.6 ± 178.4 μg/g tissue weight (N = 5) respectively.

Chemotaxonomic studies of nine Paris species from China based on ultra-high performance

liquid chromatography tandem mass spectrometry and Fourier transform infrared spectroscopy

Yuanzhong Wang, Ehu Liu, Ping Li

ABSTRACT

Paris species, which contain steroid saponins, have been used as herb folk medicines in Asia. In the

present study, a comprehensive strategy based on liquid chromatography-tandem mass spectrometry

(LC–MS/MS) and Fourier transform infrared (FT-IR) spectroscopy was firstly proposed to evaluate

the chemotaxonomic relationships of nine Paris species sampled from different geographical regions in

China. Principle component analysis (PCA) based on FT-IR data revealed chemical similarities in term

of the nine species and geographical regions, indicating the accumulation of metabolites affected by

the combination of geographical factors and species. The chemotaxonomic relationships of four

species supported the morphological taxonomy and implied ancestry from P. polyphylla. After high-

efficiency chromatographic separation, ions trap/time-of-flight mass spectrometry (IT-TOFMS) and

triple quadrupole mass spectrometry (QQQ-MS) were used to identify unknown metabolites and

simultaneously determine six key compounds (polyphyllin I, II, V, VI, VII and gracillin) in Paris

species, respectively. The tentative identification of 22 steroid saponins was indicative of a common

biosynthetic pathway in Paris species. Phytoecdysones, gracillin and open-chain steroid saponins were

considered as key precursors.

133

Rapid profiling and pharmacokinetic studies of major compounds in crude extract from

Polygonum multiflorum by UHPLC-Q-TOF-MS and UPLC–MS/MS

Linlin Wang, Mangmang Sang, Erwei Liu, Prince Osei Banahene, Xiumei Gao

ABSTRACT

A reliable, rapid analytical method was established for characterization of constituents in the ethanol

extract of Polygonum multiflorum by combining an ultra-high performance liquid chromatography

with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). 131 constituents which

including phenolic acids, stilbenes, flavones, anthraquinones, naphthalenes and their derivatives were

identified or tentatively identified by using characteristic diagnostic fragment ions and references. The

established method was further applied to analyze blood samples, and successfully identified 41

compounds which were absorbed through the gastrointestine in rats after administration the extract of

P. multiflorum. Moreover, the pharmacokinetic studies of some major compounds in blood were

investigated by using ultra performance liquid chromatography with tandem mass spectrometry

(UPLC–MS/MS) method. This study showed a comprehensive research of P. multiflorum, which

could provide a meaningful basis for further quality control, pharmacological as well as toxicological

researches.

Metabolic profiling of nuciferine in rat urine, plasma, bile and feces after oral administration

using ultra-high performance liquid chromatography-diode array detection-quadrupole time-of-

flight mass spectrometry

Xiao-Lei Wu, Ming-Jiang Wu, Xin-Ze Chen, Hao-Ling Ma, De-Qin Zhang

ABSTRACT

Nuciferine, a major alkaloid found in Nelumbinis Folium, exhibits a broad spectrum of bioactivities,

such as antiobesity, anti-diabetes and anti-inflammatory. However, many research regarding nuciferine

focused on the extraction, isolation and biological activity, the metabolism is not comprehensively

explained in vivo. Thence, the present of this paper is to establish a simple method for speculating

metabolites of nuciferine. A total of 15 metabolites were detected and tentatively identified through

ultra high performance liquid chromatography-diode array detection-quadrupole time-of-flight mass

spectrometry (UHPLC-DAD-QTOF-MS), including 7 new metabolites. Among them, we also

discovered a previously unmentioned metabolically active site at the C1-OCH3 position. These

metabolites suggested that demethylation, oxidation, glucuronidation and sulfation were major

metabolic pathways. This study provided significant experiment basis for its safety estimate and

valuable information about the metabolism of nuciferine, which will be advantageous for new drug

development.

134

Development and validation of liquid chromatography tandem mass spectrometry method

quantitative determination of polymyxin B1, polymyxin B2, polymyxin B3 and isoleucine-

polymyxin B1 in human plasma and its application in clinical studies

Kim H. Hee, Yee K.J. Leaw, Jun L. Ong, Lawrence S. Lee

ABSTRACT

Polymyxin B (PB) is an antibiotic consisting of a cyclic heptapeptide and a tripeptide side chain used

in treatment of infections caused by Gram-negative bacteria. Commercial formulations of PB contain

multiple structurally related components with major constituents of PB1, PB2, PB3 and ile-PB1. To

understand the pharmacokinetics of these major components, we have developed and validated a LC–

MS/MS method to quantify PB1, PB2, PB3 and ile-PB1 in human plasma. PB was extracted from

plasma by protein precipitation using trichloroacetic acid followed by chromatographic separation on

Zorbax Bonus-RP column (100 mm × 2.1 mm, 1.8 μm) using stepwise gradient elution of water

containing 0.1% of formic acid and 0.1% of trichloroacetic acid (mobile phase A) and 90% acetonitrile

with 0.1% formic acid (mobile phase B). Despite of structural similarities, these PBs were completely

resolved in the analytical run time of 6.5 min. Detection and quantification of PBs were performed by

selected reaction monitoring (SRM) under positive ionization mode in the mass spectrometer.

Separation of PB1 and ile-PB1, as well as PB2 and PB3, before quantification is crucial because they

are structural isomers detected based the same SRM. Excellent linearity was achieved (r2 > 0.99) in

the calibration curves of PB. The developed method was accurate (95.3–111.7%) and precise (CV <

5.1%). Recovery of PB from the plasma extraction was between 53 and 76% and reproducible (CV <

4.5%). Matrix effect was not observed by post-column infusion of PB in the mass spectrometer. This

methodology has been successfully applied to clinical study of patients dosed with intravenous

infusions of PB.

Human exposure to Bisphenol A and liver health status: Quantification of urinary and

circulating levels by LC–MS/MS

Carla Nicolucci, Sonia Errico, Alessandro Federico, Marcello Dallio, Nadia Diano

ABSTRACT

A selective and highly sensitive analytical methodology for determination of Bisphenol A in human

plasma was developed and validated. The method was based on selective liquid/solid extraction,

combined with liquid chromatography–electrospray ionization tandem mass spectrometry in the

multiple reaction monitoring mode and negative ionization. The linearity of the detector response was

verified in human plasma over the concentration range 0.100–200 ng mL−1. The detection limit was

0.03 ng mL−1 and the quantification limit was 0.100 ng mL−1. The analytical features of the proposed

in-house validated method were satisfactory: precision was <10% and recoveries were around 84–

104%. The matrix effect was studied and compensated using deuterated labeled standard. The

applicability of the proposed method was demonstrated analyzing human plasma samples from

individuals affected by non-alcoholic fatty liver disease. Bisphenol A was detected above the detection

limit in all samples. The data show a persistence of unconjugated Bisphenol A levels in plasma and

indicate a chronic Bisphenol A exposure of the target organ, suggesting an association between liver

health status and Bisphenol A exposure. The results from our study are valuable for further

investigation with large sample size and longitudinal study designs, necessary to confirm the observed

association.

135

Determination of prodrug treosulfan and its biologically active monoepoxide in rat plasma, liver,

lungs, kidneys, muscle, and brain by HPLC–ESI–MS/MS method

Michał Romański, Anna Kasprzyk, Artur Teżyk, Agnieszka Widerowska, Franciszek Główka

ABSTRACT

A prodrug treosulfan (TREO) is currently investigated in clinical trials for conditioning prior to

hematopoietic stem cell transplantation. Bioanalysis of TREO and its active derivatives, monoepoxide

(S,S-EBDM) and diepoxide, in plasma and urine underlay the pharmacokinetic studies of these

compounds but cannot explain an organ pharmacological action or toxicity. Recently, distribution of

TREO and S,S-EBDM into brain, cerebrospinal fluid, and aqueous humor of the eye has been

investigated in animal models and the obtained results presented clinical relevance. In this paper, a

selective and rapid HPLC–ESI–MS/MS method was elaborated and validated for the studies of

disposition of TREO and S,S-EBDM in rat plasma, liver, lungs, kidneys, muscle, and brain. The two

analytes and codeine, internal standard (IS), were isolated from 50 μL of plasma and 100 μL of

supernatants of the tissues homogenates using ultrafiltration Amicon vials. Chromatographic

resolution was accomplished on C18 column with isocratic elution. The limits of quantitation of TREO

and S,S-EBDM in the studied matrices ranged from 0.11 to 0.93 μM. The HPLC–MS/MS method was

adequately precise and accurate within and between runs. The IS-normalized matrix effect differed

among the tissues and was the most pronounced in a liver homogenate supernatant (approximately

0.55 for TREO and 0.35 for S,S-EBDM).

Profiles of amino acids and biogenic amines in the plasma of Cri-du-Chat patients

Danielle Zildeana Sousa Furtado, Fernando Brunale Vilela de Moura Leite, Cleber Nunes Barreto,

Bernadete Faria, Nilson Antonio Assunção

ABSTRACT

Cri-du-chat syndrome (CDCS) is a rare innate disease attributed to chromosome 5p deletion

characterized by a cat-like cry, craniofacial malformation, and altered behavior of affected children.

Metabolomic analysis and a chemometric approach allow description of the metabolic profile of

CDCS as compared to normal subjects. In the present work, UHPLC/MS was employed to analyze

blood samples withdrawn from CDCS carriers (n = 18) and normal parental subjects (n = 18), all aged

0–34 years, aiming to set up a representative CDCS profile constructed from 33 targeted amino acids

and biogenic amines. Methionine sulfoxide (MetO) was of particular concern with respect to CDCS

redox balance. Increased serotonin (3-fold), methionine sulfoxide (2-fold), and Asp levels, and a little

lower Orn, citrulline, Leu, Val, Ile, Asn, Gln, Trp, Thr, His, Phe, Met, and creatinine levels were found

in the plasma of CDCS patients. Nitrotyrosine and Trp did not differ in normal and CDCS

individuals.The accumulated metabolites may reflect, respectively, disturbances in the redox balance,

deficient purine biosynthesis, and altered behavior, whereas the amino acid abatement in the latter

group may affect the homeostasis of the urea cycle, citric acid cycle, branched chain amino acid

synthesis, Tyr and Trp metabolism and amino acid biosynthesis. The identification of enzymatic

deficiencies leading to the amino acid burden in CDCS is further required for elucidating its molecular

bases and eventually propose specific or mixed amino acid supplementation to newborn patients

aiming to balance their metabolism.

136

Online microdialysis-ultra performance liquid chromatography–mass spectrometry method for

comparative pharmacokinetic investigation on iridoids from Gardenia jasminoides Ellis in rats

with different progressions of type 2 diabetic complications

Xueju Zhang, Lu Wang, Zhong Zheng, Zifeng Pi, Fengrui Song

ABSTRACT

Iridoid glycosides consist the main bioactive constituents of Gardenia jasminoides Ellis (G.

jasminoides), which is used alone or in combination with other medicine herbs for treatment of

diabetes mellitus in China. This paper investigated for the first time comparative pharmacokinetics

(PKs) of four unbound iridoids (including genipin, geniposide, gardenoside and geniposidic acid) in rat

blood between healthy and type 2 diabetic groups with different disease progressions (9 or 13 weeks).

Online microdialysis-ultra performance liquid chromatography-mass spectrometry (MD- UPLC–

MS/MS) method was used after oral administration of iridoid extracts obtained from the fruits of G.

jasminoides. Student's t-test was used for statistical comparison of PK parameters. Results showed that

genipin, geniposide and geniposidic acid feature higher Cmax, larger area under the curve (AUC),

lower clearance (CL), longer Tmax and mean residence time (MRT) in type 2 diabetic rats than those

in healthy rats (p < 0.05). However, no significant difference was observed in PK parameters between

the two diabetic groups with different complication progressions (p > 0.05). Type 2 diabetic rats

showed significantly altered PK behaviors of genipin, geniposide and geniposidic acid (especially

systemic exposure, AUCs of genipin, geniposide and geniposidic acid). Online MD-UPLC–MS/MS

provides real-time sampling and monitoring, these functions can be applied to PKs of iridoids from G.

jasminoides.

Optimization of a new methodology for trace determination of elements in biological fluids:

Application for speciation of inorganic selenium in children’s blood

Reza Akramipour, Mitra Hemati, Nazir Fattahi, Meghdad Pirsaheb, Toraj Ahmadi-Jouibari

ABSTRACT

The continuous sample drop flow microextraction (CSDFME) joined with the iridium-modified tube

graphite furnace atomic absorption spectrometry (GFAAS) has been developed as a highly sensitive

technique for the speciation of selenium in blood samples. In this method 32.0 μl carbon tetrachloride

is transferred to the bottom of a conical sample cup. Then the 5.0 ml of aqueous solution transforms to

fine droplets while passing through the organic solvent. At this stage, Se(IV)-APDC hydrophobic

complex is extracted into the organic solvent. After extraction, the conical sample cup is transferred to

the GFAAS and 20 μl of extraction solvent was injected into the graphite tube by the aim of

autosampler. Under the optimum conditions, the calibration graph was linear in the range of 0.06–3.0

μg l−1 with detection limit of 0.02 μg l−1. The enrichment factor and enhancement factor were 106

and 91, respectively. Repeatability (intra–day) and reproducibility (inter–day) of method based on

seven replicate measurements of 2.5 μg l−1 of selenium were 3.7% and 4.2%, respectively. Total

inorganic Se(IV, VΙ) was measured after reduction of Se(VΙ) with gentle boiling in 5 M HCl medium

for 50 min and adjusting pH to 3, and the concentration of Se(VΙ) was calculated by subtracting the

Se(IV) concentration from the total selenium concentration.

137

Detailed analysis of cortisol, cortisone and their tetrahydro- and allo-tetrahydrometabolites in

human urine by LC–MS/MS

Katarzyna Kosicka, Anna Siemiątkowska, Dąbrówka Pałka, Agata Szpera-Goździewicz, Franciszek K.

Główka

ABSTRACT

Cortisol (F) and cortisone (E) are metabolized to A-ring reduced metabolites in the reactions catalyzed

by 5α- and 5β-reductase. 5α-tetrahydrocortisol (alloTHF) and 5β-tetrahydrocortisol (THF) are

produced from F. The metabolism of E takes place in analogy to form alloTHE and THE. Up to now,

the analysis of endogenous glucocorticoids did not consider alloTHE, limiting the metabolism of E to

THE only. Nevertheless, such simplification can generate inaccuracy in the assessment of the function

of enzymes crucial for glucocorticoids metabolism: 11β-hydroxysteroid dehydrogenase type 1 and

type 2 (11β-HSD1 and 11β-HSD2), as well as 5α- and 5β-reductase. The paper presents the new LC–

MS/MS method for the simultaneous analysis of F and E with their tetrahydro- (THF and THE) and

allo-tetrahydrometabolites (alloTHF and alloTHE) in urine. The method was fully validated and allows

determining both the unconjugated and total concentrations of urinary glucocorticoids. The method

meets the EMA‘s recommendations and was proved to be useful in the analysis of clinical samples.

The LLOQ of 1 ng/mL allows the determination of free urinary F, E, THF and THE, but not alloTHF

and alloTHE, in samples obtained from pregnant women.

Urinary metabonomics study of the hepatoprotective effects of total alkaloids from Corydalis

saxicola Bunting on carbon tetrachloride-induced chronic hepatotoxicity in rats using 1H NMR

analysis

Fang Wu, Hua Zheng, Zheng-Teng Yang, Bang Cheng, Zhi-Heng Su

ABSTRACT

Chronic liver injury has been shown to cause liver fibrosis due to the sustained pathophysiological

wound healing response of the liver, and eventually progresses to cirrhosis. The total alkaloids of

Corydalis saxicola Bunting (TACS), a collection of important bioactive ingredients derived from the

traditional Chinese folk medicine Corydalis saxicola Bunting (CS), have been reported to have

protective effects on the liver. However, the underlying molecular mechanisms need further

elucidation. In this study, the urinary metabonomics and the biochemical changes in rats with carbon

tetrachloride (CCl4)-induced chronic liver injury due to treatment TACS or administration of the

positive control drug-bifendate were studied via proton nuclear magnetic resonance (1H NMR)

analysis. Partial least squares-discriminate analysis (PLS-DA) suggested that metabolic perturbation

caused by CCl4 damage was recovered with TACS and bifendate treatment. A total of seven

metabolites including 2-oxoglutarate, citrate, dimethylamine, taurine, phenylacetylglycine, creatinine

and hippurate were considered as potential biomarkers involved in the development of CCl4-induced

chronic liver injury. According to pathway analysis using identified metabolites and correlation

network construction, the tricarboxylic acid (TCA) cycle, gut microbiota metabolism and taurine and

hypotaurine metabolism were recognized as the most affected metabolic pathways associated with

CCl4 chronic hepatotoxicity. Notably, the changes in 2-oxoglutarate, citrate, taurine and hippurate

during the process of CCl4-induced chronic liver injury were significantly restored by TACS

treatment, which suggested that TACS synergistically mediated the regulation of multiple metabolic

pathways including the TCA cycle, gut microbiota metabolism and taurine and hypotaurine

metabolism.

138

Pharmacokinetic properties of the synthetic cannabinoid JWH-018 and of its metabolites in

serum after inhalation

Stefan W. Toennes, Anna Geraths, Werner Pogoda, Alexander Paulke, Johannes G. Ramaekers

ABSTRACT

Each year, synthetic cannabinoids are occurring in high numbers in the illicit drug market, but data on

their pharmacology and toxicology are scarcely available. Therefore, a pilot study was performed to

assess adverse effects of JWH-018, which is one of the oldest and best known synthetic cannabinoids.

Six subjects inhaled smoke from 2 and 3 mg JWH-018. The drug and nine of its metabolites were

analyzed in their blood samples taken during the following 12 h by liquid chromatography–mass

spectrometry (LC–MSMS). The maximum concentration of JWH-018 reached 2.9–9.9 ng/ml after

inhalation and markedly decreased during the next 1.5 h, followed by a multiexponential decline (t1/2

in median 1.3 h and 5.7 h). The concentration of the pentanoic acid metabolite was slightly higher than

that of the 3-, 4- and 5-hydroxypentyl metabolites and of the 6-hydroxyindol metabolite. The data also

suggest a multiexponential decline and slow terminal elimination of JWH-018 and all metabolites. The

detection of JWH-018 and of its metabolites in serum requires high analytical sensitivity. The

pharmacokinetic properties of inhaled JWH-018 are similar to that of THC. A slow terminal

elimination of drug and metabolites may lead to accumulation in chronic users.

Charged derivatization and on-line solid phase extraction to measure extremely low cortisol and

cortisone levels in human saliva with liquid chromatography–tandem mass spectrometry

Balázs Magda, Zoltán Dobi, Katalin Mészáros, Éva Szabó, Pál T. Szabó

ABSTRACT

The aim of this study was to develop a sensitive, reliable and high-throughput liquid chromatography –

electrospray ionization – mass spectrometric (LC-ESI–MS/MS) method for the simultaneous

quantitation of cortisol and cortisone in human saliva. Derivatization with 2-hydrazino-1-

methylpyridine (HMP) was one of the most challenging aspects of the method development. The

reagent was reacting with cortisol and cortisone at 60 °C within 1 h, giving mono- and bis-hydrazone

derivatives. Investigation of derivatization reaction and sample preparation was detailed and discussed.

Improvement of method sensitivity was achieved with charged derivatization and use of on-line solid

phase extraction (on-line SPE). The lower limit of quantitation (LLOQ) was 5 and 10 pg/ml for

cortisol and cortisone, respectively. The developed method was subsequently applied to clinical

laboratory measurement of cortisol and cortisone in human saliva.

139

Comparability study of Rituximab originator and follow-on biopharmaceutical

Othman Montacir, Houda Montacir, Murat Eravci, Andreas Springer, Maria Kristina Parr

ABSTRACT

Immunglobolin G (IgG)-based biopharmaceuticals are emerging on the pharmaceuticals market due to

their high target selectivity in different diseases. In parallel, a growing interest by other companies to

produce similar or highly similar follow-on biologics exits, once the patent of blockbuster

biotherapeutics is about to expire. In correlation to their complex structure, an analytical challenge is

facing the approval of these biosimilars. Health authorities (e.g. FDA and EMA) have issued several

guidelines to define critical quality attributes during manufacturing process changes. In the current

study, physicochemical characterization using state-of-the-art analytics was applied to analyse intact

mass, post-translational modifications (PTMs) and higher order structure of Rituximab and one of its

biosimilars. Intact mass analysis, middle-up approach as well as subunit analysis revealed similar

glycoforms but additional lysine variants in the biosimilar. The N-glycosylation site was confirmed for

both, the originator and the biosimilar. PTMs and higher order structure were confirmed to be similar.

A special focus was given to N-glycosylation due to its potential to monitor the batch-to-batch

consistency and alteration during the production bioprocess. Comparison of the N-glycosylation

profiles obtained from three batches of the biosimilar and the reference product showed quantitative

variations, although the N-glycans were qualitatively similar. Furthermore, a head-to-head

comparability of functional properties was performed to investigate the impact of glycosylation

alteration and PTMs on potency within the biosimilar batches and between originator and follow-on

biodrug. The data affirm that the difference is still in the acceptable range for biosimilarity.

Whole blood microsampling for the quantitation of estetrol without derivatization by liquid

chromatography-tandem mass spectrometry

Gwenaël Nys, Anne Gallez, Miranda G.M. Kok, Gaël Cobraiville, Marianne Fillet

ABSTRACT

Quantitative bioanalysis and especially pharmacokinetic studies are challenging since only low

volumes of biological material are available and low concentrations (ng/ml) are often expected. In this

context, volumetric absorptive microsampling (VAMS) devices were developed to accurately collect

10 or 20 μl of whole blood from tested subjects. In this study, we present the development and

validation of ultra-high performance liquid chromatography coupled to tandem mass spectrometry

method after VAMS sampling for the quantitation of estetrol (E4), a potentially new medicine for

hormone replacement, contraception and osteoporosis therapies. Interestingly, a very simple sample

preparation procedure was developed without any derivatization step. Even if lack of sensitivity is a

common consideration when using negative ionization mode, we demonstrated in this work that an

excellent sensitivity could be reached by carefully optimizing the nature and concentration of the

mobile phase additive. After the optimization of every experimental parameter, the stability,

selectivity, trueness, precision and accuracy of the final method were successfully demonstrated. In

addition, the excellent performances of the method were confirmed by two independent proof-of-

concept pharmacokinetic studies of E4 after VAMS collection in a murine model.

140

Meropenem, levofloxacin and linezolid in human plasma of critical care patients: A fast semi-

automated micro-extraction by packed sorbent UHPLC-PDA method for their simultaneous

determination

Vincenzo Ferrone, Roberto Cotellese, Lorenzo Di Marco, Simona Bacchi, Giuseppe Carlucci

ABSTRACT

An ultra high-performance liquid chromatographic (UHPLC) method with PDA detection was

developed and validated for the simultaneous quantification of meropenem, linezolid, and levofloxacin

in human plasma and applied in human plasma of critical care patients. A semi-automated

microextraction by packed sorbent (MEPS) for sample preparation was used. All parameters in the

extraction step (pH, sample volume, sample dilution and number of aspiration – ejection cycles) and in

the desorption step (percentage of acetonitrile in the solvent of elution and number of aspirations of

elution solvent through the device) were statistically significant when the recovery was used as

response. The method showed good linearity with correlation coefficients, r2 > 0.9991 for the three

drugs, as well as high precision (RSD%< 10.83% in each case). Accuracy ranged from −7.8% to

+6.7%. The limit of quantification of the three drugs was established at 0.01 μg/mL for linezolid and

levofloxacin and 0.02 μg/mL for meropenem. Linezolid, meropenem, levofloxacin and the internal

standard were extracted from human plasma with a mean recovery ranged from 92.4% to 97.4%.

During validation, the concentration of meropenem, linezolid and levofloxacin was found to be stable

after 3 freeze-thaw cycles and for at least 24 h after extraction. This method will be subsequently used

to quantify the drugs in patients to establish if the dosage regimen given is sufficient to eradicate the

infection at the target site.

Investigation on the combined effect of cocaine and ethanol administration through a liquid

chromatography–mass spectrometry metabolomics approach

Elena Sánchez-López, Alberto Marcos, Emilio Ambrosio, Oleg A. Mayboroda, Antonio L. Crego

ABSTRACT

Alcohol is the most widely consumed legal drug, whereas cocaine is the illicit psychostimulant most

commonly used in Europe. The combined use of alcohol and cocaine is frequent among drug-abuse

consumers and leads to further exacerbation of health consequences compared to individual

consumption. The pharmacokinetic and metabolic interactions leading to an increase in their combined

toxicity still remains poorly understood. Here, the first metabolomics study of combined cocaine and

ethanol chronic exposure effects is reported. A Liquid Chromatography strategy based on sample

derivatization with 9-fluorenylmethyloxycarbonyl chloride and using a C18 column coupled to high

resolution Mass Spectrometry (time of flight analyzer) was employed to analyze plasma from rats

exposed intravenously to these drugs in a 52-min analysis. Using a combination of non-supervised and

supervised multivariate analysis the metabolic differences between our experimental groups were

explored and unraveled. A comparative analysis of the individual models and their variable importance

in the projection values have shown that every experiment intervention includes a subset of specific

metabolites. Eleven of these metabolites were annotated, where eight were unequivocally identified

using standards and three were tentatively identified by matching the MS/MS spectra to libraries. The

results demonstrated that the affected metabolic pathways were mainly those related to the metabolism

of different amino acids.

141

Validation of a dried blood spot method for therapeutic drug monitoring of citalopram,

mirtazapine and risperidone and its active metabolite 9-hydroxyrisperidone using HPLC–MS

Johanna Weber, Stefanie Oberfeld, Andrea Bonse, Klaus Telger, Georg Hempel

ABSTRACT

Citalopram, mirtazapine and risperidone are frequently prescribed for psychiatric illnesses such as

depression and psychosis or for aggressive behavior in elderly patients with dementia. The plasma

concentrations vary greatly between patients, especially in elderly patients. Thus, therapeutic drug

monitoring (TDM) increases the safety of antipsychotic treatment and a more rapid response to

treatment may be achieved. To facilitate TDM, the objectives of this study were to develop and

validate a reliable dried blood spot method to simultaneously quantify citalopram, mirtazapine and

risperidone including its active metabolite 9-hydroxyrisperidone. The blood punches were extracted by

methanol using an ultrasonic bath, purified by liquid–liquid extraction and analyzed by liquid

chromatography/mass spectrometry (LC–MS). All acceptance criteria of the EMA and FDA guidelines

for method validation were fulfilled. Linearity was shown over the range of 2.5–300 μg/L for all

substances. The analytes were stable for at least one month at all investigated storage conditions,

including storing at room temperature exposed to light. Retrieving capillary blood by finger-pricking

the assay was successfully applied in elderly patients. Venous serum samples were drawn

simultaneously to compare capillary blood with serum concentrations. Given the validated results and

the calculated capillary blood:serum ratio, the studied dried blood spot method offers an excellent

application in TDM and can be applied in ambulatory care.

Characterization of an unknown impurity in doxofylline using LC–MS and NMR

Peixi Zhu, Jingxian Lu, Liya Hong, Weike Su, Erwin Adams

ABSTRACT

During quality control of doxofylline, a novel impurity was detected, which was above the

identification threshold defined by ICH. First, a liquid chromatographic method compatible with mass

spectrometric (MS) detection was developed. Based on tandem multistage MS and high resolution MS

data, the unknown impurity was found to consist of two theophylline groups connected by a methylene

group. The structure was further confirmed by 1D and 2D nuclear magnetic resonance (NMR)

experiments after semi-preparative isolation. In addition, the formation of the impurity was also

discussed.

142

Determination of AB-CHMINACA and its metabolites in human hair and their deposition in

hair of abusers

Juhyun Sim, Han Soo Cho, Jaesin Lee, Sangwhan In, Eunmi Kim

ABSTRACT

Despite global efforts to control the abuse of synthetic cannabinoids, the high-level of turnover from

the market impedes regulation, endangering public health. N-[(1S)-1-(aminocarbonyl)-2-

methylpropyl]-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (AB-CHMINACA) is the most

popular synthetic cannabinoid in South Korea since its introduction in 2014. Nonetheless, few studies

have been carried out on AB-CHMINACA and its metabolites, and its deposition in human hair. The

purpose of this study was to develop and validate an analytical method for detection of AB-

CHMINACA and its six metabolites in hair using a liquid chromatography tandem mass spectrometry

(LC–MS/MS) system, for forensic applications. The methanol extracts of hair samples were

evaporated, filtered, and analyzed by LC–MS/MS with electrospray ionization in positive ion mode.

The limits of detection and quantification ranged from 0.5 to 10 pg/mg and 2 to 50 pg/mg,

respectively. Good linearity was achieved within the range of 5–1000 pg/mg or 10–1000 pg/mg

depending on the analyte. Intra- and inter-assay precision and accuracy values were below 15%. No

significant variation was observed using different sources of hair matrices. These validation results

proved the selectivity, accuracy and reproducibility of the method. The established method was

applied to 37 authentic samples from suspected synthetic cannabinoid users. AB-CHMINACA and its

two metabolites, AB-CHMINACA M2 and AB-CHMINACA M4, were detected. The concentration of

the parent drug was much higher than those of its metabolites, and the amount of AB-CHMINACA

M2 was greater than that of AB-CHMINACA M4 in all samples. No other metabolites were detected

in the samples.

Development of a new chlorogenic acid certified reference material for food and drug analysis

Dezhi Yang, LingTai Jiao, Baoxi Zhang, Guanhua Du, Yang Lu

ABSTRACT

This paper reports the preparation and characterization of a new chlorogenic acid (CHA) certified

reference material (CRM), which is unavailable commercially. CHA is an active ingredient found in

many geo-authentic Chinese medicinal materials and developed as an anti-cancer drug. In this work,

trace impurities were isolated and identified through various techniques. CHA CRM was quantified

with two analytical methods, and their results were in good agreement with each other. The certified

value and corresponding expanded uncertainty of CHA CRM reached 99.4% ± 0.2%, which was

calculated by multiplying the combined standard uncertainty by the coverage factor (k = 2), at a

confidence level of 95%. This CRM can be used to calibrate measurement system, evaluate or validate

measurement procedures, assign traceable property values to non-CRMs, and conduct quality control

assays.

143

Automation of plasma protein binding assay using rapid equilibrium dialysis device and Tecan

workstation

Zhengqi Ye, Craig Zetterberg, Hong Gao

ABSTRACT

Binding of drug molecules to plasma proteins is an important parameter in assessing drug ADME

properties. Plasma protein binding (PPB) assays are routinely performed during drug discovery and

development. A fully automated PPB assay was developed using rapid equilibrium dialysis (RED)

device and Tecan workstation coupled to an automated incubator. The PPB assay was carried out in

unsealed RED plates which allowed the assay to be fully automated. The plasma pH was maintained at

7.4 during the 6-h dialysis under 2% CO2 condition. The samples were extracted with acetonitrile and

analyzed by liquid chromatography tandem mass spectrometry. The percent bound results of 10

commercial drugs in plasma protein binding were very similar between the automated and manual

assays, and were comparable to literature values. The automated assay increases laboratory

productivity and is applicable to high-throughput screening of drug protein binding in drug discovery.

―Ghost peaks‖ of ezetimibe: Solution degradation products of ezetimibe in acetonitrile induced

by alkaline impurities from glass HPLC vials

Jianyang Jin, Zhiying Wang, Jinsheng Lin, Wenquan Zhu, Min Li

ABSTRACT

Unpredictable degradation of Ezetimibe solutions in pure acetonitrile occurs when they are stored in

glass HPLC vials. The occurrence of the two main degradation peaks and one minor peak was

unpredictable at the time of each sample preparation and over time, it appeared that approximately

15% of the sample solutions in glass HPLC vials would eventually show the degradation peaks. Once

the degradation peaks occurred in a particular vial, typically within 24 h, they would keep growing

until reaching a total yield of about 4–5%. Through a comprehensive investigation, it is determined

that the solution degradation is caused by a base-catalyzed process, during which ezetimibe undergoes

(1) dimerization to form two dimeric impurities, which have not been reported in the literature, and (2)

to a less degree, isomerization to produce an isomeric impurity that has been reported before.

144

Metabolite quantification by NMR and LC-MS/MS reveals differences between unstimulated,

stimulated, and pure parotid saliva

João Figueira, Sandra Gouveia-Figueira, Carina Öhman, Pernilla Lif Holgerson, Anders Öhman

ABSTRACT

Saliva is a readily available biofluid that is sensitive to metabolic changes and can be collected through

rapid and non-invasive collection procedures, and it shows great promise for clinical metabolomic

studies. This work studied the metabolite composition of, and the differences between, saliva samples

collected by unstimulated spitting/drooling, paraffin chewing-stimulated spitting, and parotid gland

suction using targeted nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography

coupled to tandem mass spectrometry (LC-MS/MS) for metabolite quantification. As applied here,

these two analytical techniques provide complementary metabolite information and together extend the

metabolome coverage with robust NMR quantification of soluble metabolites and sensitive targeted

LC-MS/MS analysis of bioactive lipids in specific metabolic pathways. The NMR analysis was

performed on ultrafiltrated (3 kDa cutoff) saliva samples and resulted in a total of 45 quantified

metabolites. The LC-MS/MS analysis was performed on both filtered and unfiltered samples and

resulted in the quantification of two endocannabinoids (AEA and PEA) and 22 oxylipins, which at

present is the most comprehensive targeted analysis of bioactive lipids in human saliva. Important

differences in the metabolite composition were observed between the three saliva sample collection

methods, which should be taken into consideration when designing metabolomic studies of saliva.

Furthermore, the combined use of the two metabolomics platforms (NMR and LC-MS/MS) proved to

be viable for research and clinical studies of the salivary metabolome.

Determination of AZD3759 in rat plasma and brain tissue by LC–MS/MS and its application in

pharmacokinetic and brain distribution studies

Shan Xiong, Mingxing Xue, Yanling Mu, Zhipeng Deng, Ruican Zhou

ABSTRACT

A simple and sensitive high performance liquid chromatography with tandem mass spectrometry (LC–

MS/MS) method for determination of AZD3759, a novel epidermal growth factor receptor tyrosine

kinase inhibitor, in rat plasma and brain homogenate was developed and validated over the range of

1.0–1000 ng/mL. Chromatographic separation was carried out on a C18 column with acetonitrile and

0.1% formic acid in water as mobile phase with gradient elution at a flow rate of 0.4 mL/min. The

lower limits of quantification (LLOQs) were 1.0 ng/mL for AZD3759 in both rat plasma and brain

homogenate. The intra-day and inter-day precision and accuracy of AZD3759 were well within the

acceptable limits of variation. The simple and sensitive LC–MS/MS method was successfully applied

to the pharmacokinetic and brain distribution studies following an oral administration of AZD3759 to

rats.

145

Studying the effects of natural extracts with metabolomics: A longitudinal study on the

supplementation of healthy rats with Polygonum cuspidatum Sieb. et Zucc.

Gregorio Peron, Jalal Uddin, Matteo Stocchero, Stefano Mammi, Stefano Dall‘Acqua

ABSTRACT

Background: A longitudinal study was performed to evaluate the effects of Polygonum cuspidatum

extract (standardized at 20% resveratrol) supplementation on healthy rats. The effects were explored

by monitoring urinary metabolome changes using UPLC-HRMS and 1H NMR-based approaches. The

aim of the study was to explore the effects of P. cuspidatum supplementation on a healthy animal

model using metabolomics, in order to determine possible modes of action and obtain information on

bioactivity.

Methods: Healthy Sprague-Dawley rats were orally supplemented with 100 mg/kg of dried P.

cuspidatum extract for 49 days and 24-h urinary outputs were collected. Samples were analysed by

untargeted UPLC-HRMS and 1H NMR approaches and the obtained data sets were modelled by an

adaptation of post-transformation of PLS2 to longitudinal studies. Putative markers were discovered

by a stability selection procedure and specific oxidative stress markers were monitored by a targeted

HPLC–MS/MS analysis to assess the in vivo antioxidant activity of P. cuspidatum extract.

Results: UPLC-HRMS and 1H NMR platforms showed two different but complementary patterns of

metabolites describing the changes ascribable to P. cuspidatum supplementation and using both

approaches, a comprehensive resveratrol metabolism and urinary excretion could be observed.

Markers of P. cuspidatum supplementation effects identified by UPLC-HRMS were mainly related to

its antioxidant activity and to a possible ―adaptogenic‖ activity. Urinary changes observed by 1H NMR

were mainly related to energy metabolism. UPLC-HRMS and 1H NMR metabolomics approaches

allowed the effects of a prolonged supplementation with P.

Identification and characterization of a thermally cleaved fragment of monoclonal antibody-A

detected by sodium dodecyl sulfate-capillary gel electrophoresis

Kei Kubota, Naoki Kobayashi, Masayuki Yabuta, Motomu Ohara, Koji Otsuka

ABSTRACT

This report describes a novel, comprehensive approach to identifying a fragment peak of monoclonal

antibody-A (mAb-A), detected by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-cGE).

The fragment migrated close to the internal standard (10 kDa marker) of SDS-cGE and increased

about 0.5% under a 25 °C condition for 6 months. Generally, identification of fragments observed in

SDS-cGE is challenging to carry out due to the difficulty of collecting analytical amounts of

fractionations from the capillary. In this study, in-gel digestion peptide mapping and reversed phase

liquid chromatography-mass spectrometry (RPLC–MS) were employed to elucidate the structure of

the fragment. In addition, a Gelfree 8100 fractionation system was newly introduced to collect the

fragment and the fraction was applied to the structural analysis of a mAb for the first time. These three

analytical methods showed comparable results, proving that the fragment was a fraction of heavy chain

HC1-104. The fragment contained complementarity determining regions (CDRs), which are significant

to antigen binding, and thus would affect the efficacy of mAb-A. In addition, SDS-cGE without the 10

kDa marker was demonstrated to clarify the increased amount of the fragment, and the experiment

revealed that the fragment increases 0.2% per year in storage at 5 °C.

146

Synchronous determination with double-wavelength by RP-HPLC-UV and optimization of

ultrasound-assisted extraction of phenolic acids from Caragana species using response surface

methodology

Zhi Zeng, Zhongyin Ji, Na Hu, Shasha Chen, Yourui Suo

ABSTRACT

The utilization of Caragana korshinskii Kom (CK) is currently concentrated on its ecological and fuel

functions. Little attention has been devoted to the analysis of their phenolic acid (PA) components. To

obtain more data for further utilization of CK, a new analysis protocol was tested to determine PAs

synchronously by RP-HPLC-UV with double-wavelength (280 nm and 320 nm) detection.

Specifically, separation of PA components was performed on a Hypersil Gold C18 reverse phase

column with gradient elution. A four-factor-three-level Box-Behnken design was implemented for

optimization of PA extraction. The results demonstrated that CK were rich primarily in chlorogenic

acid, vanillic acid, caffeic acid and rosmarinic acid. The total content of PAs in CK leaves was the

highest compared with its other parts. The distribution of total flavonoid content of CK was leaves >

flowers > bark, while that of the total phenolic content of CK was flowers > leaves > bark.

Untargeted metabolite analysis-based UHPLC-Q-TOF-MS reveals significant enrichment of p-

hydroxybenzyl dimers of citric acids in fresh beige-scape Gastrodia elata (Wutianma)

Chang-Jiang-Sheng Lai, Yuan Yuan, Da-Hui Liu, Chuan-Zhi Kang, Lu-Qi Huang

ABSTRACT

In order to comprehensively elucidate the chemical biosynthesis process of the beige-scape Gastrodia

elata Blume (Wutianma) as a traditional herbal medicines, the untargeted analysis–based UHPLC-

PDA-ESI-Q-TOF-MS reveals the metabolites ranging from the skeletons to novel dimers of citric

acids in fresh and dried immature/mature stem tubers. Interestingly, two novel types of dimers for

citric acids with the anhydride groups at sn-1 and/or sn-5 were discovered in fresh samples. Moreover,

the classical mono- versus novel di-mers, and the aglycons versus the glycosides could be easily

discriminated by signature fragmentation patterns and some novel adduct ions. The heat map of

contents demonstrated more p-hydroxybenzyl metabolites than gastroxyl ones were determined in

fresh Wutianma revealing a significant specificity with the lack of the sufficient gastrodin and

gastroxyl products in biosynthetic pathway.

147

Volume 141 July 2017

Matrix-assisted laser-desorption/ionization mass spectrometric imaging of olanzapine in a single

hair using esculetin as a matrix

Hang Wang, Ying Wang, Ge Wang, Lizhi Hong

ABSTRACT

Matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) for the analysis

of intact hair is a powerful tool for monitoring changes in drug consumption. The embedding of a low

drug concentration in the hydrophobic hair matrix makes it difficult to extract and detect, and requires

an improved method to increase detection sensitivity. In this study, an MSI method using MALDI-

Fourier transform ion cyclotron resonance was developed for direct identification and imaging of

olanzapine in hair samples using the positive ion mode. Following decontamination, scalp hair samples

from an olanzapine user were scraped from the proximal to the distal end three times, and 5 mm hair

sections were fixed onto an Indium-Tin-Oxide (ITO)-coated microscopic glass slide. Esculetin (6,7-

dihydroxy-2H-chromen-2-one) was used as a new hydrophobic matrix to increase the affinity,

extraction and ionization efficiency of olanzapine in the hair samples. The spatial distribution of

olanzapine was observed using five single hairs from the same drug user. This matrix improves the

affinity of olanzapine in hair for molecular imaging with mass spectrometry. This method may provide

a detection power for olanzapine to the nanogram level per 5 mm hair. Time course changes in the

MSI results were also compared with quantitative HPLC–MS/MS for each 5 mm segment of single

hair shafts selected from the MALDI target. MALDI imaging intensities in single hairs showed good

semi-quantitative correlation with the results from conventional HPLC–MS/MS. MALDI-MSI is

suitable for monitoring drug intake with a high time resolution.

A novel liquid chromatography/tandem mass spectrometry (LC–MS/MS) based bioanalytical

method for quantification of ethyl esters of Eicosapentaenoic acid (EPA) and Docosahexaenoic

acid (DHA) and its application in pharmacokinetic study

Sekarbabu Viswanathan, P.R.P. Verma, Muniyandithevar Ganesan, Jeganathan Manivannan

ABSTRACT

Omega-3 fatty acids are clinically useful and the two marine omega-3 fatty acids eicosapentaenoic acid

(EPA) and docosahexaenoic acid (DHA) are prevalent in fish and fish oils. Omega-3 fatty acid

formulations should undergo a rigorous regulatory step in order to obtain United States Food and Drug

Administration (USFDA) approval as prescription drug. In connection with that, despite quantifying

EPA and DHA fatty acids, there is a need for quantifying the level of ethyl esters of them in biological

samples. In this study, we make use of reverse phase high performance liquid chromatography coupled

with mass spectrometry (RP-HPLC–MS)technique for the method development. Here, we have

developed a novel multiple reaction monitoring method along with optimized parameters for

quantification of EPA and DHA as ethyl esters. Additionally, we attempted to validate the bio-

analytical method by conducting the sensitivity, selectivity, precision accuracy batch, carryover test

and matrix stability experiments. Furthermore, we also implemented our validated method for

evaluation of pharmacokinetics of omega fatty acid ethyl ester formulations.

148

Identification of a host cell protein impurity in therapeutic protein, P1

Deepti Ahluwalia, Harbhajan Dhillon, Thomas Slaney, Hangtian Song, Girija Krishnamurthy

ABSTRACT

Residual host cell proteins (HCPs) are process-related impurities present in biotherapeutics that can

pose safety health risks to patients. An adequate control of HCP levels in the final product, and

demonstration of HCP clearance throughout a product manufacturing process is critical for all

biotherapeutic products. Developing effective downstream purification processes can be challenging as

HCPs and product proteins may possess an affinity for each other or have similar physicochemical

properties, resulting in co-purification. In the current study, we identified the presence of CHO-

catalase subunit protein as an impurity present in purified P1 protein. This previously unreported HCP

impurity, was detected in P1 protein generated in Chinese hamster ovary (CHO) cells. Purified drug

substance samples contained elevated CHO HCP levels when measured using a commercial anti-CHO

HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit. This finding, prompted further

characterization of the HCP profile using 1D and 2D gels/ western blots using an anti-human IgG

antibody as well as a commercial anti-CHO HCP antibody (Cygnus 813) for the detection of host cell

proteins. The CHO-catalase protein has been characterized using a combination approach of one-

dimensional (1D) and two-dimensional (2D) gels and western blotting techniques, and the identity

confirmed using liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Western blot

analyses using the anti-CHO HCP antibody detected a potential HCP band at ∼60 kDa and a pI of ∼8

in the purified P1 sample. The 60 kDa HCP band was excised from 1D SDS-PAGE gels and LC-

MS/MS analysis identified it to be CHO-catalase subunit. The identity of catalase monomer was

further confirmed by western blot analysis using a specific anti-catalase antibody.

Simultaneous separation and determination of four uncaria alkaloids by capillary

electrophoresis using dual cyclodextrin system

Lou Li, Liying Xu, Meng Chen, Guangbin Zhang, Anjia Chen

ABSTRACT

The purpose of this study was to develop a simple, quick and precise capillary zone electrophoresis

method (CZE) for the separation and determination of uncaria alkaloids using dual cyclodextrins as

additives for the separation. The four analytes were baseline separated within 15 min at the applied

voltage of 15 kV with a running buffer (pH 5.7) consisting of 40.0 mM phosphate buffer, 161.7 mM 2-

hydroxypropyl-β-cyclodextrin (HP-β-CD) and 2.21 mM mono-(6-ethylenediamine-6-deoxy)-β-

cyclodextrin (ED-β-CD). Under the optimum conditions, a good linearity was achieved with

correlation coefficients from 0.9989 to 0.9992. The detection limits and the quantitation limits ranged

from 0.63 to 0.98 μg/mL and from 2.08 to 3.28 μg/mL, respectively. Excellent accuracy and precision

were obtained. Recoveries of the analytes varied from 97.1 to 103.2%. This method was suitable for

the quantitative determination of these alkaloids in the stem with hook of Uncaria rhynchophylla and

the formulations of Uncaria rhynchophylla.

149

CE method for the in-process control of the synthesis of active substances conjugated with gold

nanoparticles

Wioleta Maruszak, Elżbieta U. Stolarczyk, Krzysztof Stolarczyk

ABSTRACT

A fast capillary electrophoresis method was developed and validated for the in-process control (IPC)

of the synthesis of active substances (APIs) with gold nanoparticles (AuNPs). The capillary

electrophoresis method was key to ensure that the reaction step conducted in order to obtain AuNP and

API conjugates will produce the expected product without the presence of free APIs, which is a critical

parameter determining the quality of the synthetic material. Capillary electrophoresis was performed

using uncoated fused-silica capillaries with the effective length of 40 cm, 50 μm i.d. and the

background electrolyte consisted of 20 mM borate buffer (pH 8.5) with the application of

hydrodynamic injection 50 mbar/5s, voltage 20 kV, temperature of the capillary cassette 25 °C and UV

detection at 261 nm for GE, 541 nm for AuNP-GE, 227 nm for PE and 535 nm for AuNP-PE. During

validation the specificity, linearity, accuracy, precision, range, and stability of the sample solution

were confirmed. The linear regression (R2 = 0.999) between the corrected peak areas of the analytes

and their amount was fulfilled in the range from 2.4 μg/mL to 0.3 mg/mL for genistein and from 4.6

μg/mL to 0.6 mg/mL for pemetrexed. Within this range the method was proved to be accurate (99.0%

for genistein and 99.9% for pemetrexed) and precise for both analytes with the intra-day RSD values

of 0.77% and 0.97% for the migration time of genistein and pemetrexed, respectively. The inter-day

RSD values were 1.90% and 2.27% for the migration time of genistein and pemetrexed, respectively.

The LOD and LOQ values for pemetrexed were 1.4 μg/mL and 4.6 μg/mL, respectively, and for

genistein 0.72 μg/mL and 2.4 μg/mL, respectively. The results obtained during the validation indicate

that the method is sufficient to be applied for the IPC of the synthesis of APIs with gold nanoparticles.

Achievable separation performance and analysis time in current liquid chromatographic

practice for monoclonal antibody separations

Szabolcs Fekete, Jean-Luc Veuthey, Davy Guillarme

ABSTRACT

The separation performance of a chromatographic system is often described in terms of column

efficiency and peak capacity. Thanks to the new developments in column technology over the past few

years, the achievable peak capacity drastically improved and the analysis time can be significantly

shortened. Indeed, highly efficient wide-pore reversed-phase (RPLC) materials packed with small fully

porous and superficially porous particles can be successfully used for the analytical characterization of

therapeutic proteins. For non denaturating chromatographic approaches, such as ion exchange (IEX)

and size-exclusion chromatography (SEC), non-porous ion-exchanger as well as sub −3 μm size

exclusion supports are commercially available and open new avenues in protein separations. In this

study, the current possibilities offered by chromatography for the characterization of monoclonal

antibody (mAb) are discussed. For this purpose, recently published data have been reviewed and

calculations were performed to compare the maximum achievable peak capacity and related analysis

times using typical samples under RPLC, IEX and SEC conditions. Carefully chosen realistic column

pressure, mobile phase temperature, flow rate and column dimensions were considered for the case

studies discussed through the paper.

150

Identification and characterization of process-related substances and degradation products in

apremilast: Process optimization and degradation pathway elucidation

Yuting Lu, Xiaoyue Shen, Taijun Hang, Min Song

ABSTRACT

This study aims at investigating the separation, identification and characterization of related substances

in apremilast by LC–MS hyphenated techniques, as well as the synthesis optimization and the

degradation pathways elucidation. Forced degradation studies were conducted under the ICH

prescribed stress conditions. The chromatographic separation was achieved on XBridge C18 column

(4.6mm × 150 mm, 3.5 μm) using a mobile phase consisting of water adjusted to pH 3.0 with formic

acid as solvent A and acetonitrile as solvent B in linear gradient elution program. Twelve related

substances were detected all together in apremilast and its stress samples. Their structures were

identified mainly through positive ESI high-resolution TOF-MS analysis of the parent ions' accurate

masses and elemental compositions, and the corresponding MS/MS spectra elucidation. There were

three process-related substances and nine degradation products, seven of them were first reported. Two

degradation products and one process-related substance were further verified by semi-preparation and

NMR determination. Their origins and formation mechanisms were also discussed, based on which

effective approaches for the synthesis optimization were conducted. Therefore, the related substances

investigation are valuable for apremilast manufacturing process optimization and quality control.

Antiproliferative hydroxy-fatty acids from the fodder legume Stylosanthes guianensis

Marco Clericuzio, Bruno Burlando, Barbara Borghesi, Annalisa Salis, Laura Cornara

ABSTRACT

Stylosanthes guianensis is a fodder legume native from South America and widely grown worldwide.

Dried plant material was purchased on the web and taxonomically identified by light and SEM

microscopy, and morphological analysis of plants germinated from seeds. The plant was extracted with

dichloromethane:2-propanol (9:1). Bioguided fractionation using calcein-AM cytotoxicity assay on

HeLa and A431 tumor cells allowed to isolate a lipophilic fraction, endowed with strong cytotoxicity.

By means of 1- and 2-D NMR, HPLC–MS, and HR-ESIMS it could be seen that the fraction was an

inseparable mixture of complex lipids, mainly consisting of esterified 3-hydroxy fatty acids. Acidic

methanolysis of the mixture yielded 3-OH C10 and C12 carboxylic acids, together with palmitic,

stearic, and arachidonic acids. Mass values indicate the presence of dimeric and trimeric combinations

of 3-hydroxy, C10/C12 acids, and C16/C18/C20 acids, linked via ester bond. Monomeric hydroxyl-

fatty acids were also observed, in particular derivatives of mono-hydroxy and di-hydroxy linolenic,

linoleic, and oleic acids. 3-O-acylated, esterified fatty acids are unusual in higher plants, and recall

motifs of Gram-negative endotoxin lipid A. These oxylipins are likely to be responsible for the

antiproliferative activity of S. guianensis, suggesting possible use of the plant in the development of

antitumor drugs.

151

Development and validation of a liquid chromatographic method for the analysis of squaric acid

dibutyl ester and its impurities

Marwa F. Mansour, Peixi Zhu, Ann Van Schepdael, Erwin Adams

ABSTRACT

A simple, fast and selective stability indicating liquid chromatographic method has been described for

the simultaneous determination of squaric acid dibutyl ester and its impurities. The chromatographic

separation was achieved on a C2 column (250 mm × 4.6 mm i.d., 5 μm) using a mobile phase

consisting of 0.15% phosphoric acid – acetonitrile – methanol (30:60:10, v/v/v). Isocratic elution was

performed at a flow rate of 1.0 mL min−1. The analytes were detected by UV at 252 nm. The method

was validated according to the ICH guidelines and satisfactory results were obtained. The specificity

of the developed method was tested using forced degradation solutions of the drug substance.

Characterization of squaric acid dibutyl ester and its forced degradation products was achieved by

coupling mass spectrometry (MS) to the liquid chromatographic (LC) system. The method was

successfully applied for quality control purposes including assay and determination of related

compounds as required by regulatory guidelines to ensure its safety and efficacy since no monograph

is available in official compendia.

Macro-Raman spectroscopy for bulk composition and homogeneity analysis of multi-component

pharmaceutical powders

Hui Wang, David Barona, Sulayman Oladepo, Lisa Williams, Reinhard Vehring

ABSTRACT

A new macro-Raman system equipped with a motorized translational sample stage and low-frequency

shift capabilities was developed for bulk composition and homogeneity analysis of multi-component

pharmaceutical powders. Different sampling methods including single spot and scanning measurement

were compared. It was found that increasing sample volumes significantly improved the precision of

quantitative composition analysis, especially for poorly mixed powders. The multi-pass cavity of the

macro-Raman system increased effective sample volumes by 20 times from the sample volume

defined by the collection optics, i.e., from 0.02 μL to about 0.4 μL. A stochastic model simulating the

random sampling process of polydisperse microparticles was used to predict the sampling errors for a

specific sample volume. Comparison of fluticasone propionate mass fractions of the commercial

products Flixotide® 250 and Seretide® 500 simulated for different sampling volumes with

experimentally measured compositions verified that the effective sample volume of a single point

macro-Raman measurement in the multi-pass cavity of this instrument was between 0.3 μL and 0.5 μL.

The macro-Raman system was also successfully used for blend uniformity analysis. It was concluded

that demixing occurred in the binary mixture of l-leucine and d-mannitol from the observation that the

sampling errors indicated by the standard deviations of measured leucine mass fractions increased

during mixing, and the standard deviation values were all larger than the theoretical lower limit

determined by the simulation. Since sample volume was shown to have a significant impact on

measured homogeneity characteristics, it was concluded that powder homogeneity analysis results, i.e.,

the mean of individual test results and absolute and relative standard deviations, must be presented

together with the effective sample volumes of the applied testing techniques for any measurement of

powder homogeneity to be fully meaningful.

152

Isolation, identification and characterization of potential impurities of anidulafungin

Lanning Zhao, Qilong Wang, Yi Bie, Xiaoxia Lu

ABSTRACT

Eight impurities ranging from 0.03 to 0.97% in anidulafungin bulk drug were detected by HPLC. Four

impurities (Imp-I, Imp-II, Imp-III and Imp-VIII) among impurities were isolated from the self-

prepared or marketed samples of anidulafungin bulk drug by means of preparative HPLC. A thorough

study was undertaken to characterize these impurities and based on 1D (1H, 13C, H-D, DEPT 90 and

135) and 2D (COSY, TOCSY, HSQC, HMBC) NMR and ESI–MS spectral data. Based on the

characterization data, Imp-I was found to be known open-chain hydrolysis product formed during the

synthesis and degradation. Imp-II and Imp-III was lacked a methyl group at the C-4 and C-8 in

anidulafungin, respectively, whereas Imp-VIII contained a methoxy group at the C-23. The latter three

new impurities were identified as process-related substances.

Dialkyl anionic surfactant in field-amplified sample injection and sweeping-micellar

electrokinetic chromatography for determination of eight leanness-promoting β-agonists in

animal feeds

Sung-Yu Hsieh, Chun-Chi Wang, Hwang-Shang Kou, Shou-Mei Wu

ABSTRACT

The beta-adrenergic agonists (β-agonists) working as repartitioning agents that make the carcass leaner

and enhance the feeding efficiency in animals have been banned in the European Union, China and

Taiwan. Here, traditional anionic surfactants, such as sodium dodecyl sulfate (SDS) were replaced

with sodium di-(2-ethylhexyl)-sulfosuccinate (AOT) in field-amplified sample injection and sweeping-

micellar electrokinetic chromatography (FASI-sweeping MEKC) for simultaneous analysis of eight β-

agonists in animal feeds. The AOT vesicles provided a better resolution of β-agonists than micelles of

SDS. The detection limits of the eight β-agonists were above 5 ng/mL by using this stacking capillary

electrophoresis (CE) method. In comparison of traditional MEKC method (sample injection, 1 psi for 5

s), the stacking strategy provided 400–2000 fold sensitivity enhancement. After method validation, this

method was successfully applied for analyzing four animal feeds, and none β-agonist was detected.

This strategy possessing good resolution of eight β-agonists was suitable for serving as a tool for

routine analysis of animal feeds.

153

Isolation and structural characterization of glucosylceramides from Ethiopian plants by

LC/APCI-MS/MS

Efrem N. Tessema, Tsige Gebre-Mariam, Christian E.H. Schmelzer, Reinhard H.H. Neubert

ABSTRACT

Chronic skin conditions such as atopic dermatitis, psoriasis and aged skin are characterized by

defective skin barrier and dryness which are associated with reduced levels of skin ceramides (CERs).

The beneficial effects of plant-derived CERs for skin hydration and skin barrier recovery have been

shown in several studies. Although plenty of glucosylceramide (GlcCER)-based dietary supplements

meant for skin barrier improvement have been marketed, there are limited commercial sources of plant

GlcCERs. In an attempt to explore alternative GlcCER sources, a reversed phase LC–MS/MS method

with atmospheric pressure chemical ionization (APCI) interface was developed for separation and

structural identification of GlcCERs isolated from three plants. The GlcCERs were extracted from the

seeds of grass pea (Lathyrus sativus L.), Ethiopian mustard (Brassica carinata) and haricot bean

(Phaseolus vulgaris) and purified by column chromatography and preparative LC–MS. The individual

GlcCER species were further separated and qualitatively analyzed by LC/APCI-MS/MS. The amount

of GlcCERs in each plant was quantified by HPTLC. All GlcCER species detected in the three plants

consisted of C18 di/trihydroxy sphingoid bases amide linked with hydroxy fatty acids (C14-C24). The

trihydroxy SBs were acylated with very long chain FAs (C22-C24). The major GlcCERs derived from

grass pea, Ethiopian mustard and haricot bean are composed of sphingenine (d18:1) linked to

hydroxypalmitic acid (h16:0), 4-hydroxy-8-sphingenine (t18:1) coupled with hydroxynervonic acid

(h24:1) and sphingadienine (d18:2) joined with h16:0, respectively. The GlcCERs contents in haricot

bean (161.2 mg/kg) and grass pea (130.0 mg/kg) were found to be higher compared to Ethiopian

mustard (71.8 mg/kg).

Drug-protein binding mechanism of juglone for early pharmacokinetic profiling: Insights from

ultrafiltration, multi-spectroscopic and molecular docking methods

Pan Zhao, Guihua Gao, Lianjun Zhang, Qian Cai, Xiaohong Hou

ABSTRACT

Juglone (JL), as one of the major bioactive components present in the bark of Juglans mandshruica

Maxim, exhibits versatile bioactivities, especially anti-cancer activity. To better understand the

pharmacokinetic properties of juglone, the protein binding rate of juglone was determined by

ultrafiltration method, and the binding affinity and mechanism between JL and human serum albumin

(HSA) was investigated in vitro through multi-spectroscopic, thermodynamic, and molecular modeling

methods. The binding degree of JL was measured more than 99.0% which suggested that JL had high

binding ability to serum albumin. Fluorescence data showed that juglone quench the intrinsic

fluorescence of HSA upon forming the JL–HSA nonfluorescent complex at 1:1 stoichiometric

proportion, and the complex formation had a high affinity of 104 L·mol−1. Meanwhile, the site marker

competitive experiments and the thermodynamic parameters (ΔG = -26.08 kJ·mol−1, ΔH = –16.34

kJ·mol−1, ΔS = 32.69 J·mol−1·K−1) indicated that juglone could spontaneously bound to the site I

(subdomain IIA) of HAS through hydrophobic and hydrogen bonding interactions. As further revealed

by the synchronous fluorescence, three-dimensional fluorescence, Fourier transform infrared (FT-IR)

and circular dichroism (CD) spectroscopy, JL could cause conformational and structural alterations of

HSA. Additionally, molecular docking was employed to further define the specific binding site and the

result was in accordance with the conclusion of experimental analysis.

154

Quantification of alprenolol and propranolol in human plasma using a two-dimensional liquid

chromatography (2D-LC)

Virgínia M.F. Gonçalves, Patrícia Rodrigues, Cláudia Ribeiro, Maria E. Tiritan

ABSTRACT

A new two-dimensional liquid chromatography (2D-LC) method using a column switching valve, with

a restricted-access media (RAM) column in the first dimension was developed and validated for the

quantification of two β-blockers in human plasma. Several parameters, such as sample collection,

mobile phase composition and flow rate for sample cleanup, transference and analytical separation of

the β-blockers were investigated and optimized. The developed method allowed for the simultaneous

pre-treatment and quantification of alprenolol (ALP) and propranolol (PRO) in human plasma in less

than 25 min. The method consisted in the injection of 100 μL of plasma samples on the RAM alkyl-

diol-silica column (Lichrospher® RP-18 ADS, 25 μm) with water/acetonitrile (98:2, v/v; at a flow rate

of 2.0 mL/min) and then transferred (via a six-port valve) to the analytical column (Luna PFP (2), 150

× 4.6 mm ID, 100 Å, 3 μm) with 0.1% (v/v) triethylamine in water acidified with trifluoroacetic acid

(pH = 3)/acetonitrile (74:26, v/v) at a flow rate of 0.6 mL/min in a back-flush mode. The column oven

temperature was optimized to 42 °C and the fluorescence detector set at 280 nm and 310 nm

(excitation and emission, respectively). The method was validated according to the European

Medicines Agency‘s guidelines and was linear (r2 > 0.999) over a dynamic range of 5.0 – 200 ng/mL.

Recoveries ranged from 90.2 and 107% and the lower limit of quantification was 5.0 ng/mL for both

compounds. Precision was expressed as a percentage of relative standard deviation and did not exceed

11%. Finally, the method was successfully applied to determine the plasma concentration of PRO in

four healthy volunteers.

An LC–MS/MS method for quantification of AC0010, a novel mutant-selective epidermal

growth factor receptor (EGFR) inhibitor, and its metabolites in human plasma and the

application to a pharmacokinetic study

Lu Wang, Xin Zheng, Weicong Wang, Pei Hu, Ji Jiang

ABSTRACT

AC0010 is an irreversible, mutant-selective EGFR inhibitor that effectively inhibits EGFR active and

T790 M resistance mutations in non-small cell lung cancer (NSCLC). A sensitive ultra-performance

liquid chromatography – tandem mass spectrometry (UPLC–MS/MS) method was developed and fully

validated for determining AC0010 and its metabolites in human plasma. The samples were purified by

solid-phase extraction (SPE) columns and separated on a BEH C18 column. Electrospray ionization

(ESI) in positive ion mode and multiple reaction monitoring (MRM) were used to monitor the ion

transitions of AC0010 (m/z 488 → 257) and its metabolites M1 (m/z 474 → 403), M2 (m/z 504 →

487), M4 (m/z 434 → 377), M7 (m/z 490 → 405), MII-1 (m/z 651 → 434) and MII-2 (m/z 609 →

434). The results revealed that the method had excellent selectivity and linearity. Intra-day and inter-

day precisions (in terms of relative standard deviation, RSD) were lower than 14.4% and the

accuracies (in terms of relative error, RE) were within the range of ±10.3% for all the analytes. The

lower limit of quantification (LLOQ), stability, matrix effect and extraction recovery were also

validated and satisfied the criteria of validation. Finally, the method was successfully applied to a

pharmacokinetic study of NSCLC patients after a single oral dose of 350 mg of AC0010.

155

Validation and application of an ultrahigh-performance liquid chromatographic-Orbitrap mass

spectrometric method for the simultaneous detection and quantification of volatile and non-

volatile organic acids in human faecal samples

Claudio Gardana, Cristian Del Bo‘, Paolo Simonetti

ABSTRACT

A simple and selective ultrahigh-performance liquid chromatographic-Orbitrap mass spectrometric

(UHPLC-HR-MS) method was developed and validated for the simultaneous detection and

quantification of short-chain fatty acids (SCFAs) such as acetic, propionic, butyric, isobutyric, valeric,

isovaleric, 2-methyl-butyric (IS) and lactic, pyruvic and succinic acid in human faecal samples. A

simple extraction procedure with 0.001% formic acid in water was performed on 40 samples. The

extracts were centrifuged and analyzed by UHPLC-HR-MS on a sub-2 μm column using gradient

elution; meanwhile, the same samples were analyzed by GC-FID and HPLC-UV as reference methods

The UHPLC-HR-MS method showed a recovery of 83–105%, a repeatability of 2.2–8.3% and an

intermediate precision of 2.9–9.4%. The LOD and LLOQ were in the range of 0.04–0.23 and 0.2–0.5

μg/ml, respectively. Regarding the SCFAs, statistical analysis showed a good correlation between the

data obtained by UHPLC-HR-MS and those provided by GC-FID (p > 0.05). On the contrary, the LC-

UV data were not in agreement with those obtained by UHPLC-HR-MS determination (p < 0.05). To

the best of our knowledge, this is the first method available for the simultaneous extraction and

quantification of SCFAs, lactic, pyruvic and succinic in faecal samples by UHPLC-HR-MS.

Validation of a SPE HPLC–UV method for the quantification of a new ER-specific

photosensitizer OR-141 in blood serum using total error concept

Emilie Bony, Yohan Mace, Adán Pinto, David Delvaux, Joëlle Quetin-Leclercq

ABSTRACT

The aim of this work is to develop the first validated HPLC–UV method quantification in blood serum

for a new endoplasmic reticulum (ER)-specific benzophenazine photosensitizer (OR-141). A fast solid

phase extraction (SPE) cleaning sample procedure was achieved on C18 encapped (ec) SPE cartridges

and the separation was performed on a RP-18e column (5 μM) using an isocratic elution with

methanol. The method has been fully validated according to accuracy profiles based on total error and

tolerance intervals. Calibration was performed in the matrix and trueness (<4.25% relative bias),

repeatability (<4.75% relative standard deviation (RSD)), intermediate precision (<5.37% RSD),

selectivity, response function, linearity, and dilution effect were evaluated for both OR-141 regio-

isomers. Afterwards the developed method was successfully applied to perform the quantitative

determination of OR-141 in mouse blood serum samples in a preliminary pharmacokinetic study.

156

Determination of a novel anticancer AMPK activator hernandezine in rat plasma and tissues

with a validated UHPLC–MS/MS method: Application to pharmacokinetics and tissue

distribution study

Yang Song, Zhibin Wang, Baozhen Zhang, Yujia Zhang, Xuesong Feng

ABSTRACT

Hernandezine, a novel anticancer AMPK activator, is a major active constituent of Thalictrum

Ranunculaceae. A simple, specific and sensitive liquid chromatography tandem mass spectrometry

(LC–MS/MS) method has been developed and validated for the quantification of hernandezine in rat

plasma and tissues after intravenous administration. Sample preparation was carried out through a

protein-precipitation extraction with acetonitrile using tetrandrine as internal standard (IS). The

chromatographic separation was achieved by using an Agilent ZORBAX Eclipse Plus C18 column

with a mobile phase of acetonitrile and water (containing 10 mM ammonium acetate) in an isocratic

elution way. The mass spectrometry (MS) analysis was conducted in positive ionization mode with

multiple reaction monitoring (MRM) transitions at m/z 653.4 → 411.2 for hernandezine and m/z 623.3

→ 381.3 for tetrandrine (IS). Calibration curves were linear over the ranges of 20.0–4000 ng/ml f or

both plasma samples and tissue samples (r > 0.991). The lower limit of quantification (LLOQ) was

20.0 ng/ml. The intra-day and inter-day precision (RSD%) were less than 14.0%, while the accuracy

was ranged from 85.2% to 114.9%. Finally, this developed method was successfully applied in the

pharmacokinetics and tissue distribution study of hernandezine after intravenous administration.

LC–MS/MS determination of tranexamic acid in human plasma after phospholipid clean-up

Nicolas Fabresse, Fanta Fall, Isabelle Etting, Philippe Devillier, Stanislas Grassin-Delyle

ABSTRACT

Tranexamic acid is a widely used antifibrinolytic drug but its pharmacology and pharmacokinetics

remains poorly understood. Owing to the recent knowledge on phospholipid-induced matrix effects

during human plasma analysis, our aim was to develop a liquid chromatography-mass spectrometry

method for the quantitation of tranexamic acid after efficient sample clean-up. Sample preparation

consisted in phospholipid removal and protein precipitation. Hydrophilic interaction liquid

chromatography was used and the detection was achieved with multiple reaction monitoring. The

method was validated according to the European Medicine Agency guideline in the range 1.0–1000.0

μg/mL. The performance of the method was excellent with a precision in the range 1.2-3.0%, an

accuracy between 88.4 and 96.6% and a coefficient of variation of the internal standard-normalized

matrix factor below 6.7%. This method is suitable for the quantification of tranexamic acid in the wide

range of concentrations observed during clinical studies, with all the advantages related to

phospholipid removal.

157

Metabolic profiles of neotuberostemonine and tuberostemonine in rats by high performance

liquid chromatography/quadrupole time-of-flight mass spectrometry

Yongbin Tong, Weitong Xu, Yan Wu, Liting Ou, Chaofeng Zhang

ABSTRACT

Neotuberostemonine (NS) and tuberostemonine (TS), a pair of stereoisomers, are the active

components contained in Stemona tuberosa, an antitussive herbal medicine in China. Two isomers

have different pharmacological efficacies, which will be related with their in vivo disposition.

However, the metabolic fates of NS and TS remain unknown. A method of high performance liquid

chromatography/quadrupole time-of-flight mass spectrometry coupled with mass detect filter

technique was established to investigate the metabolites in rat plasma, bile, urine, and feces after oral

administration of the equal doses of NS and TS. The results showed that NS produced 48 phase I

metabolites, including NS, 3 hydrolyzed, 14 hydroxylated, 20 monohydrolyzed + hydroxylated and 10

dihydrolyzed + hydroxylated metabolites. The number of detected NS metabolites was 11, 39, 22 and

30 in plasma, bile, urine and feces. TS yielded 23 phase I metabolites, including TS, 3 hydrolyzed, 7

hydroxylated, 9 monohydrolyzed + hydroxylated and 3 dihydrolyzed + hydroxylated metabolites.

Besides, TS yielded 9 phase II metabolites, including 1 glucuronic acid and 2 glutathione conjugates,

and the later further degraded and modified into cysteine-glycine, cysteine and N-acetylcysteine

conjugates. The number of detected TS metabolites was 9, 24, 24 and 15 in plasma, bile, urine and

feces. Different metabolic patterns may be one of the main reasons leading to different

pharmacological effects of NS and TS.

Comprehensive profiling and characterization of coumarins from roots, stems, leaves, branches,

and seeds of Chimonanthus nitens Oliv using ultra-performance liquid

chromatography/quadrupole-time-of-flight mass spectrometry combined with modified mass

defect filter

Ting Tan, Yun Luo, Chen-Cong Zhong, Xu Xu, Yulin Feng

ABSTRACT

Chimonanthus nitens Oliv (CNO), having been studied and developed as the tea beverages, tea raw

materials and preparations, an unique species in China, have been extensively used for treating colds

and influenza for centuries. In the present study, a method based on the modified mass defect filter

(MDF) was firstly developed and validated for comprehensive profiling of coumarins in the different

parts of CNO via ultra-performance liquid chromatography tandem quadrupole time-of-flight mass

spectrometry (UPLC-QTOF/MS). The five-point screening approach based on MDF and the visual

isotopic ion technique could rapidly screen the interested precursor ions. The fragmentation behavior

of coumarins was systematically investigated and a total of 42 coumarins including 27 potential new

ones were unambiguously or tentatively identified in the CNO. Collectively, the results obtained in the

present work may provide useful information for future utilization of CNO.

158

New analytical method for determination of epimer metabolites in rat plasma after oral

administration of Paeoniflorin by UPLC-TOF-MS following picolinoyl derivatization

Zhigang Wang, Shuhan Tang, Masao Hattori, Hailong Zhang, Ning Zhang

ABSTRACT

A highly sensitive analytical method was developed to study the in vivo metabolism of paeoniflorin,

one of the most principal components in traditional Chinese medicine. After hydrolyzation with

sulfatase, the epimer metabolites 7S-paeonimetabolin I and 7R-paeonimetabolin I of paeoniflorin in rat

plasma were successfully detected and well separated by LC–MS following picolinoyl derivatization

for the first time. Borneol was used as the internal standard to quantify 7S-paeonimetabolin I and 7R-

paeonimetabolin I in rat plasma. 7S-paeonimetabolin I and 7R-paeonimetabolin I show similar but

different pharmacokinetic behavior. 7S-paeonimetabolin I reached the maximum mean plasma

concentration of 45.7 ± 4.6 ng/mL at about 1.5 h after oral administration of paeoniflorin at a dose of 5

mg/kg, while 7R-paeonimetabolin I reached the maximum mean plasma concentration of 39.2 ± 3.5

ng/mL at about 1.5 h. The full metabolic pathway of paeoniflorin in rats was proposed. The

monoterpene compound paeoniflorin was found to be metabolized to the sulfate of 7S-

paeonimetabolin I and 7R-paeonimetabolin I in vivo which maybe responsible for the pharmacological

effect of paeoniflorin.

Application of 2D-NMR with room temperature NMR probes for the assessment of the higher

order structure of filgrastim

Robert G. Brinson, Houman Ghasriani, Derek J. Hodgson, Kristie M. Adams, John P. Marino

ABSTRACT

The higher order structure (HOS) of biotherapeutics is a critical quality attribute that can be evaluated

by nuclear magnetic resonance (NMR) spectroscopy at atomic resolution. NMR spectral mapping of

HOS can be used to establish HOS consistency of a biologic across manufacturing changes or to

compare a biosimilar to an innovator reference product. A previous inter-laboratory study performed

using filgrastim drug products demonstrated that two-dimensional (2D)-NMR 1HN-15NH

heteronuclear correlation spectroscopy is a highly robust and precise method for mapping the HOS of

biologic drugs at natural abundance using high sensitivity NMR ‗cold probes.‘ Here, the applicability

of the 2D-NMR method to fingerprint the HOS of filgrastim products is demonstrated using lower

sensitivity, room temperature NMR probes. Combined chemical shift deviation and principal

component analysis are used to illustrate the performance and inter-laboratory precision of the 2D-

NMR method when implemented on room temperature probes.

159

Influence of sulfur fumigation on the chemical profiles of Atractylodes macrocephala Koidz

evaluated by UFLC–QTOF–MS combined with multivariate statistical analysis

Xue Sun, Xiao-bing Cui, Hong-mei Wen, Chen-xiao Shan, Wei Li

ABSTRACT

In the present study, the chemical compositions of Atractylodes macrocephala Koidz. (AMK) were

analyzed systematically and influence of sulfur fumigation on the chemical profiles was evaluated by

ultrafast flow liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry

(UFLC–QTOF–MS) combined with multivariate statistical analysis. 52 components were detected

from non-fumigated AMK (NF-AMK) and 28 components were newly produced after sulfur

fumigation, out of which 59 major peaks were identified. The concentrations of 20 compounds

significantly decreased and 37 compounds obviously increased. The potential structural transformation

mechanism of terpenoids was explored to illustrate the correlation of the components contents before

and after sulfur fumigation. Eight sulfur-containing/dehydrated-integrated atractylenolides that

evolved from the NF-AMK were screened out as potential characteristic chemical markers to examine

the post-harvest handling procedures of commercial AMK with excessive sulfur fumigation and

maintain consistent quality.

A sandwich immunoassay for brucellosis diagnosis based on immune magnetic beads and

quantum dots

Li Li, Dehui Yin, Kun Xu, Yushen Liu, Juan Li

ABSTRACT

Brucellosis is a serious zoonosis with a rapid increase in incidence across epidemic regions. Currently,

there are numerous methods for diagnosing brucellosis. However, these studies often have a few

defects, such as low sensitivity, poor specificity, time consuming, laborious, and even potential

biological risk. To overcome these shortcomings, we have developed a rapid, sensitive and accurate

diagnosis procedure for brucellosis based on the immune magnetic beads (IMB) probe and quantum

dots (QDs) – staphylococcal protein A (SPA) probe. With the presence of Brucella antibody in the

tested serum, the QDs-SPA probe links to the IMB probe and an immune-sandwich complex is

formed. As a result, the fluorescence intensity from QDs increased significantly and was correlated

with the amount of Brucella antibody. Under the optimized conditions, 248 blood samples were

detected and the diagnosis effect was evaluated. The area under the receiver-operating characteristic

(ROC) curve was 0.970 (95% confidence interval (CI), 0.9479–0.9920). The diagnostic sensitivity was

96.15% (95% CI, 91.82–98.58%), the diagnostic specificity was 94.12% (95% CI, 87.64–97.81%)

with a fluorescence intensity cutoff value of 150.4 and the detection time was only 100 min. This

diagnostic procedure can be satisfactorily applied to the diagnosis of brucellosis.

160

Systematically characterize the absorbed effective substances of Wutou Decoction and their

metabolic pathways in rat plasma using UHPLC-Q-TOF-MS combined with a target network

pharmacological analysis

Tengfei Xu, Shizhe Li, Yufei Sun, Zifeng Pi, Zhiqiang Liu

ABSTRACT

Wu-tou Decoction (WTD) is a famous traditional Chinese medicine (TCM) formula which is applied

to treat arthritis and pain of joints. In this study, a sensitive and rapid method was developed for the

separation and identification of the absorbed constituents and metabolites of WTD in the rats plasma

by ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass

spectrometry (UHPLC-Q-TOF-MS). A total of 22 absorbed prototype constituents and 49 metabolites

were identified or tentatively characterized in dosed plasma. The possible metabolic pathways of these

constituents involved sulfation, glucuronidation, demethylation, hydroxylation and so on. What‘s

more, we optimized the conventional process ways of network pharmacology and proposed a new

concept called target-network pharmacology (T-NP). T-NP method used the absorbed constituents and

the corresponding target proteins to generate compound-target network, and compare to the

conventional method indifferent using the compounds collected from herbs, it could reduce the false

positive results. We found that the following proteins were related to the WTD therapeutic effects,

such as PTGS2, PTGS1, MAPK3, PPARG, TNF, IL4 and IL6. On the whole, the proposed method

clearly presented the metabolic processes of WTD and the results gave a comprehensive metabolic

profile of WTD in vivo for the first time.

Nontargeted metabolomics approach for the differentiation of cultivation ages of mountain

cultivated ginseng leaves using UHPLC/QTOF-MS

Xiangwei Chang, Juanjuan Zhang, Dekun Li, Dazheng Zhou, Zhengliang Ye

ABSTRACT

The adulteration or falsification of the cultivation age of mountain cultivated ginseng (MCG) has been

a serious problem in the commercial MCG market. To develop an efficient discrimination tool for the

cultivation age and to explore potential age-dependent markers, an optimized ultra high-performance

liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS)-based

metabolomics approach was applied in the global metabolite profiling of 156 MCG leaf (MGL)

samples aged from 6 to 18 years. Multivariate statistical methods such as principal component analysis

(PCA) and partial least squares discriminant analysis (PLS-DA) were used to compare the derived

patterns between MGL samples of different cultivation ages. The present study demonstrated that 6–

18-year-old MGL samples can be successfully discriminated using two simple successive steps,

together with four PLS-DA discrimination models. Furthermore, 39 robust age-dependent markers

enabling differentiation among the 6–18-year-old MGL samples were discovered. The results were

validated by a permutation test and an external test set to verify the predictability and reliability of the

established discrimination models.

161

Studies on the metabolites difference of psoralen/isopsoralen in human and six mammalian liver

microsomes in vitro by UHPLC–MS/MS

Aihong Yang, Junxiu Chen, Yetao Ma, Lili Wang, Xin He

ABSTRACT

Psoralen and isopsoralen are found in many fruits, vegetables and traditional Chinese medicines

(TCM), such as Ficus carica L., Celery, Fructus Psoraleae etc. Modern pharmacological studies found

that psoralen and isopsoralen can show estrogen-like activity, antitumor, and antibacterial activities

etc. However, some research results also show some liver damage associated with the use of

psoralen/isopsoralen or related medicines in human. Many studies focus on the pharmacological

activities of psoralen/isopsoralen, while it is important to choose the suitable pharmacological models

which are relevant to human in drug metabolism and pharmacokinetic process. The aim of this study is

to identify the metabolites of psoralen/isopsoralen by human and six mammalian liver microsomes,

and compare the metabolites difference of different species. Psoralen/isopsoralen are metabolized by

liver microsomes of different animals to form five and seven metabolites, respectively. The

metabolism of psoralen/isopsoralen undergoes hydroxylation, hydrogenation and hydrolysis, and

oxidation of the furan ring to generate a furanoepoxide or γ-ketoenal intermediate. Furanoepoxide then

forms a dihydrodiol, while γ-ketoenal forms 6-(7-hydroxycoumaryl)-acetic acid (in psoralen)/8-(7-

hydroxycoumaryl)-acetic acid (in isopsoralen). By comparing the types of metabolites in the seven

liver microsomes, it shows that the metabolic behaviors of psoralen by Beagle dog is most relevant to

human, while the metabolic behaviors of isopsoralen by Sprague-Dawley rat is most similar to human.

By comparing the relative amounts of the main metabolites, it shows that the metabolic capabilities of

Sprague-Dawley rat and Rhesus monkey for psoralen are most similar to human, while the metabolic

capabilities of Mouse, Dunkin-Hartley guinea pig, Sprague-Dawley rat, and human for isopsoralen are

similar. Furthermore, the results show that the metabolic capability of human for psoralen and

isopsoralen are weaker than other mammal species.

Al cation induces aggregation of serum proteins

P. Chanphai, L. Kreplak, H.A. Tajmir-Riahi

ABSTRACT

Al cation is known to induce protein fibrillation and causes several neurodegenerative disorders. We

report the spectroscopic, thermodynamic analysis and AFM imaging for the Al cation binding process

with human serum albumin (HSA), bovine serum albumin (BSA) and milk beta-lactoglobulin (b-LG)

in aqueous solution at physiological pH. Hydrophobicity played a major role in Al–protein interactions

with more hydrophobic b-LG forming stronger Al–protein complexes. Thermodynamic parameters

ΔS, ΔH and ΔG showed Al–protein bindings occur via hydrophobic and H-bonding contacts for b-LG,

while van der Waals and H-bonding interactions prevail in HSA and BSA adducts. AFM clearly

indicated that aluminum cations are able to force BSA and b-LG into larger or more robust aggregates

than HSA, with HSA 4 ± 0.2 (SE, n = 801) proteins per aggregate, for BSA 17 ± 2 (SE, n = 148), and

for b-LG 12 ± 3 (SE, n = 151). Thioflavin T test showed no major protein fibrillation in the presence of

Al cation. Al complexation induced major alterations of protein conformations with the order of

perturbations b-LG > BSA > HSA.

162

Volume 142 August 2017

Fighting falsified medicines: The analytical approach

Hervé Rebiere, Pauline Guinot, Denis Chauvey, Charlotte Brenier

ABSTRACT

Given the harm to human health, the fight against falsified medicines has become a priority issue that

involves numerous actors. Analytical laboratories contribute by performing analyses to chemically

characterise falsified samples and assess their hazards for patients. A wide range of techniques can be

used to obtain individual information on the organic and inorganic composition, the presence of an

active substance or impurities, or the crystalline arrangement of the formulation‘s compound. After a

presentation of these individual techniques, this review puts forward a methodology to combine them.

In order to illustrate this approach, examples from the scientific literature (products used for erectile

dysfunction treatment, weight loss and malaria) are placed in the centre of the proposed methodology.

Combining analytical techniques allows the analyst to conclude on the falsification of a sample, on its

compliance in terms of pharmaceutical quality and finally on the safety for patients.

Lipophilicity estimation of statins as a decisive physicochemical parameter for their hepato-

selectivity using reversed-phase thin layer chromatography

Azza H. Rageh, Noha N. Atia, Hamdy M. Abdel-Rahman

ABSTRACT

Lipophilicity plays a crucial role in determining the hepato-selectivity and hence, the biological

activity and the associated side effects of statins. Herein, the employment of RP-TLC for estimation of

lipophilicity of six statins namely; atorvastatin, simvastatin, pravastatin, lovastatin, rosuvastatin and

fluvastatin is examined. A very good correlation between the chromatographically-determined

retention parameters (relative lipophilicity (RM0) or lipophilic parameter (C0)) and both experimental

and computed log P values were obtained. However, the results indicate that the type of organic

modifier in the mobile phase system (methanol, acetonitrile and acetone) has a small influence on

RM0 or C0 values. Higher values of RM0 or C0 are ascribed to lipophilic statins and lower values of

RM0 or C0 are attributed to hydrophilic ones. Therefore, RM0 or C0 could be effectively used as

simple practical predictors of extra-hepatic distributions of statins and thus their expected side effects.

Furthermore, three QSPR (quantitative structure-property relationship) models were constructed to

describe the relationship between RM0 with log P and log D of the statins under investigation. These

models can be very useful to predict the lipophilicity of other members of statin drugs and might be

expanded to newly synthesized compounds with the same structural features.

163

Metabolite profiling of flavonols and in vitro antioxidant activity of young shoots of wild

Humulus lupulus L. (hop)

Annalisa Maietti, Virginia Brighenti, Gianpiero Bonetti, Paola Tedeschi, Federica Pellati

ABSTRACT

Humulus lupulus L., commonly named hop, is well-known for its sedative and estrogenic activity.

While hop cones are widely characterized, only few works have been carried out on the young shoots

of this plant. In the light of this, the aim of this study was to identify for the first time the flavonoids

present in young hop shoots and to compare the composition of samples harvested from different

locations in Northern Italy with their antioxidant activity. The samples were extracted by means of

dynamic maceration with methanol. The HPLC-UV/DAD, HPLC-ESI-MS and MS2 analysis were

carried out by using an Ascentis C18 column (250 × 4.6 mm I.D., 5 μm), with a mobile phase

composed of 0.1 M formic acid in both water and acetonitrile, under gradient elution. Quercetin and

kaempferol glycosides were the main compounds identified and quantified in hop shoot extracts. Total

flavonols ranged from 2698 ± 185 to 517 ± 48 μg/g (fresh weight). The antioxidant activity was

determined by means of the radical scavenging activity assay against diphenylpicrylhydrazyl (DPPH)

and by using a photochemiluscence assay with a Photochem® apparatus. The results showed that hop

shoots represent a new source of flavonols; therefore, they can be useful for a possible incorporation in

the diet as a functional food or applied in the nutraceutical ambit.

Development of assay for determination of eletriptan hydrobromide in loaded PLGA

nanoparticles

Ozgur Esim, Ayhan Savaser, Sevinc Kurbanoglu, Cansel K. Ozkan, Yalcin Ozkan

ABSTRACT

Eletriptan Hydrobromide is a serotonin 5-HT1 receptor agonist and it used for the treatment of

migraine headaches with or without aura. Even if the drug is well absorbed after oral administration, it

has some drawbacks like first pass metabolism and decrease in bioavailability after migraine attacks.

Encapsulation of drug into polymeric nanoparticles is one of the methods for protecting the drug

against degradation. The present work described a preparation of Eletriptan Hydrobromide loaded poly

(d,l-lactide-co-glycolide) nanoparticles prepared using o/w single emulsion solvent evaporation

method. In order to determine the factors affecting the physicochemical properties of the nanoparticles

on the particle size of poly (d,l-lactide-co-glycolide) nanoparticles, D-Optimal design is used.

Moreover, novel, simple, sensitive, selective, and fully validated chromatographic technique for the

quantification of Eletriptan Hydrobromide from Eletriptan Hydrobromide loaded poly(d,l-lactide-co-

glycolide) nanoparticles was developed. Poly(d,l-lactide-co-glycolide) concentration, sonication time

and sonication energy were found as significant factors (p < 0.05) on particle size of nanoparticles.

Limit of detection and limit of quantification values were calculated as 0.28 μg mL−1and 0.86 μg

mL−1, respectively.

164

Characterization of flavonol mono-, di-, tri- and tetra-O-glycosides by ultra-performance liquid

chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry and its

application for identification of flavonol glycosides in Viola tianschanica

Yan Qin, Boyan Gao, Haiming Shi, Jie Cao, Zhihong Cheng

ABSTRACT

In this study, 21 flavonol O-glycoside standards including flavonol mono-, di-, tri- and tetra-O-

glycosides have been systematically studied by ultra-high performance liquid chromatography-

electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS/MS) in

the negative ionization mode to analyze their fragmentation patterns. Here for the first time, the Z23−

fragment (corresponding to the loss of C-2‖ terminal sugar moiety) was observed in MS/MS spectra of

flavonol 3-O-triglycosides. The intensity ratio of [Y0-H]−/Y0− was proposed as a criterion to

distinguish the interglycosidic 1 → 2 and 1 → 6 linkages in flavonol 3-O-diglycosyl-7-O-

monoglycosides. The established fragmentation behaviors have been successfully applied to

characterization of flavonol glycosides in Viola tianschanica. A total of 30 flavonoid glycosides

including 3 flavonol mono-O-glycosides, 10 di-O-glycosides, 10 tri-O-glycosides, 4 tetra-O-glycosides

and 3 flavone di-C-glycosides were identified or tentatively identified on the base of their UV profiles,

MS/MS data and/or by comparing with reference substances. Among these 15 flavonoid glycosides

were reported from V. tianschanica for the first time.

Development and validation of a rapid reversed-phase HPLC method for the quantification of

monoclonal antibody bevacizumab from polyester-based nanoparticles

Flávia Sousa, Virgínia M.F. Gonçalves, Bruno Sarmento

ABSTRACT

Bevacizumab is a powerful human monoclonal antibody approved worldwide for treatment of several

types of cancer and ocular diseases due to its potential as antiangiogenic drug. Nowadays, in order to

improve the monoclonal antibody-based therapy, attempts have been focused in the formulation of

these biomacromolecules into nanoparticles. Thus, the aim of this work was to develop and validate a

reversed-phase high-performance liquid chromatography with fluorescence detection method for the

determination of bevacizumab from nanoparticulate systems, according to the International

Conference on Harmonization guidelines. Chromatographic analysis were performed on a RP-C8

column with a mobile phase composed by water-0.1% (v/v) TFA and acetonitrile-0.1% (v/v) TFA in

gradient mode at a flow rate of 1 mL min−1. Results showed that the proposed method is specific,

linear in the range of 10–100 μg mL−1 (r2 = 0.9997), accurate (recovery rate 100.50 ± 0.85%), precise

at the intraday and inter-day (relative standard deviation less than 1.79%) and robust. The detection

and quantification limits were calculated by specific linear calibration curve with less concentrated

standard (range of 1–20 μg mL−1). The LOD was 2.16 μg mL−1 and LOQ was 6.55 μg mL−1. This

method was also successfully used, for the first time, to quantify and compare the content of

bevacizumab encapsulated into poly(lactic-co-glycolic acid)-based nanoparticles before and after

lyophilization.

165

Quantification of active ingredients in semi-solid pharmaceutical formulations by near infrared

spectroscopy

Lisa B. Schlegel, Manfred Schubert-Zsilavecz, Mona Abdel-Tawab

ABSTRACT

Near infrared (NIR) spectroscopy is increasingly gaining significance in the pharmaceutical industry

for quality and in-process control. However, the potential of this method for quantitative quality

control in pharmacies has long been neglected and little data is available on its application in analysis

of creams and ointments. This study evaluated the applicability of NIR spectrometer with limited

wavelength range (1000–1900 nm) for quantitative quality control of six different dermatological

semi-solid pharmaceutical preparations. Each contained a frequently used active ingredient in a

common concentration either in a water-free lipid base or in an aqueous cream matrix. Based on direct

NIR transflectance measurements through standardized glass beakers and partial least squares (PLS)

multivariate calibration, quantitative models were generated comparing several data pre-processing

methods Whereas difficulties were observed for mixtures containing 2% (w/w) metronidazole or 4%

(w/w) erythromycin, content determination was possible with sufficient accuracy for salicylic acid (5

% (w/w)) and urea (10% (w/w)) in hydrophilic as well as in lipophilic formulations meeting the limit

of a maximum deviation of ± 5% (relative) from the reference values. Exemplarily, one of the methods

was successfully validated according to the EMA Guideline, determining several figures of merit such

as specificity, linearity, accuracy, precision and robustness.

Detection of regulated herbs and plants in plant food supplements and traditional medicines

using infrared spectroscopy

E. Deconinck, C.A. Sokeng Djiogo, J.L. Bothy, P. Courselle

ABSTRACT

The identification of a specific toxic or regulated plant in herbal preparations or plant food

supplements is a real challenge, since they are often powdered, mixed with other herbal or synthetic

powders and compressed into tablets or capsules. The classical identification approaches based on

micro- and macroscopy are therefore not possible anymore. In this paper infrared spectroscopy,

combined with attenuated total reflectance was evaluated for the screening of plant based preparations

for nine specific plants (five regulated and four common plants for herbal supplements). IR and NIR

spectra were recorded for a series of self-made triturations of the targeted plants. After pretreatment of

the spectral data chemometric classification techniques were applied to both data sets (IR and NIR)

separately and the combination of both. The results show that the screening of herbal preparations or

plant food supplements for specific plants, using infrared spectroscopy, is feasible. The best model was

obtained with the Mid-IR data, using SIMCA as modelling technique. During validation of the model,

using an external test set, 21 of 25 were correctly classified and six of the nine targeted plants showed

no misclassifications for the selected test set. For the other three a success rate of 50% was obtained.

Mid-IR combined with SIMCA can therefore be applied as a first step in the screening of unknown

samples, before applying more sophisticated fingerprint approaches or identification tests described in

several national and international pharmacopoeia. As a proof of concept five real suspicious samples

were successfully screened for the targeted regulated plants.

166

UV-induced electron transfer between triethylamine and 5-bromo-2′-deoxyuridine A puzzle

concerning the photochemical debromination of labeled DNA

Paweł Wityk, Magdalena Zdrowowicz, Justyna Wiczk, Janusz Rak

ABSTRACT

5-Bromo-2′-deoxyuridine (BrdU) photosensitizes DNA to strand break formation. However, this type

of photodamage is completely quenched by the presence of triethylamine (TEA) which originates from

RP-HPLC purification commonly employed by oligonucleotide providers. While the presence of TEA

in oligonucleotide samples does not interfere with PCR or other molecular biology applications, the

mechanism of photochemical reaction proceeding in the labeled DNA is dramatically changed due to

the photoinduced electron transfer (PET) between the photoexcited BrdU and the ground state TEA.

For the first time, we demonstrated that the latter process produces 2′-deoxyuridne2′-deoxyuridine

(debromination) in the labeled DNA instead of the expected strand break. PET between TEA and

BrdU was additionally confirmed by the UV irradiations of aqueous solutions containing both species.

Indeed, the efficient formation of 2′-deoxyuridine was observed in the studied photolytes. Moreover,

we showed the formation of an additional product in these binary mixtures, i.e. imidazole derivative,

that is not formed in DNA and was reported in the literature in the context of dark rather than

photochemical processes. Using mass spectrometry we demonstrated that the amount of TEA impurity

in the commercial samples of oligos exceeds up to 3 orders of magnitude that of the purchased DNA.

Development of an in vivo-relevant drug product performance method for an amorphous solid

dispersion

Brian W. Pack, Yelizaveta Babayan, Mark A. Schrad, Paul A. Stroud, Aktham Aburub

ABSTRACT

The purpose of this work was to develop a meaningful in vitro dissolution method for evacetrapib

spray-dried dispersion (SDD) tablets that is discriminating for crystalline drug substance (DS) content.

Justification of the method conditions included evaluation of dissolution media, rotation speed,

surfactant selection and level of surfactant to achieve sink conditions. Discrimination was illustrated

by testing SDD tablets spiked with 10%, 20%, and 30% crystalline DS. The results demonstrated a

13%, 22% and 32% drop in the dissolution end point, respectively, as compared to unspiked SDD

tablets. Additionally, tablets containing crystalline DS and tablets containing SDD were tested in a

relative bioavailability (RBA) study. Utilizing the proposed dissolution method, the dissolution end

point of SDD tablets was determined to be approximately 4 fold higher than that of the tablets

containing crystalline DS. These results compare favourably to the in vivo RBA study results where

SDD tablets had a 4.6 fold increase in exposure compared to tablets containing crystalline DS.

167

Ultrasound-assisted low-density solvent dispersive liquid–liquid microextraction for the

simultaneous determination of 12 new antidepressants and 2 antipsychotics in whole blood by

gas chromatography–mass spectrometry

Xiujuan Chen, Shuiqing Zheng, Jian Le, Zheyuan Qian, Yifeng Chai

ABSTRACT

Antidepressant drugs are widely used in the treatment of different psychiatric disorders, as well as in

conjunction with antipsychotics for the treatment of major depressive disorder. In this study, a simple

and rapid ultrasound-assisted low-density solvent dispersive liquid–liquid microextraction (UA-LDS-

DLLME) method was developed for the simultaneous determination of 12 new antidepressants

(norfluoxetine, fluoxetine, fluvoxamine, agomelatine, mirtazapine, moclobemide, melitracen, N-

desmethylmirtazapine, maprotiline, sertraline, citalopram, paroxetine) and 2 antipsychotics (clozapine

and haloperidol) in human whole blood by gas chromatography–mass spectrometry (GC–MS).

Different parameters affecting the UA-LDS-DLLME were optimized and the optimal conditions were

as follows: 100 μL of toluene as extraction solvent, extraction pH 12 and 3 min of ultrasound stirring.

Good linearity (R2 ≥ 0.991) was obtained at the concentration range of 15–1500 ng/mL for

norfluoxetine, fluoxetine, fluvoxamine, melitracen, maprotiline and citalopram, and 5–500 ng/mL for

agomelatine, mirtazapine, moclobemide, N-desmethylmirtazapine, sertraline, paroxetine, clozapine

and haloperidol. The intra-day and inter-day precision were all less than 10%, and accuracy of intra-

day and inter-day were in the range of −12.7% to 7.9% and −13.9 to 11.8%, respectively. The

extraction recoveries of most analytes were more than 60%. The UA-LDS-DLLME/GC–MS method

was demonstrated with acceptable precision, accuracy and good specificity for the simultaneous

determination of 12 antidepressants and 2 antipsychotics, and has been successfully applied in a real

case.

Application of protein A-modified capillary-channeled polymer polypropylene fibers to the

quantitation of IgG in complex matrices

Hung K. Trang, R. Kenneth Marcus

ABSTRACT

Polypropylene (PP) capillary-channeled polymer (C-CP) fibers loaded with recombinant

Staphyloccocus aureus protein A (rSPA) were used as an affinity chromatography stationary phase for

the quantitation of immunoglobulin G (IgG) in complex biological matrices. Optimization of the

chromatographic method regarding mobile phase components and load/elution conditions was

performed. The six-minute analysis, including a load step with 12 mM phosphate at pH 7.4, an elution

step with 0.025% phosphoric acid and a re-equilibration step, was employed for quantitation of IgG1

from 0.075 to 3.00 mg mL−1 in an IgG-free CHO cell supernatant matrix. Quantification of IgG1

content in a different CHO cell line was accomplished using the external calibration curve as well as

using a standard addition approach. The high level of agreement between the two approaches suggests

that the protein A-modified C-CP fiber phase is immune from matrix effects due to concomitant

species such as host cell proteins (HCPs), host cell DNA, media components and other leachables and

extractables. The inter-day and intra-day precision of the method were 3.1 and 3.5%RSD respectively

for a single column. Column-to-column variability was 1.31 and 6.62%RSD for elution time and peak

area, respectively, across columns prepared in different batches. The method reported here is well-

suited for IgG analysis in complex harvest cell culture media in both the development and production

environments.

168

Simple and rapid quantification of vancomycin in serum, urine and peritoneal/pleural effusion

via UHPLC–MS/MS applicable to personalized antibiotic dosing research

Lenka Javorska, Lenka Kujovska Krcmova, Petr Solich, Milan Kaska

ABSTRACT

Management of the therapy of life-threatening bacterial infection is extremely based on an optimal

antibiotic treatment. Achieving the correct vancomycin dosage in blood and target tissues can be

complicated in special situations, e.g., where large fluid sequestration and/or acute renal failure occur.

A UHPLC–MS/MS method operating in electrospray (ESI) positive ion mode was applied for the

determination of vancomycin in serum, urine and peritoneal/pleural effusion. Sample pretreatment was

composed of dilution and simple protein precipitation where only a small volume (50 μL) of serum,

urine or peritoneal/pleural effusion was required. The separation of vancomycin was performed on a

Meteoric Core C18 BIO column (100 × 4.6 mm, 2.7 μm) by gradient elution with 0.1% formic acid in

water and acetonitrile. The total time of analysis was 4.5 min. The method was found to be linear in

the range of 2–60 μM (or 0.5–10 μM) for serum, 0.27–10 μM (or 2–60 μM) for peritoneal/pleural

effusion and 25–300 μM for urine, which was adequate for the determination of vancomycin in patient

samples. The intra- and inter-day precision was below 8% RSD, and accuracy was from 89 to 104%.

The UHPLC/MS–MS method offers a fast and reliable approach to determine vancomycin

concentrations in three different human body fluid samples (serum, urine and peritoneal/pleural

effusion) with a simple sample pretreatment that was the same for all selected specimens.

Biphenyl based stationary phases for improved selectivity in complex steroid assays

Johanna M. Lindner, Michael Vogeser, Stefanie H. Grimm

ABSTRACT

The measurement of steroid hormones and their corticoid precursors is an important aspect in

endocrinology since these analytes are biomarkers for several endocrine disorders. Over the last few

years, HPLC–MS/MS has become the method of choice to analyze these compounds. There are

already several methods using stationary phases modified with C18 groups. However, since these

columns sometimes do not enable sufficient separation of some isobaric steroids, we investigated the

potential of a different RP modification using biphenyl groups for the separation of challenging isobars

such as corticosterone, 11- and 21-deoxycortisol. The aim of our work was the development of an

isotope dilution UHPLC–MS/MS assay for clinical research that combines simple and effective sample

preparation with a powerful MS method quantifying a broad steroid panel (aldosterone, corticosterone,

cortisol, cortisone, 11‐deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol,

dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17-OH-progesterone, progesterone, and

testosterone) in human serum. After a manual protein precipitation step using zinc trifluoroacetate

(ZnTFA) in methanol, the supernatants were directly injected into the UHPLC–MS system.

Chromatographic baseline separation of all isobaric compounds (corticosterone ↔ 11-deoxycortisol ↔

21-deoxycortisol, 17-OH-progesterone ↔ 11‐deoxycorticosterone, and aldosterone ↔ cortisone) was

achieved using a Kinetex Biphenyl column (150 × 2.1 mm, 1.7 μm) with a mobile phase consisting of

0.2 mM ammonium fluoride in water and methanol. The total run time was 10 min. For detection we

used a Xevo TQ-S mass spectrometer operating in the ESI positive and negative modes. The method

was validated according to the EMA guideline for bioanalytical method validation. The results for

accuracy (within-run: 92.3%–115%, between-run: 92.4 %–113%) and imprecision (within-run:

0.80%–9.05%, between-run: 1.98 %–15.2%) were satisfying.

169

Ultra-sensitive and selective quantification of endothelin-1 in human plasma using ultra-

performance liquid chromatography coupled to tandem mass spectrometry

Yosuke Suzuki, Lukas Witt, Walter Mier, Gerd Mikus, Jürgen Burhenne

ABSTRACT

Endothelin-1 (ET-1) is a potent endogenous vasoconstrictor peptide and the plasma concentrations are

commonly quantified by immunoassays such as enzyme-linked immuno-sorbent assays (ELISA) with

the disadvantage of possible cross-reactivity with closely related endothelin derivatives. The aim of

this study was to develop and validate an ultra-sensitive and selective assay for the quantification of

ET-1 in human plasma, using ultra-performance liquid chromatography with tandem mass

spectrometry (UPLC-MS/MS) after solid phase extraction. The assay fulfilled the requirements of the

US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) guidelines for

assay validation, with a lower limit of quantification of 1.5 pg/mL for ET-1. Recovery rates from

plasma ranged between 80.8% and 93.6%, and matrix effect varied between 121% and 135%. The

assay was successfully applied to assess the time course of plasma ET-1 concentrations in two human

volunteers after co-administration of bosentan and clarithromycin. In this trial, the concentrations

measured by UPLC-MS/MS were slightly lower than those measured by ELISA, with a strong positive

correlation between the two methods. Our novel UPLC-MS/MS method is applicable to the clinical

setting and may have better selectivity for ET-1 than ELISA.

Serum metabolomics reveals the mechanistic role of functional foods and exercise for obesity

management in rats

Naglaa M. Ammar, Mohamed A. Farag, Tahani E. Kholeif, Nadia S. Metwally, Abdel- Hamid Z.

Abdel- Hamid

ABSTRACT

Obesity is one of the independent risk factors for several health problems, leading to metabolic

perturbations and for which analytical approaches i.e., ―metabolomics‖ is needed to monitor the

underlying metabolic changes. In this study, obesity associated changes were assessed via serum

metabolites analysis of obese rats fed on high fat diet. Obese rats were subsequently treated with

different functional foods used for obesity management including pomegranate, grapefruit, and red

cabbage in parallel to swimming exercise. Serum samples were analyzed using gas chromatography-

mass spectrometry (GC–MS) followed by multivariate data analysis to classify samples and determine

if such treatments can help revert obesity related metabolic changes back to normal status. Results led

to the identification of several novel metabolites biomarkers for obesity related to lipids, amino acids

and central tricarboxylic acid (TCA) pathways. Distinct variations in metabolite levels were recorded

in obese rats compared to normal ones including l-aspartic, l-alanine, l-glutamine, l-glycine,

phenylethanolamine, α-aminobutyric acid and β-hydroxybutyric acid. Metabolomics approach

developed herein provides novel insight onto the metabolic disturbances associated with obesity,

which will assist in future drug design that can help mitigate against such changes.

170

Systematic screening and characterization of prototype constituents and metabolites of total

astragalosides using HPLC-ESI-IT-TOF-MSn after oral administration to rats

Hong-Fu Li, Feng Xu, Ping Yang, Guang-Xue Liu, Shao-Qing Cai

ABSTRACT

Astragalosides (AGs) are the main bioactive constituents in Astragali Radix (AR), and have a wide

range of pharmacological properties, including immunoregulatory, cardioprotective, neuroprotective,

antioxidative, antidiabetic, and antinociceptive effects. However, the metabolism of total AGs remains

unclear. To clarify the metabolic fate of AGs after oral administration to rats, total AGs were isolated

from AR extracts using AB-8 macroporous resin chromatography and preparative HPLC, and then

analyzed using HPLC-DAD-ELSD and LC–MS. HPLC-ESI-IT-TOF-MSn was used to systematically

screen and characterize prototype constituents and metabolites of total AGs in rat feces, urine, and

plasma samples. As a result, 123 AG-related compounds from feces were detected and structurally

characterized. Among the 123 compounds, 107 were phase I metabolites, of which 91 were new

metabolites, and 73 were new compounds. In addition, six prototype constituents in urine, and one in

plasma were detected. The main metabolic sites in the structure of cycloastragenol (CAG), the

aglycone of AGs, were found to be the 9, 19-cyclopropane ring (E ring) and the 20, 24-furan ring (F

ring). The cleavage mode of CAG derivatives in negative ion mode was identified, and was found to

be highly dependent on the integrity of the E ring. Mono- to tetra-hydroxylated and carboxyl

substituted metabolites were tentatively identified. Deglycosylation, hydroxylation, dehydrogenation,

isomerization, ring cleavage, and carboxyl substitution were considered to be the major metabolic

reactions involved in the formation of the metabolites, among which carboxyl substitution was a novel

metabolic reaction.

Development, validation and application of a novel liquid chromatography tandem mass

spectrometry assay measuring uracil, 5,6-dihydrouracil, 5-fluorouracil, 5,6-dihydro-5-

fluorouracil, α-fluoro-β-ureidopropionic acid and α-fluoro-β-alanine in human plasma

Ottiniel Chavani, Berit Packert Jensen, R. Matthew Strother, Christopher M. Florkowski, Peter M.

George

ABSTRACT

The plasma 5,6-dihydrouracil/uracil (UH2/U) ratio is a possible phenotypic marker of

dihydropyrimidine dehydrogenase (DPD) activity, hence an index of 5-fluorouracil (5-FU) response

and toxicity. Studies have re-affirmed the value of 5-FU and 5,6-dihydro-5-fluorouracil (FUH2) for

therapeutic drug monitoring (TDM). However, FUH2 has limited stability in plasma, necessitating

expedited plasma separation and freezing, where routine compliance may not be easy. The metabolites

α-fluoro-β-ureidopropionic acid (FUPA) and α-fluoro-β-alanine (FβAL) are stable in plasma and are

probable candidates for TDM. This paper describes development, validation and application of an LC–

MS/MS assay quantifying U, UH2, 5-FU, FUH2, FUPA and FβAL in human plasma. Extraction was

by salt-assisted liquid–liquid extraction (LLE) in two-stages with pH adjustment. The supernatants

were mixed, dried and reconstituted. Analytes were resolved on the Luna PFP (2) (150 × 2.00 mm, 3

μ) column by gradient elution and analyzed by tandem mass spectrometry via electrospray ionisation

in positive polarity. The analytical response was linear (r2 ≥ 0.99) in the concentration (ng/mL) ranges:

50–10 000 for FβAL and FUH2, 50–5 000 for FUPA, 50–100 000 for 5–FU, 5–200 for U and 10–400

for UH2. Within- and between-run accuracy and precision were ≤ 10.2% and ≤ 9.8% respectively

across the QC range and inclusive of LLOQ.

171

Multi-platform metabolomics and a genetic approach support the authentication of agarwood

produced by Aquilaria crassna and Aquilaria malaccensis

Huy Truong Nguyen, Jung-Eun Min, Nguyen Phuoc Long, Ma Chi Thanh, Sung Won Kwon

ABSTRACT

Agarwood, the resinous heartwood produced by some Aquilaria species such as Aquilaria crassna,

Aquilaria malaccensis and Aquilaria sinensis, has been traditionally and widely used in medicine,

incenses and especially perfumes. However, up to now, the authentication of agarwood has been

largely based on morphological characteristics, a method which is prone to errors and lacks

reproducibility. Hence, in this study, we applied metabolomics and a genetic approach to the

authentication of two common agarwood chips, those produced by Aquilaria crassna and Aquilaria

malaccensis. Primary metabolites, secondary metabolites and DNA markers of agarwood were

authenticated by 1H NMR metabolomics, GC–MS metabolomics and DNA-based techniques,

respectively. The results indicated that agarwood chips could be classified accurately by all the

methods illustrated in this study. Additionally, the pros and cons of each method are also discussed. To

the best of our knowledge, our research is the first study detailing all the differences in the primary and

secondary metabolites, as well as the DNA markers between the agarwood produced by these two

species.

Apoptosis induction activity and molecular docking studies of survivin siRNA carried by Fe3O4-

PEG-LAC-chitosan-PEI nanoparticles in MCF-7 human breast cancer cells

Sanam Arami, Majid Mahdavi, Mohammad-Reza Rashidi, Reza Yekta, Nasser Samadi

ABSTRACT

Delivery of small interfering RNAs (siRNAs) into cells still remains a challenge in gene delivery

studies. Here, we investigated the ability of synthesized Fe3O4-PEG-LAC-chitosan-PEI nanoparticles

for siRNA delivery of survivin as the model gene into cells. The cellular uptake of survivin siRNA

carried by synthesized nanoparticles into MCF-7 breast cancer cell line was evaluated by florescent

microscopy and flowcytometry, both proving the efficacy of nanoparticles in delivery of up to 64.7%

in comparison with lipofectamine 2000. Furthermore, the delivery of survivin siRNA by the

nanoparticles (nanoplex) induced apoptosis that was assessed through DAPI staining and Annexin

V/PI assays. In addition, we evaluated the efficacy of treatment with nanoplexes in the presence of

mitoxantrone, as a chemotherapeutic agent. Our data indicated that inhibition of survivin expression

increased the cell sensitivity to mitoxantrone. Real-time PCR and western blotting analysis revealed a

significant reduction in mRNA and protein levels of survivin upon delivery of siRNA. Molecular

docking studies showed that nanoparticles can bind to centeral BIR domain of survivin, exactly above

zinc ion location with high affinity (ΔG: −10.3 Kcal/mol). Also, thermodynamic studies proved the

experimental results theoretically, revealing that the siRNA-loaded nanoparticles have a suppressing

effect on survivin mRNA. Therefore, delivery of survivin siRNA into MCF-7 cells using Fe3O4-PEG-

LAC-chitosan-PEI nanoparticles as a carrier enhances the cell death.

172

Exploring the neuroprotective effects of ginkgolides injection in a rodent model of cerebral

ischemia–reperfusion injury by GC–MS based metabolomic profiling

Jian-liang Geng, Ji-ye Aa, Si-qi Feng, Shu-yao Wang, Guang-ji Wang

ABSTRACT

Cerebral ischemia–reperfusion (I/R) injury usually contributes to mortality and disability after

ischemic stroke. Ginkgolides injection (GIn), a standard preparation composed of ginkgo diterpene

lactones extract, is clinically used for neuroprotective treatment on reconvalescents of cerebral

infarction. However, the understanding about its therapeutic mechanism is still lacking. In this study, a

gas chromatography–mass spectrometry (GC–MS) based metabolomic approach coupled with

multivariate data analysis (MVDA) was applied to explore the neuroprotective effects of GIn in a

rodent model of focal ischemic stroke induced by transient middle cerebral artery occlusion (tMCAO).

Metabolomic profiling revealed a series of metabolic perturbations that underlie the cerebral I/R

pathological events. GIn can reverse the I/R induced brain metabolic deviations by modulating

multiple metabolic pathways, such as glycolysis, Krebs cycle, pentose phosphate pathway (PPP), γ-

aminobutyrate (GABA) shunt and lipid metabolism. Moreover, the main bioactive components of GIn

were distributed to brain tissue much more easily in tMCAO rats than in normal rats after an

intravenous administration, suggesting that the increased cerebral exposure to ginkgolides in I/R

pathological condition potentially facilitated the neuroprotective effects of GIn by directly targeting at

brain. The present study provided valuable information for our understanding about metabolic changes

of cerebral I/R injury and clinical application of GIn.

Quantitative LC–HRMS determination of selected cardiovascular drugs, in dried blood spots, as

an indicator of adherence to medication

Dennis Bernieh, Graham Lawson, Sangeeta Tanna

ABSTRACT

Dried blood spot (DBS) sampling was investigated as a means of obtaining micro-volume blood

samples for the quantitative analyses of ten commonly UK prescribed cardiovascular drugs as an

indicator of medication adherence. An 8 mm disc was punched out from each DBS from calibration,

quality control and volunteer samples and extracted using methanol containing the internal standard.

Each extract was evaporated to dryness, the residue reconstituted in methanol:water (40:60 v/v)

containing 0.1% formic acid and analysed by LC–HRMS. Chromatography was performed using

gradient elution on a Zorbax Eclipse C18 HD 100 mm × 2.1 mm, 1.8 μm pore size column with the

column oven temperature at 40 °C. Flow rate of the mobile phase was 0.6 ml/min with a run time of

2.5 min. Electrospray positive ionization was used for MS detection. Drug recoveries from spiked

blood spots were 68% for simvastatin and ≥87% for all other target drugs. Compound specificity was

obtained operating the MS with a 5 ppm mass window. The LC–HRMS method was validated, with

results for accuracy and precision within acceptable limits; analytes were stable at room temperature

for at least 10 weeks and different blood spot volumes and haematocrit values had no significant

effect. The LC–HRMS assay was used to analyse DBS samples from volunteers, some of whom were

prescribed one or more of the target drugs. In results from 37 volunteers the assay successfully

identified volunteers who were known to be either adherent or nonadherent; confirmed the correct

drug/drugs for multiple prescriptions; demonstrated no false positives from other cardiovascular drugs;

revealed several examples of unsuspected non-adherence. These results indicated that the developed

assay was suitable for trials with patients.

173

Determination of thiopurine S-methyltransferase activity by hydrophilic interaction liquid

chromatography hyphenated with mass spectrometry

Daniel Pecher, Svetlana Dokupilová, Zuzana Zelinková, Maikel Peppelenbosch, Peter Mikuń

ABSTRACT

Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurines used

in the therapy of inflammatory bowel diseases (IBD). In this work a new progressive method for the

determination of TPMT activity in red blood cells lysates was developed. Analysis was carried out by

means of hydrophilic interaction liquid chromatography (HILIC) hyphenated with mass spectrometry

(MS). In comparison with reversed-phase high-performance liquid chromatography (RP-HPLC), that

has been typically applied in determination of TPMT activity, the HILIC significantly improved the

analytical signal provided by MS, shortened analysis time, and improved chromatographic resolution.

The HILIC-HPLC-MS method was optimized and validated, providing favorable parameters of

detection and quantitation limits (5.5 and 16.5 pmol/mL, respectively), linearity (coefficient of

determination 0.9999 in the range of 0.01–1.0 nmol/mL), recovery and precision (93.25–100.37% with

RSD 1.06-1.32% in the whole concentration range of QC samples). Moreover, in contrast to the

conventional RP-HPLC-UV approach, the complex phenotype TPMT profiles can be reliably and

without interferences monitored using the HILIC-HPLC-MS method. Such advanced monitoring can

provide valuable detail information on the thiopurines (e.g. evaluating ratio of methylated and non-

methylated 6-mercaptopurine) and, by that, TPMT action in biological systems before and during the

therapy of IBD.

Workflow methodology for rat brain metabolome exploration using NMR, LC–MS and GC–MS

analytical platforms

Binta Diémé, Antoine Lefèvre, Lydie Nadal-Desbarats, Laurent Galineau, Sylvie Mavel

ABSTRACT

We developed a multi-platform approach for the metabolome exploration of rat brain tissue, using

liquid chromatography coupled with mass spectrometry (LC–MS), nuclear magnetic resonance

spectroscopy (NMR) and gas-chromatography coupled with mass spectrometry (GC–MS). The critical

steps for metabolite exploration of cerebral tissues are tissue lysis and metabolites extraction. We first

evaluated the impact of freeze-drying compared to wet tissue metabolites extraction using NMR and

LC–MS with a reversed phase liquid chromatography. Then, we compared four metabolite extraction

methods Based on the number of metabolites extracted, their intensity and their coefficient of variation

(%CV), the most reproducible protocol (one-step extraction with acetonitrile on lyophilized material)

was chosen to further evaluate the impact of sample mass on method performance (3, 6, and 9 mg were

essayed). GC–MS analysis was also investigated by analyzing four different methoximation/silylation

derivatization combinations. The optimal analytical protocols were proposed to establish the reliability

required to realize untargeted brain tissue metabolomics exploration. The most reliable workflow was

then exemplified by analyzing three rat brain regions (cerebellum, frontal and parietal cortices, n = 12)

by 1H NMR, LC–MS and GC–MS, allowing their clustering based on their metabolic profiles. We

present here an example of development of methodology that should be done before running analysis

campaigns.

174

Bioelectrical impedimetric sensor for single cell analysis based on nanoroughened quartz

substrate; suitable for cancer therapeutic purposes

Milad Gharooni, Mohammad Abdolahad

ABSTRACT

Single cells analysis has been interested in recent decade. Apart from scientific benefits to achieve new

biological phenomena in cell study, many diagnostic and therapeutic protocols in non-communicable

diseases were introduced by single cell analysis. Moreover, non-invasive methods to maintain the

investigated cell for time dependent monitoring has been widely studied because of its importance in

some crucial cases such as drug resistance in cancer. Bioelectrical monitoring is one of such methods

Although the procedures reported based on electrical probing might not induce cell disruption, indirect

connection between recording electrodes and cell membrane (mostly in microfluidic approaches)

reduced the quality of response and limited the precision of the results. Here, a bioelectronic sensor for

monitoring the effect of anticancer drugs on single breast cancer cells was fabricated based on nano-

roughened gold electrodes on a quartz substrate applied direct contacts to cell membrane. Whole of the

surface except a microcircle surrounded the sensing region was passivated by overbaked photoresist

layer. Cells were dropped on the sensor without the assistance of any micropipette or microfluidic

systems and just individual regions for attachment of one cell has been opened on the sensing region

arrays. MCF-7 cancer cells were time tracked under the effect of Paclitaxel and Mebendazole anti-

tubulin drugs in low and high doses. Inducing non regulated depolymerization and polymerization in

tubulin structures of the single cancer cells were monitored by the electrical signals recorded before

and after drug treatment.

Highly sensitive UHPLC–MS/MS method for the simultaneous estimation of propafenone and its

metabolites 5-hydroxypropafenone and N-depropylpropafenone on human dried blood spots

technique and application to a pharmacokinetic study

Adinarayana Andy, Raja Reddy Kallem, Ramesh Mullangi, Divya Andy, J.V.L.N. Seshagiri Rao

ABSTRACT

A highly sensitive, rapid and selective UHPLC–MS/MS method has been developed and validated for

quantification of the propafenone (PF), 5-hydroxypropafenone (5-OHPF) and N-depropylpropafenone

(N-DPF) on human dried blood spot (DBS). The assay procedure involves a solid–liquid extraction of

PF, 5-OHPF and N-DPF and amlodipine (internal standard, I.S.) from dried human DBS cards using

water and acetonitrile. The chromatographic resolution was achieved on a BEH C18 column using a

gradient mobile phase consisting of 0.1% formic acid in water and acetonitrile with 0.1% formic acid

at flow rate of 0.6 mL/min. The UHPLC–MS/MS was operated under the multiple-reaction monitoring

mode using electrospray ionization. Total run time of analysis was 1.1 min and elution of PF, 5-OHPF,

N-DPF and I.S. occurred at 0.69, 0.6, 0.68 and 0.73 min, respectively. A detailed method validation

was performed as per the regulatory guidelines and the standard curves found to be linear in the range

of 5.11–1000 ng/mL for PF and 5-OHPF and 0.51–100 ng/mL for N-DPF with a correlation

coefficient of ≥0.99 for all the drugs. The intra- and inter-day accuracies were in the range of 95.6–107

and 93.5–103; 93.4–106 and 96.3–107 and 87.9–103 and 96.5–102%, for PF, 5-OHPF and N-DPF,

respectively. The intra- and inter-day precisions were in the range of 2.50–5.52 and 3.38–5.18; 2.16–

6.34 and 3.23–4.94 and 2.63–7.55 and 1.56–10.2%, for PF, 5-OHPF and N-DPF, respectively. The

validated assay method was successfully applied to a pharmacokinetic study in humans.

175

Simple determination of L-hydroxyproline in idiopathic pulmonary fibrosis lung tissues of rats

using non-extractive high-performance liquid chromatography coupled with fluorescence

detection after pre-column derivatization with novel synthetic 9-acetylimidazol-carbazole

Yan Ren, Juanjuan Zhao, Yanan Shi, Caiyun Chen, Changjun Lv

ABSTRACT

L-Hydroxyproline (L-Hyp) is an important biomarker for idiopathic pulmonary fibrosis (IPF). The

quantitative methods based on high-performance liquid chromatography coupled with fluorescence

detection after pre-column derivatization typically requires complicated derivatization conditions and

obtains unstable derivatives. Here, a novel derivatization reagent, 9-acetylimidazol-carbazole, was

synthesized for the first time to efficiently and rapidly label the amino groups of L-Hyp. The high-

performance liquid chromatography method with pre-column derivatization was performed on an

Agilent ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm). The product was measured using

fluorescence detection at excitation and emission wavelengths of 232 and 370 nm, respectively. The

method was validated in specificity, linearity, limit of detection (66.7 fmol), limit of quantification

(333.3 fmol), intra-day precision (0.75%), inter-day precision (3.82%), stability (3.15%), and recovery

(90.7–109.4%). The validated method was successfully applied to the determination of L-Hyp in the

lung tissues of healthy and IPF rats. The results showed that the concentration of L-Hyp (3.64 mg/g) in

the IPF model was significantly higher than the concentration (2.33 mg/g) in the healthy control group

with P < 0.01. This is a new method for the determination of L-Hyp and can be used for other amino

acid-related studies in the future.

A simple method for assaying colistimethate sodium in pharmaceutical aerosol samples using

high performance liquid chromatography

Adam P. Metcalf, Lucy E.A. Hardaker, Ross H.M. Hatley

ABSTRACT

A rapid and simple reversed-phase high performance liquid chromatography (HPLC) method for the

quantitation of colistimethate sodium in pharmaceutical formulations has been developed. The

chromatographic separation was performed using a Phenomenex Kinetex XB-C18 column with

gradient elution using a mobile phase containing acetonitrile and 32 mM sodium sulphate. Quantitation

is based on the sum of the areas of two prominent peaks in the chromatogram, which produces a total

peak area that is stable for 120 sample injections. The HPLC method was validated over the range

0.05–7 mg/mL, and was shown to be suitable for the analysis of aerosolised pharmaceuticals in terms

of aerosol output onto filter and for the analysis of samples from a cascade impactor, which is used for

the determination of aerosol particle size.

176

Quantification of apigenin trimethyl ether in rat plasma by liquid chromatography–tandem

mass spectrometry: Application to a pre-clinical pharmacokinetic study

Mai Gamal Elhennawy, Hai-Shu Lin

ABSTRACT

Apigenin trimethyl ether (5,7,4′-trimethoxyflavone, ATE) is a naturally occurring polymethoxyflavone

with a wide range of health-promoting activities. In this study, a sensitive liquid chromatography–

tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of

ATE in rat plasma. Protein precipitation was applied as plasma clean-up procedure; the electrospray

ionization was operated in its positive ion mode while ATE and formononetin (internal standard) were

measured by multiple reactions monitoring (ATE: m/z 313.1 → 298.1; formononetin: 269.2 → 213.3).

This LC–MS/MS method displayed good selectivity, sensitivity (lower limit of quantification = 2.5

ng/ml), accuracy (both intra- and inter-day analytical recovery within 100 ± 10%) and precision (both

intra- and inter-day RSD <10%). The matrix effect was found to be insignificant. The pharmacokinetic

profiles of ATE were subsequently examined in Sprague-Dawley rats after single oral administration

(10 mg/kg). When given in an aqueous suspension, ATE was slowly absorbed with quite low plasma

exposure (AUC). Fasting further attenuated its oral absorption and led to ∼70% drops in average

maximal plasma concentration (Cmax) and AUC. When dosed in a solution formulated with 2-

hydroxypropyl-β-cyclodextrin, the oral absorption of ATE was substantially improved with ∼500%

increases in average Cmax and AUC. Clearly, aqueous solubility has been identified as a barrier to the

oral absorption of ATE. The information obtained from this study will facilitate further medicinal

exploration on ATE.

Simultaneous analysis of regorafenib and sorafenib and three of their metabolites in human

plasma using LC–MS/MS

Marie Allard, Nihel Khoudour, Benoît Rousseau, Charlotte Joly, Anne Hulin

ABSTRACT

A new liquid chromatography-tandem mass spectrometry (LC–MS/MS) method, performed by

electrospray ionization in positive mode using a triple quadrupole mass spectrometry, has been

developed and validated for the simultaneous determination of regorafenib (REGO), its two

metabolites regorafenib-M2 and regorafenib-M5, sorafenib (SORA), and its N-oxide metabolite in

human plasma. Separation is achieved on an Hypersil Gold® column using a gradient elution of 10

mM ammonium formate containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid

(B) at a flow rate of 0.3 mL/min. After addition of two internal standards and a protein precipitation,

the supernatant is diluted two-fold in a 0.1% (v/v) formic acid solution. Two selected reaction

monitoring transitions are used, for each analyte, one for quantitation and the second one for

confirmation. The standard curves are ranged from 50 to 5 000 ng/mL for REGO and its metabolites

and 80 to 5 000 ng/mL for SORA and its metabolite and were fitted to a 1/x weighted linear regression

model. The method also showed satisfactory results in terms of sensitivity, specificity, precision (intra-

and inter-day CV from 2.4 to 10.2%), accuracy (from 91.0 to 111.7%), recovery as well as stability of

the analytes under various conditions. The method is usually used in clinical practice in order to

improve the SORA treatment for renal carcinoma, REGO treatment for colorectal cancer and both for

hepatocellular carcinoma.

177

Stability behavior of antiretroviral drugs and their combinations 7: Comparative degradation

pathways of lamivudine and emtricitabine and explanation to their differential degradation

behavior by density functional theory

Moolchand Kurmi, Saranjit Singh

ABSTRACT

The interest in this study was to establish comparative degradation behavior of lamivudine (3TC) and

emtricitabine (FTC) under solution and solid state stress conditions. Structurally, the two drugs differ

only in terms of additional fluorine at 5 position in FTC. Along with the known degradation products

of both the drugs, one additional degradation product was observed in each case, which was

characterized by mass spectrometry. Both the drugs degraded via the same route, but at a differential

rate in acid, base and oxidative stress conditions. The variable rate of degradation in acid and base

conditions was justified by the application of density functional theory (DFT).

An atmospheric pressure ionization source using a high voltage target compared to electrospray

ionization for the LC/MS analysis of pharmaceutical compounds

Arnaud Lubin, Ronald De Vries, Deirdre Cabooter, Patrick Augustijns, Filip Cuyckens

ABSTRACT

The type and design of an ionization source can have a significant influence on the performances of a

bioanalytical method. It is, therefore, of high interest to evaluate the performances of newly introduced

sources to highlight their benefits and limitations in comparison to other well established sources. In

this paper, liquid chromatography – mass spectrometry (LC/MS) performances of a new atmospheric

pressure ionization (API) source, commercialized as UniSpray, is evaluated. The dynamic range of 24

pharmaceutical and biological compounds is compared between the new API source and electrospray

ionization (ESI) for 3 different mobile phase conditions. Matrix effects are also compared with ESI on

a refined selection of 19 pharmaceutical and biological compounds in 4 matrices commonly

encountered in bioanalysis. A slightly better dynamic range towards lower concentrations was often

observed with the new API source. Matrix effects were quite similar between the two sources with a

small, but statistically significant, lower percentage of matrix effects observed for the new API source

in plasma and bile in the positive ion mode, and bile in negative ion mode for ESI. Finally, the

sensitivity of late eluting compounds could be improved on the new API source by post-column

addition of water.

178

UHPLC–MS/MS method with sample dilution to test therapeutic adherence through

quantification of ten antihypertensive drugs in urine samples

Amedeo De Nicolò, Valeria Avataneo, Franco Rabbia, Mauro Sciandra, Antonio D‘Avolio

ABSTRACT

Nowadays, hypertension represents an important health problem, particularly in developed countries.

In some cases the standard therapeutic approaches are not able to reestablish the normal blood pressure

values: this condition is called ―resistant hypertension‖. However, a fraction of cases of resistant

hypertension are actually due to poor adherence to the prescribed therapy. Therapeutic Drug

Monitoring could represent a direct and useful tool to correctly identify non-compliant patients.

Nevertheless, high throughput methods for the simultaneous monitoring of a wide panel of drugs in the

same analysis are lacking and, furthermore, there is not a generally acknowledged ―standard‖ matrix

for this test (plasma or urine). In this work, we validated a UHPLC–MS/MS assay to quantify ten

among the most used antihypertensive agents in urine samples, covering all the current classes:

amlodipine, atenolol, clonidine, chlortalidone, doxazosin, hydrochlorothiazide, nifedipine, olmesartan,

ramipril and telmisartan. Both standards and quality controls were prepared in urine matrix. Only 100

μL of each sample were added with 40 μL of internal standard and 860 μL of water:acetonitrile 90:10,

acidified with 0.05% formic acid. Chromatographic separation was performed on an Acquity® UPLC

HSS T3 1.8 μm 2.1 × 150 mm column, with a gradient of water and acetonitrile, both added with

0.05% formic acid. Accuracy, intra-day and inter-day precision fitted FDA guidelines for all analytes,

while matrix effects resulted reproducible among different urine lots. Method performances were

tested on urine samples from hypertensive patients with good results. This simple analytical method

could represent a useful tool for the management of antihypertensive therapy.

A novel carbohydrate labeling method utilizing transfer hydrogenation-mediated reductive

amination

Zsuzsanna Kovács, Gábor Papp, Henrietta Horváth, Ferenc Joó, András Guttman

ABSTRACT

One of the most frequently used high-resolution glycan analysis methods in the biopharmaceutical and

biomedical fields are capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection.

Glycans are usually labeled by reductive amination with a charged fluorophore containing a primary

amine, which reacts with the aldehyde group at the reducing end of the glycan structures. In this

reaction, first a Schiff base is formed that is reduced to form a stable conjugate by a hydrogenation

reagent, such as sodium cyanoborohydride. In large scale biopharmaceutical applications, such as

clone selection for glycoprotein therapeutics, hundreds of reactions are accomplished simultaneously,

so the HCN generated in the process poses a safety concern. To alleviate this issue, here we propose

catalytic hydrogen transfer from formic acid catalyzed by water-soluble iridium(III)- and

ruthenium(II)-phosphine complexes as a novel alternative to hydrogenation. The easily synthesized

water-soluble iridium(III) and the ruthenium(II) hydrido complexes showed high catalytic activity in

carbohydrate labeling. This procedure is environmentally friendly and reduces the health risks for the

industry. Using carbohydrate standards, oligosaccharides released from glycoproteins with highly

sialylated (fetuin), high mannose (ribonuclease B) and mixed sialo and neutral (human plasma) N-

glycans, we demonstrated similar labeling efficiencies for iridium(III) dihydride to that of the

conventionally used sodium cyanoborohydride based reaction. The derivatization reaction time was

less than 20 min with no bias towards the above mentioned specific glycan structures.

179

Non-volatile extractable analysis of prefilled syringes for parenteral administration of drug

products

Noemí Dorival-García, Iben Larsson, Jonathan Bones

ABSTRACT

The determination of extractable profiles for single-use technologies represents an important aspect of

pharmaceutical production to minimize any possible compromise in drug product quality or potential

risk to patients by identifying substances that may potentially leach from such devices. An approach

for the extractable assessment of prefilled syringes, a promising alternative for parenteral

administration of pharmaceutical products, is described herein. Four extraction solvents were selected:

a mixture 2-propanol:water (1:1), was intended to represent aggressive conditions to extract a broad

spectrum of extractables, including organic additives and substances which are poorly water-soluble.

Extractions with buffers at three different working pH values spanning a range standardly used in

pharmaceutical formulations were also evaluated to identify substances that require specific conditions

for their extraction due to their individual chemical properties. Syringes from two different brands

were analysed along with their corresponding plunger stoppers. Syringes were extracted at 40 °C for 4

days, the plunger stoppers were extracted with 2-propanol at 70 °C for 24 h according to ISO 10993-

12:2012. Extractables were identified by UHPLC–MS on a quadrupole time of flight instrument using

a non-targeted discovery strategy. A total of 25 compounds were identified, mostly polymer additives

and their degradation products. The presented methodology represents a reference point for further

studies focused on the characterisation of extractables and leachables from prefilled syringes.

A rapid microextraction by packed sorbent − liquid chromatography tandem mass spectrometry

method for the determination of dexamethasone disodium phosphate and dexamethasone in

aqueous humor of patients with uveitis

Federica Bianchi, Monica Mattarozzi, Nicolò Riboni, Paolo Mora, Maria Careri

ABSTRACT

A new method based on microextraction by packed sorbent (MEPS) coupled with liquid

chromatography-tandem mass spectrometry (LC–MS/MS) was developed and validated for the

determination of dexamethasone and dexamethasone disodium phosphate in human aqueous humor. A

central composite design was applied to investigate the effects of both loading and eluting cycles in the

MEPS procedure; subsequently the multicriteria method of the desirability functions allowed to find

the best conditions for the simultaneous extraction of both the analytes. Detection was performed on a

LTQ XL linear ion trap mass spectrometer operating in the positive electrospray ionization mode

applying multiple reaction monitoring mode. The assay was validated in accordance with the

guidelines bioanalytical method validation obtaining quantitation limits in the low μg/L range, a

precision characterized by RSD ≤ 16% and recovery rates in the 91–119% range.

180

Isotope-coded derivatization based LC/ESI-MS/MS methods using a pair of novel reagents for

quantification of hydroxycinnamic acids and hydroxybenzoic acids in fermented brown rice

product

Shoujiro Ogawa, Kiriko Takafuji, Sumi Tsubuku, Yukiko Horie, Tatsuya Higashi

ABSTRACT

Hydroxycinnamic acids (HCAs) and hydroxybenzoic acids (HBAs) are antioxidant phytochemicals

found in rice and effective for the prevention of human diseases including cancer. FBRA, which is a

functional food manufactured by fermenting brown rice and rice bran with Aspergillus oryzae, has

been demonstrated to have chemopreventive effects against carcinogenesis in various organs. In this

study, we developed methods for the relative and absolute quantification of ferulic acid, sinapic acid,

caffeic acid, protocatechuic acid and syringic acid in the FBRA and raw material (RM; unfermented

brown rice and rice bran) samples by LC/ESI-MS/MS combined with derivatization using a newly

developed reagent, N-(2-aminoethyl)-4-(diethylamino)benzamide (ADB) and its deuterium-coded

analog, d-ADB. For the relative quantification, the FBRA and RM samples were derivatized with

ADB and d-ADB, respectively, then the resulting derivatives were mixed and subjected to LC/ESI-

MS/MS; by this method, we found that the fermentation process significantly increased the free HCA

and HBA contents. The HCA and HBA contents in the FBRA were also determined, in which the d-

ADB-derivatized standards of known amounts were used as the internal standards. The ADB-

derivatization enabled the sensitive and specific detection, and the use of d-ADB significantly

improved the assay precision.

Rapid discovery of cyclopamine analogs from Fritillaria and Veratrum plants using LC-Q-TOF-

MS and LC-QqQ-MS

Yuan Du, Zu-Guo Zheng, Yue Yu, Zi-Tian Wu, Hui-Jun Li

ABSTRACT

Cyclopamine, an inhibitor of the Hedgehog (Hh) signaling pathway, has been paid much attention in

treating a wide variety of tumors. However, isolation and purification of cyclopamine analogs from

medicinal plants remain challengeable. We herein proposed an efficient strategy using liquid

chromatography quadrupole-time-of-flight mass spectrometry (LC-Q-TOF-MS) and liquid

chromatography triple-quadrupole mass spectrometry (LC-QqQ-MS) for rapid screening and targeted

isolation of cyclopamine analogs in Fritillaria and Veratrum plants. Firstly, fifteen reference

compounds were characterized by LC-Q-TOF-MS and their characteristic fragment ions were

summarized. Secondly, according to the characteristic fragment ions at m/z 67.1, 84.1, 109.1 and

114.1, rapid chemical screening of plant extracts was carried out by LC-QqQ-MS using precursor ion

scan mode and 69 pre-target compounds were screened out. Thirdly, 24 real target compounds were

verified by LC-Q-TOF-MS based on relative abundances (over 20%) of characteristic fragment ions.

Fourthly, the targeted isolation of Fritillaria ussuriensis bulb and Veratrum dahuricum rhizome

afforded a novel cyclopamine analog namely 15β-hydroxy-23-isopengbeisine B as well as four known

ones, whose structures were determined by nuclear magnetic resonance (NMR) analysis. Additionally,

these five analogs were evaluated for the inhibitory activity of Hh signaling pathway in NIH/3T3 cell

and cytotoxicity in PANC-1 and HepG2 cells. These results indicated that the proposed strategy was

reliable for rapid discovery and targeted isolation of important natural products from chemically

complex plant matrices.

181

Development and validation of a HS/GC–MS method for the simultaneous analysis of diacetyl

and acetylpropionyl in electronic cigarette refills

Sophia Barhdadi, Michaël Canfyn, Patricia Courselle, Vera Rogiers, Eric Deconinck

ABSTRACT

The use of e-cigarettes as alternative for tobacco cigarettes has become increasingly popular, even

though their safety has not yet been scientifically established. One of the frequently raised concerns is

the potential toxicity of certain flavours present in the e-liquids, such as diacetyl and acetylpropionyl.

It is therefore important to be able to identify and quantify both compounds. Numerous analytical

methods have been published for determining e-liquid compositions, but concerns exist with respect to

the lack of analytical evaluation. Hence in this study, a new HS/GC–MS-based method was developed

for the screening and quantification of diacetyl and acetylpropionyl in e-liquids. This method was fully

validated using the ‗total error‘ approach. The LOQ of the analytical method was 5 ppm for diacetyl

and acetylpropionyl. The obtained accuracy profiles show that the β-expectation tolerance intervals did

not exceed the acceptance limits of ± 10%, meaning that 95% of future measurements will be included

in the [-10%, 10%] bias limits. As proof of applicability, the validated method was successfully

applied on a small set of e-liquid samples, indicating that this methodology could be used for routine

quality control analyses of e-liquids.

Structural characterization and discrimination of the Paris polyphylla var. yunnanensis and

Paris vietnamensis based on metabolite profiling analysis

Li-ping Kang, Yuan-yuan Huang, Zhi-lai Zhan, Da-hui Liu, Lu-qi Huang

ABSTRACT

This study aimed to distinguish the rhizomes of Paris polyphylla var. yunnanensis (Franch) Hand

Mazz (PPY) and Paris veitnamensis (Takht.) H. Li (PV) using metabolomics-based ultra high-

performance liquid chromatography coupled with quadrupole time-of-fligh mass spectrometry

(UHPLC/Q-TOF MS). First, the UHPLC/Q-TOF MS approach was optimized for metabolite profiling.

Then, the MS data were processed using UNIFI™ combined with an in-house library to automatically

characterize the metabolites. Based on the exact mass information, the fragmentation characteristics,

and the retention time of compounds, and the fragmentation mechanism and retention behavior of

steroidal glycosides in the references, the structures identified by UNIFI were further verified. Overall,

146 metabolites, including 42 potential new compounds, were identified or tentatively identified.

Pattern recognition analysis of the PPY and PV MS data revealed that they were clearly separated, and

15 potential biomarkers for differentiating between them were selected. These biomarkers were

subsequently used to successfully predict the genus of PPY and PV samples. These results indicated

that metabolite profiling by UHPLC/Q-TOF MS is an effective, robust approach for determining the

characteristic biomarkers that differentiate between TCM species with multiple botanical origins.

182

Volume 143 September 2017

Reliability of point-of-collection testing devices for drugs of abuse in oral fluid: A systematic

review and meta-analysis

Juliana Nichterwitz Scherer, Taís Regina Fiorentin, Bruna Tassi Borille, Graciela Pasa, Flavio

Pechansky

ABSTRACT

Point-of-collection testing (POCT) devices for drugs of abuse are used to screen for the presence of

psychoactive substances (PAS) in different types of settings and environments. However, these quick

and advantageous tools also present disadvantages, including low-reliability measures in comparison

to chromatographic assays. Therefore, this article presents a systematic review and meta-analysis of

studies evaluating the reliability of measurements of PAS detection in oral fluid using POCT devices.

The reliability measures for detection of the five most important drug classes – cocaine,

amphetamines, benzodiazepines, cannabinoids and opioids, are reported. The article also presents a

subgroup analysis considering the reliability estimates for the different POCT devices that were

evaluated by the studies contemplated in the review. A discussion considering the strengths and

limitations of POCT techniques was performed in order to guide policymakers, traffic agents and other

professionals who also conduct such tests. The use of POCT devices often involves legal and moral

aspects of the subjects tested, which demands critical evaluation of these devices before they are

implemented in different settings.

High-throughput thermofluor-based assays for inhibitor screening of STAT SH2 domains

Elvin D. de Araujo, Pimyupa Manaswiyoungkul, Johan Israelian, Jisung Park, Patrick T. Gunning

ABSTRACT

The development of STAT protein-specific inhibitors has been the focus of a number of drug

discovery programs. STAT activation occurs through phosphorylation at the STAT SH2 domain,

resulting in dimerization, translocation to the nucleus, and transcription of proliferative genes. Due to

the functional significance of the SH2 domain in mediating multiple components of the STAT

signalling cascade, many libraries of inhibitors have been designed to target the SH2 domain. This has

triggered the requirement for effective high-throughput screening platforms for analyzing binding by

larger chemical libraries to STAT proteins. Herein, we present strategies for the development of a

high-throughput thermal denaturation-based assay for identifying STAT inhibitors as well as high-

yielding recombinant expression and purification of untagged STAT1, STAT3, and STAT5 proteins.

This assay reports changes in the fluorescence of a labelled peptide bound to the STAT protein as a

function of increasing temperature. STAT inhibitors which displace the labelled peptide elicit a change

in the melt profile, which is quantitatively determined as a change in the area under the curve. This

assay offers an alternative, but complimentary, high-throughput screening strategy for identifying new

inhibitors of STAT proteins as well as characterizing further, the mode of inhibition by existing

libraries of compounds.

183

Trace level determination of 5-hydroxytryptamine and its related indoles in amniotic fluid by

gas chromatography–mass spectrometry

Hongmei Shi, Bo Wang, Lingmei Niu, Mengsi Cao, Pingping Zhang

ABSTRACT

5-hydroxytryptamine (5-HT) and its derivatives are endogenously active substances involved in

multiple physiological and pathological processes. A novel method of detetermining 5-hydroxyindole

ethanol (5-HTOL), 5-hydroxyindole acetic acid (5-HIAA), 5-hydroxytryptophan (5-HTP) and 5-HT in

amniotic fluid by gas chromatography-mass spectrometry (GC–MS) was established based on a

modified method of derivatization by silanization, in combination with solid-phase extraction

pretreatment. Good linearity was achieved in the tested calibration range. The limits of detection

(LOD) were 0.05, 0.08, 0.56, 0.43 μg/L for 5-HTOL, 5-HIAA, 5-HTP and 5-HT, respectively.

Accuracy (92.4-103.3) and precision (RSD < 5.4%) for all analytes was also determined. Then the

method was used to analyze samples of amniotic fluid from 12 patients carrying foetuses with trisomy

21 and 12 healthy controls. Compared with normal fetuses, the levels of 5-HTOL, 5-HTP and 5-HT in

the amniotic fluid were significantly altered in the fetuses with trisomy 21 (P < 0.01); the level of 5-

HIAA showed no significant difference between the two groups (P>0.05). This is a rapid, sensitive and

reliable method for the determination of 5-HTOL, 5-HIAA, 5-HTP and 5-HT, and the study provide

both potential trisomy 21 markers and elucidation of the physiological and pathological roles of 5-HT.

Identification of related substances in tofacitinib citrate by LC-MS techniques for synthetic

process optimization

Xiao Wu, Xuefang Zeng, Lei Wang, Taijun Hang, Min Song

ABSTRACT

A specific LC-MS method was developed for separation, identification and characterization of the

process-related substances and degradation products in tofacitinib citrate. The separation was achieved

on a LiChrospher C18 column (250 mm × 4.6 mm, 5 μm) by linear gradient elution of 0.1%

ammonium acetate solution (pH adjusted to 4.0 by formic acid) and acetonitrile at a flow rate of 1.0

mL/min. Forced degradation studies were conducted under hydrolytic (acidic, basic), oxidative,

photolytic and thermal stress conditions as described in ICH. It was found that tofacitinib was stable

under photolytic condition, but degraded obviously in acidic, basic, thermal and oxidative conditions.

The high resolution TOF-MS and MS/MS were used for determination and structural identification of

the related substances. Eleven major related substances were detected and identified as five process-

related substances and six degradation products, and three of them were further synthesized and

characterized by NMR spectroscopy. The most plausible mechanisms involved in the formation of the

related substances were also proposed. Since related substances have a significant impact on drug

safety, quality and efficacy, the data obtained are valuable for process monitoring and quality

assurance of tofacitinib citrate.

184

Macro- and microstructural tracking of ageing-related changes of papaverine hydrochloride-

loaded electrospun nanofibrous buccal sheets

Adrienn Kazsoki, Péter Szabó, Károly Süvegh, Tamás Vörös, Romána Zelkó

ABSTRACT

Electrospun papaverine hydrochloride-loaded nanofibrous sheets consist of hydroxypropyl

cellulose/poly(vinyl alcohol) composite were prepared for buccal administration for cerebral ischemia.

The nanofibrous drug delivery system was subjected to accelerated stability test for four weeks in

order to scrutinize the solid state changes relating to the stress induced (40 ± 2 °C/75 ± 5% relative

humidity) physical ageing. Micro- and macrostructural alterations were detected using scanning

electron microscopy (SEM), Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR) and

positron annihilation lifetime spectroscopy (PALS). Significant changes were revealed at both

supramolecular and macroscopic levels. Microscopic morphology uncovered major morphological

transitions. Subtle variations of Raman and FTIR spectra indicated that the local chemical environment

of papaverine was altered suggesting a partial phase transition of the active. Discrete o-Ps lifetimes and

lifetime-distributions unveiled a two-step ageing process of the drug carrier. In addition to the tracking

of the glassy-to-rubbery transition of the fiber forming polymers, the Raman spectroscopy enabled

monitoring the kinetics of the phase transition observed.

A simplified guide for charged aerosol detection of non-chromophoric compounds—Analytical

method development and validation for the HPLC assay of aerosol particle size distribution for

amikacin

Arianne Soliven, Imad A. Haidar Ahmad, James Tam, Nani Kadrichu, Andrei Blasko

ABSTRACT

Amikacin, an aminoglycoside antibiotic lacking a UV chromophore, was developed into a drug

product for delivery by inhalation. A robust method for amikacin assay analysis and aerosol particle

size distribution (aPSD) determination, with comparable performance to the conventional UV detector

was developed using a charged aerosol detector (CAD). The CAD approach involved more parameters

for optimization than UV detection due to its sensitivity to trace impurities, non-linear response and

narrow dynamic range of signal versus concentration. Through careful selection of the power

transformation function value and evaporation temperature, a wider linear dynamic range, improved

signal-to-noise ratio and high repeatability were obtained. The influences of mobile phase grade and

glassware binding of amikacin during sample preparation were addressed. A weighed (1/X2) least

square regression was used for the calibration curve. The limit of quantitation (LOQ) and limit of

detection (LOD) for this method were determined to be 5 μg/mL and 2 μg/mL, respectively. The

method was validated over a concentration range of 0.05–2 mg/mL. The correlation coefficient for the

peak area versus concentration was 1.00 and the y-intercept was 0.2%. The recovery accuracies of

triplicate preparations at 0.05, 1.0, and 2.0 mg/mL were in the range of 100–101%. The relative

standard deviation (Srel) of six replicates at 1.0 mg/mL was 1%, and Srel of five injections at the limit

of quantitation was 4%. A robust HPLC-CAD method was developed and validated for the

determination of the aPSD for amikacin. The CAD method development produced a simplified

procedure with minimal variability in results during: routine operation, transfer from one instrument to

another, and between different analysts.

185

Proton dissociation properties of arylphosphonates: Determination of accurate Hammett

equation parameters

Gergő Dargó, Adrienn Bölcskei, Alajos Grün, Szabolcs Béni, György T. Balogh

ABSTRACT

Determination of the proton dissociation constants of several arylphosphonic acid derivatives was

carried out to investigate the accuracy of the Hammett equations available for this family of

compounds. For the measurement of the pKa values modern, accurate methods, such as the differential

potentiometric titration and NMR-pH titration were used. We found our results significantly different

from the pKa values reported before (pKa1: MAE = 0.16 pKa2: MAE = 0.59). Based on our recently

measured pKa values, refined Hammett equations were determined that might be used for predicting

highly accurate ionization constants of newly synthesized compounds (pKa1 = 1.70–0.894σ, pKa2 =

6.92–0.934σ).

Sub–1 min separation in sequential injection chromatography for determination of synthetic

water-soluble dyes in pharmaceutical formulation

Polina Davletbaeva, Petr Chocholouń, Andrey Bulatov, Dalibor Ńatínský, Petr Solich

ABSTRACT

Sequential Injection Chromatography (SIC) evolved from fast and automated non-separation

Sequential Injection Analysis (SIA) into chromatographic separation method for multi-element

analysis. However, the speed of the measurement (sample throughput) is due to chromatography

significantly reduced. In this paper, a sub–1 min separation using medium polar cyano monolithic

column (5 mm × 4.6 mm) resulted in fast and green separation with sample throughput comparable

with non-separation flow methods The separation of three synthetic water-soluble dyes (sunset yellow

FCF, carmoisine and green S) was in a gradient elution mode (0.02% ammonium acetate, pH 6.7 –

water) with flow rate of 3.0 mL min−1 corresponding with sample throughput of 30 h−1.

Spectrophotometric detection wavelengths were set to 480, 516 and 630 nm and 10 Hz data collection

rate. The performance of the separation was described and discussed (peak capacities 3.48–7.67, peak

symmetries 1.72–1.84 and resolutions 1.42–1.88). The method was represented by validation

parameters: LODs of 0.15-0.35 mg L−1, LOQs of 0.50–1.25 mg L−1, calibration ranges 0.50-150.00

mg L−1 (r > 0.998) and repeatability at 10.0 mg L−1 of RSD ≤ 0.98% (n = 6). The method was used

for determination of the dyes in ―forest berries‖ colored pharmaceutical cough-cold formulation. The

sample matrix – pharmaceuticals and excipients were not interfering with vis determination because of

no retention in the separation column and colorless nature. The results proved the concept of fast and

green chromatography approach using very short medium polar monolithic column in SIC.

186

Impurity profiling of liothyronine sodium by means of reversed phase HPLC, high resolution

mass spectrometry, on-line H/D exchange and UV/Vis absorption

M. Ruggenthaler, J. Grass, W. Schuh, C.G. Huber, R.J. Reischl

ABSTRACT

For the first time, a comprehensive investigation of the impurity profile of the synthetic thyroid API

(active pharmaceutical ingredient) liothyronine sodium (LT3Na) was performed by using reversed

phase HPLC and advanced structural elucidation techniques including high resolution tandem mass

spectrometry (HRMS/MS) and on-line hydrogen-deuterium (H/D) exchange. Overall, 39 compounds

were characterized and 25 of these related substances were previously unknown to literature. The

impurity classification system recently developed for the closely related API levothyroxine sodium

(LT4Na) could be applied to the newly characterized liothyronine sodium impurities resulting in a

wholistic thyroid API impurity classification system. Furthermore, the mass-spectrometric CID-

fragmentation of specific related substances was discussed and rationalized by detailed fragmentation

pathways. Moreover, the UV/Vis absorption characteristics of the API and selected impurities were

investigated to corroborate chemical structure assignments derived from MS data.

A UHPLC method for the rapid separation and quantification of anthocyanins in acai berry and

dry blueberry extracts


ABSTRACT

The presented work describes the development and validation of a rapid UHPLC-UV method using a

core-shell particle column with a pentafluorophenyl stationary phase for the separation and

quantitative analysis of the six anthocyanins in acai berry and dry blueberry extracts. The anthocyanins

(cyanidin-3-glucoside, cyanidin-3-rutenoside, delphinidin-3-galactoside, delphinidin-3-glucoside,

delphinidin-3-rutenoside, and peonidin-3-glucoside) were separated and analyzed in 5 min. The

chromatographic separation was performed on a Kinetex PFP (150 × 2.1 mm) core-shell column with a

particle size of 1.7 μm at a temperature of 50 °C. Acetonitrile was used as mobile phase B and 5%

formic acid, filtrated through a 0.22 μm filter, as mobile phase A. They were delivered at a flow rate of

0.55 mL min−1 according to the elution gradient program. The detection wavelength was set at 520

nm. A solid-liquid extraction with a solution of methanol and a 5% water solution of formic acid (25 +

75 v/v) using an ultrasonic bath was chosen for the preparation of the available commercial samples of

food supplements with a content of acai berry extract and blueberry extract. Under optimal

chromatographic conditions, the method was validated. Recoveries for all analyzed anthocyanins were

97.8–102.6% and the relative standard deviation ranged from 0.4% to 3.0% for within-day and from

0.6% to 3.1% for between-day repeatability. The limits of detection were in the range of 0.11–0.14 μg

mL−1.

187

Combined computational-experimental approach to predict blood–brain barrier (BBB)

permeation based on ―green‖ salting-out thin layer chromatography supported by simple

molecular descriptors

Krzesimir Ciura, Mariusz Belka, Piotr Kawczak, Tomasz Bączek, Joanna Nowakowska

ABSTRACT

The objective of this paper is to build QSRR/QSAR model for predicting the blood–brain barrier

(BBB) permeability. The obtained models are based on salting-out thin layer chromatography

(SOTLC) constants and calculated molecular descriptors. Among chromatographic methods SOTLC

was chosen, since the mobile phases are free of organic solvent. As consequences, there are less toxic,

and have lower environmental impact compared to classical reserved phases liquid chromatography

(RPLC). During the study three stationary phase silica gel, cellulose plates and neutral aluminum oxide

were examined. The model set of solutes presents a wide range of log BB values, containing

compounds which cross the BBB readily and molecules poorly distributed to the brain including drugs

acting on the nervous system as well as peripheral acting drugs. Additionally, the comparison of three

regression models: multiple linear regression (MLR), partial least-squares (PLS) and orthogonal partial

least squares (OPLS) were performed. The designed QSRR/QSAR models could be useful to predict

BBB of systematically synthesized newly compounds in the drug development pipeline and are

attractive alternatives of time-consuming and demanding directed methods for log BB measurement.

The study also shown that among several regression techniques, significant differences can be obtained

in models performance, measured by R2 and Q2, hence it is strongly suggested to evaluate all

available options as MLR, PLS and OPLS.

Development of a new extraction technique and HPLC method for the analysis of non-

psychoactive cannabinoids in fibre-type Cannabis sativa L. (hemp)

Virginia Brighenti, Federica Pellati, Marleen Steinbach, Davide Maran, Stefania Benvenuti

ABSTRACT

The present work was aimed at the development and validation of a new, efficient and reliable

technique for the analysis of the main non-psychoactive cannabinoids in fibre-type Cannabis sativa L.

(hemp) inflorescences belonging to different varieties. This study was designed to identify samples

with a high content of bioactive compounds, with a view to underscoring the importance of quality

control in derived products as well. Different extraction methods, including dynamic maceration

(DM), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) and supercritical-

fluid extraction (SFE) were applied and compared in order to obtain a high yield of the target analytes

from hemp. Dynamic maceration for 45 min with ethanol (EtOH) at room temperature proved to be the

most suitable technique for the extraction of cannabinoids in hemp samples. The analysis of the target

analytes in hemp extracts was carried out by developing a new reversed-phase high-performance liquid

chromatography (HPLC) method coupled with diode array (UV/DAD) and electrospray ionization-

mass spectrometry (ESI–MS) detection, by using an ion trap mass analyser. An Ascentis Express C18

column (150 mm × 3.0 mm I.D., 2.7 μm) was selected for the HPLC analysis, with a mobile phase

composed of 0.1% formic acid in both water and acetonitrile, under gradient elution. The application

of the fused-core technology allowed us to obtain a significant improvement of the HPLC performance

compared with that of conventional particulate stationary phases, with a shorter analysis time and a

remarkable reduction of solvent usage.

188

Analytical profiling of selected antioxidants and total antioxidant capacity of goji (Lycium spp.)

berries

Michele Protti, Isacco Gualandi, Roberto Mandrioli, Sergio Zappoli, Laura Mercolini

ABSTRACT

Goji berries and derived products represent a relevant source of micronutrients, most of which are

natural antioxidants and contribute to the high nutritional quality of these fruits. Three brands of dried

goji berries have been analysed by a multidisciplinary approach to get an insight into both their content

of selected antioxidants and their antioxidant capacity (AC). The former goal has been achieved by

developing a liquid chromatographic method coupled to mass spectrometry and combined to a fast

solid phase extraction. Several significant representative antioxidant compounds belonging to the

following classes: flavonoids, flavan-3-ols, phenolic acids, amino acids and derivatives, and

carotenoids have been taken into account. Quercetin and rutin were found to be the predominant

flavonoids, chlorogenic acid was the most abundant phenolic acid and zeaxanthin was the major

carotenoid. The AC of the goji berries has been evaluated by four analytical methods in order to

estimate the contributions of different reactions involved in radicals scavenging. In particular, AC has

been determined using 3 standardised methods (DPPH, ABTS, ORAC) and a recently proposed

electrochemical method, which measures the scavenging activity of antioxidants towards OH radicals

generated both by hydrogen peroxide photolysis and the Fenton reaction. The results obtained from

chemical composition and antioxidant capacity assays confirm the high nutritional and commercial

value of goji berries and highlight that the three brands do not exhibit significant differences.

Characterization and inhibition studies of tissue nonspecific alkaline phosphatase by

aminoalkanol derivatives of 1,7-dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-

trione, new competitive and non-competitive inhibitors, by capillary electrophoresis

Błażej Grodner, Mariola Napiórkowska

ABSTRACT

The article describes the inhibitory effect of two new aminoalkanol derivatives on the enzymatic

kinetic of tissue non-specific alkaline phosphatase with use of capillary zone electrophoresis to

evaluate the inhibitory effect. This technique allows to investigate of the enzymatic kinetic by the

measure of the amounts of the substrate and product in the presence of compound (I) or (II) in the

reaction mixture. The separation process was conducted using an eCAP fused-silica capillary. The

detector was set at 200 nm. The best parameters for the analysis were: 25 mM sodium dihydrogen

phosphate adjusted to pH = 2.5, temperature 25 °C, and voltage −15 kV. Lineweaver-Burk plots were

constructed and determined by comparison of the Km, of alkaline phosphatase in the presence of

inhibitor (I) or (II) with the Km in a solution without inhibitor. The influence of replacement the

propylamine group by the dimethylamine group on tissue non-specific alkaline phosphatase inhibition

activity of new derivatives (I) and (II) was investigated. The tested compounds (I) and (II) were found

to be tissue non-specific alkaline phosphatase inhibitors. Detailed kinetic studies indicated a

competitive mode of inhibition against tissue non-specific alkaline phosphatase for compound (I) and

non-competitive mode of inhibition for compound (II).

189

A validated UHPLC-QTOF-MS method for quantification of metformin and teneligliptin in rat

plasma: Application to pharmacokinetic interaction study

David Paul, Lingesh Allakonda, Nanjappan Satheeshkumar

ABSTRACT

In this study a sensitive UHPLC-QTOF-MS method was developed and validated for the quantitation

of the metformin (MET) and teneligliptin (TEN) in rat plasma using dapagliflozin as an internal

standard (IS). Chromatographic separation were carried out on a Acquity UPLC HSS Cyano column

(100 mm x 2.1 mm, 1.8 μm) using gradient mobile phase system consisting of 0.1% formic acid and

acetonitrile at a flow rate of 0.4 mL/min, within a run time of 6 min. Protein precipitation method was

used as sample preparation approach. Detection of target ions [M+H]+ at m/z 130.1085 for MET, m/z

427.2277 for TEN and m/z 409.1623 for IS was performed at positive ion electrospray ionization

mode. Linearity was assessed in the range of 0.98–1000 ng/mL for both MET and TEN. The

developed assay was validated as per US-FDA and EMA bioanalytical guidelines and successfully

applied to pharmacokinetic interaction study in SD rats. A 1.1 fold increment in the AUC levels of

MET and TEN was observed when co-administered together in rats.

Overcoming interference with the detection of a stable isotopically labeled microtracer in the

evaluation of beclabuvir absolute bioavailability using a concomitant microtracer approach

Hao Jiang, Craig Titsch, Jianing Zeng, Barry Jones, Mark E. Arnold

ABSTRACT

The oral absolute bioavailability of beclabuvir in healthy subjects was determined using a microdose

(100 μg) of the stable isotopically labeled tracer via intravenous (IV) infusion started after oral dosing

of beclabuvir (150 mg). To simultaneously analyze the concentrations of the IV microtracer

([13C6]beclabuvir) and beclabuvir in plasma samples, a liquid chromatography-triple quadrupole mass

spectrometry (LC–MS/MS) method was initially developed. Surprisingly beclabuvir significantly

interfered with the IV microtracer detection when using the selected reaction monitoring (SRM) in the

assay. An interfering component from the drug substance was observed using a high resolution mass

spectrometer (HRMS). The mass-to-charge (m/z) of the interfering component was −32 ppm different

from the nominal value for the IV microtracer and thus could not be differentiated in the SRM assay

by the unit mass resolution. To overcome this interference, we evaluated two approaches by either

monitoring an alternative product ion using the SRM assay or isolating the interfering component

using the parallel reaction monitoring (PRM) assay on the HRMS. This case study has demonstrated

two practical approaches for overcoming interferences with the detection of stable isotopically labeled

IV microtracers in the evaluation of absolute bioavailability, which provides users the flexibility in

using either LC–MS/MS or HRMS to mitigate unpredicted interferences in the assay to support

microtracer absolute bioavailability studies.

190

Ex-vivo measurement of scalp follicular infundibulum delivery of zinc pyrithione and climbazole

from an anti-dandruff shampoo

Guoqiang Chen, Chengdong Ji, Miao Miao, Kang Yang, Hans-Gerd Janssen

ABSTRACT

Efficient delivery of anti-dandruff (AD) actives into the scalp follicular infundibulum as well as onto

the scalp surface is critical for the efficacy of AD shampoos. A method involving scalp cyanoacrylate

(CA) biopsy sampling, a tailor made cutting device, ultra-high-performance liquid chromatography–

tandem mass spectrometry (UHPLC–MS/MS) analysis, scanning electron microscopy (SEM)

measurement and Raman imaging has been developed for the measurement of delivery of zinc

pyrithione (ZPT) and climbazole (CBZ) from an AD shampoo into the scalp follicular infundibulum.

Scalp CA biopsy enables the sampling of ZPT and CBZ delivered into the scalp follicular infundibula

as well as onto the scalp surface. Raman imaging of scalp CA biopsy samples allows the visualization

of the spatial distribution of ZPT and CBZ deposited on the scalp. A tailor made cutting device enables

the separation of the scalp follicular infundibulum sample (20 μm below the scalp surface) from the

scalp surface samples (including top 20 μm of infundibula). UHPLC–MS/MS was used as a sensitive

and specific methodology enabling the quantification of ZPT and CBZ without interference. Using this

method, both ZPT and CBZ were successfully quantified and spacially visualized within the scalp

follicular infundibulum, after scalp was washed with an AD shampoo.

Pooled human liver preparations, HepaRG, or HepG2 cell lines for metabolism studies of new

psychoactive substances? A study using MDMA, MDBD, butylone, MDPPP, MDPV, MDPB, 5-

MAPB, and 5-API as examples

Lilian H.J. Richter, Veit Flockerzi, Hans H. Maurer, Markus R. Meyer

ABSTRACT

Metabolism studies play an important role in clinical and forensic toxicology. Because of potential

species differences in metabolism, human samples are best suitable for elucidating metabolism.

However, in the case of new psychoactive substances (NPS), human samples of controlled studies are

not available. Primary human hepatocytes have been described as gold standard for in vitro

metabolism studies, but there are some disadvantages such as high costs, limited availability, and

variability of metabolic enzymes. Therefore, the aim of our study was to investigate and compare the

metabolism of six methylenedioxy derivatives (MDMA, MDBD, butylone, MDPPP, MDPV, MDPB)

and two bioisosteric analogues (5-MAPB, 5-API) using pooled human liver microsomes (pHLM)

combined with cytosol (pHLC) or pooled human liver S9 fraction (pS9) all after addition of co-

substrates for six phase I and II reactions. In addition, HepaRG and HepG2 cell lines were used.

Results of the different in vitro tools were compared to each other, to corresponding published data,

and to metabolites identified in human urine after consumption of MDMA, MDPV, or 5-MAPB.

Incubations with pHLM plus pHLC showed similar results as pS9. A more cost efficient model for

prediction of targets for toxicological screening procedures in human urine should be identified. As

expected, the incubations with HepaRG provided better results than those with HepG2 concerning

number and signal abundance of the metabolites. Due to easy handling without special equipment,

incubations with pooled liver preparations should be the most suitable alternative to find targets for

toxicological screening procedures for methylenedioxy derivatives and bioisosteric analogues.

191

Evaluation of pharmacokinetics and blood-brain barrier permeability of mitragynine using in

vivo microdialysis technique

Wai Mun Kong, Zahurin Mohamed, Mohammed A. Alshawsh, Zamri Chik

ABSTRACT

A microdialysis system coupled with a sensitive ultra-fast liquid chromatography–mass spectrometry

(UFLC-MS) method was developed for the pharmacokinetic analysis of mitragynine in rat blood and

striatum. Mitragynine is an active alkaloid of Mitragyna speciosa and has been proposed to be used for

opioid withdrawal therapy. In this study, chromatographic separation was performed in a gradient

elution mode with 0.1% formic acid and acetonitrile on a Zorbax Eclipse C18 column. The mass

spectrometric (MS) analysis was carried out in a positive electrospray mode and mitragynine ion (m/z

399.2) was monitored in extracted ion chromatography. A good linearity range was obtained from 10-

1000 ng/mL with acceptable accuracy and precision parameters. The microdialysate was collected

simultaneously from the striatum and the right jugular vein using microdialysis probes. After a single

intravenous administration of 10 mg/kg mitragynine, mitragynine showed a two-compartmental drug

elimination pattern with half-life (T1/2) of approximately 13 h. The percent of AUCbrain/AUCplasma

of mitragynine was calculated and shown to be 65.8 ± 4.5%. The results indicated that mitragynine

could be a suitable molecule to develop into an opioid replacement drug based on its ideal

pharmacokinetic properties, namely, small molecular size, lipophilic in nature and with excellent

blood–brain barrier (BBB) permeability.

1H NMR spectral identification of medication in cerebrospinal fluid of pediatric meningitis

Shayne Mason, Carolus J. Reinecke, Regan Solomons, Ron A. Wevers, Udo F.H. Engelke

ABSTRACT

Exploratory metabolomics studies of cerebrospinal fluid (CSF), using proton nuclear magnetic

resonance (1H NMR) spectroscopy, hold major potential application in neurodiagnostics. Such studies,

however, rely upon established databases of known metabolites. Here we address the ‗unknowns‘ in

the 1H NMR spectra of CSF from treated pediatric meningitis cases. Through knowledge of the

clinical information given by the pediatrician and analytical application of 1H NMR spectroscopy on

pure reference compounds of the medication used, we identified four of the previously unknown

compounds in the 1H NMR CSF spectra — the drugs pyrazinamide, isoniazid, acyclovir, and

sulfamethoxazole. We report on the one- and two-dimensional 1H NMR spectral data and chemical

information of these four compounds. By expanding our knowledge of 1H NMR CSF spectra from

treated meningitis cases, we are able to bring 1H NMR closer to the forefront of neurodiagnostics.

192

Therapeutic drug monitoring of beta-lactam antibiotics – Influence of sample stability on the

analysis of piperacillin, meropenem, ceftazidime and flucloxacillin by HPLC-UV

Nadine Pinder, Thorsten Brenner, Stefanie Swoboda, Markus A. Weigand, Torsten Hoppe-Tichy

ABSTRACT

Introduction: Therapeutic drug monitoring (TDM) is a useful tool to optimize antibiotic therapy.

Increasing interest in alternative dosing strategies of beta-lactam antibiotics, e.g. continuous or

prolonged infusion, require a feasible analytical method for quantification of these antimicrobial

agents. However, pre-analytical issues including sample handling and stability are to be considered to

provide valuable analytical results.

Methods: For the simultaneous determination of piperacillin, meropenem, ceftazidime and

flucloxacillin, a high performance liquid chromatography (HPLC) method including protein

precipitation was established utilizing ertapenem as internal standard. Long-term stability of stock

solutions and plasma samples were monitored. Furthermore, whole blood stability of the analytes in

heparinized blood tubes was investigated comparing storage under ambient conditions and 2–8 °C.

Results: A calibration range of 5–200 μg/ml (piperacillin, ceftazidime, flucloxacillin) and 2–200

μg/ml (meropenem) was linear with r2 > 0.999, precision and inaccuracy were <9% and <11%,

respectively. The successfully validated HPLC assay was applied to clinical samples and stability

investigations. At −80 °C, plasma samples were stable for 9 months (piperacillin, meropenem) or 13

months (ceftazidime, flucloxacillin). Concentrations of the four beta-lactam antibiotics in whole blood

tubes were found to remain within specifications for 8 h when stored at 2–8 °C but not at room

temperature.

Conclusions: The presented method is a rapid and simple option for routine TDM of piperacillin,

meropenem, ceftazidime and flucloxacillin. Whereas long-term storage of beta-lactam samples at −80

°C is possible for at least 9 months, whole blood tubes are recommended to be kept refrigerated until

analysis.

193

Development and validation of an ultra-performance liquid chromatography–tandem mass

spectrometry method for quantification of SR1001, an inverse agonist of retinoid-related orphan

receptors, and its application to pharmacokinetic studies in streptozotocin-induced diabetic mice

Cuipei Lin, Hanqing Wang, Hua Sun, Chengju Xiao, Zhijie Li

ABSTRACT

Retinoic acid receptor-related orphan receptors (RORs) play critical roles in the onset and progression

of type I diabetes, an autoimmune disease characterized by the destruction of pancreatic β-cells.

SR1001, an ROR inverse agonist, has been proven to be an effective diabetes treatment in the non-

obese diabetic (NOD) mouse model. However, optimization of this treatment is challenging because

knowledge of SR1001 pharmacokinetic (PK) behaviors in type I diabetic animals is limited. The aim

of our study was to develop and validate a specific and sensitive ultra-performance liquid

chromatography-tandem mass spectrometric (UPLC–MS/MS) method to measure the concentrations

of SR1001 in plasma and biological samples. Using the developed UPLC–MS/MS method, SR1001

linearity ranges in biological matrices were determined to be 5–1000 ng/mL, with correlation

coefficients of >0.99. The limit of detection (LOD) and limit of quantification (LOQ) values of

SR1001 were 1 and 5 ng/mL, respectively. And the intra-day and inter-day variances were less than

10%, and accuracy was within 90%–110%. The extraction recoveries of SR1001 were ≥80%, and no

significant matrix effect was observed. Using the validated UPLC–MS/MS method, levels of SR1001

in plasma and six major organs (heart, liver, spleen, lung, kidney, and brain) were determined in

streptozotocin (STZ) −induced diabetic mice. The PK parameters of SR1001 were also calculated. The

SR1001 drug concentration–time curves for organs and plasma showed similar trends, and the

elimination half-lives of SR1001 in diabetic mice were about 12 h. SR1001 was highly bound to

plasma protein, resulting in a much higher maximum concentration (Cmax = 144394 ng/mL) and area

under the concentration–time curve (AUC0-t = 2728258 ng/mL*h), but a low tissue/plasma partition

coefficient (Kp) value of <0.3.

Quantum dot-linked immunosorbent assay (QLISA) using orientation-directed antibodies

Miho Suzuki, Hikari Udaka, Takeshi Fukuda

ABSTRACT

An approach similar to the enzyme-linked immunosorbent assay (ELISA), with the advantage of

saving time and effort but exhibiting high performance, was developed using orientation-directed half-

part antibodies immobilized on CdSe/ZnS quantum dots. ELISA is a widely accepted assay used to

detect the presence of a target substance. However, it takes time to quantify the target with specificity

and sensitivity owing to signal amplification. In this study, CdSe/ZnS quantum dots are introduced as

bright and photobleaching-tolerant fluorescent materials. Since hydrophilic surface coating of quantum

dots rendered biocompatibility and functional groups for chemical reactions, the quantum dots were

modified with half-sized antibodies after partial reduction. The half-sized antibody could be bound to a

quantum dot through a unique thiol site to properly display the recognition domain for the core process

of ELISA, which is an antigen-antibody interaction. The reducing conditions were investigated to

generate efficient conjugates of quantum dots and half-sized antibodies. This was applied to IL-6

detection, as the quantification of IL-6 is significant owing to its close relationships with various

biomedical phenomena that cause different diseases. An ELISA-like assay with CdSe/ZnS quantum

dot institution (QLISA; Quantum dot-linked immunosorbent assay) was developed to detect 0.05

ng/mL IL-6, which makes it sufficiently sensitive as an immunosorbent assay.

194

Validation of liquid and gaseous calibration techniques for quantification of propofol in breath

with sorbent tube Thermal Desorption System GC–MS

Felix Maurer, Martin Geiger, Thomas Volk, Daniel I. Sessler, Sascha Kreuer

ABSTRACT

Plasma concentrations of intravenous drugs cannot currently be evaluated in real time to guide clinical

dosing. However, a system for estimating plasma concentration of the anesthetic propofol from

exhaled breath may soon be available. Developing reliable calibration and analytical validation

techniques is thus necessary. We therefore compared the established sorbent tube liquid injection

technique with a gas injection procedure using a reference gas generator. We then quantified propofol

with Tenax sorbent tubes in combination with gas-chromatography coupled mass spectrometry in the

breath of 15 patients (101 measurements). Over the clinically relevant concentration range from 10 to

50 ppbv, coefficient of determination was 0.995 for gas calibration; and over the range from 10 to 100

ng, coefficient of determination was 0.996 for liquid calibration. A regression comparing gas to liquid

calibration had a coefficient of determination of 0.89; slope 1.05 ± 0.01 (standard deviation). The limit

of detection was 0.74 ng and the lower limit of quantification was 1.12 ng for liquid; the limit of

detection was 0.90 ppbv and the lower limit of quantification was 1.36 ppbv for gas. Loaded sorbent

tubes were stable for at least 14 days without significant propofol loss as determined with either

method. Measurements from liquid or gas samples were comparably suitable for evaluation of patient

breath samples.

Development and validation of a bioanalytical method based on LC–MS/MS analysis for the

quantitation of CIGB-814 peptide in plasma from Rheumatoid Arthritis patients

Ania Cabrales-Rico, Yassel Ramos, Vladimir Besada, María del Carmen Domínguez, Luis Javier

González

ABSTRACT

CIGB-814, originally named as E18-3 APL1 or APL1 in preclinical experiments, is a novel therapeutic

peptide candidate for Rheumatoid Arthritis (RA). It is an altered peptide ligand containing a novel

CD4+ T-cell epitope of human heat shock protein 60 (83–109, MW 2988.38 g/mol) with a mutation

(D100 → L) that increases its affinity for HLA-II type molecules associated to RA. A bioanalytical

method, based on LC–MS/MS analysis, in the SRM mode was developed and fully validated to

quantify this peptide in human plasma. An internal standard with the same amino acid sequence but

labeled with three (13C615N2)-Lys residues was used for quantitation. The method provides a linear

range from 1.5 to 48 ng/mL (without matrix effect and carry over) and an accuracy and precision good

enough for monitoring more than 80% of the AUC of the PK profile in a phase I clinical trial. The

peptide was administered subcutaneously in three dose levels (1, 2.5 and 5 mg) not normalized to the

body weight of patients with RA. The low doses imposed an analytical challenge; however, a LLOQ of

1.5 ng/mL enabled the PK analysis. The Cmax, reached at 0.5 h, showed a great variability, that was

most likely due to the non-normalized doses; the proposed mechanism for this peptide; and the

variability between patients. A rapid clearance of this peptide (4–6 h) is advantageous for an

immunomodulatory drug, because the therapeutic schedule requires repeated dosages to restore

peripheral tolerance.

195

Application of volumetric absorptive microsampling device for quantification of tacrolimus in

human blood as a model drug of high blood cell partition

Kenji Kita, Yuji Mano

ABSTRACT

Volumetric absorptive microsampling device (VAMS) was evaluated for bioanalysis of tacrolimus,

which was used as a model drug with high blood cell partition. Aliquots of blood (ca. 10 μL) with

different hematocrits and fortified with tacrolimus were wicked up by VAMS and tacrolimus was

extracted with a methanol-water mixture (1:1, v/v) via sonication. After chromatography on an

AQUITY UPLC HSS T3 column (100 × 2.1 i.d., mm, 1.8 μm), tacrolimus and the internal standard

ascomycin, were detected in the positive ion mode with electrospray ionization by monitoring of

transitions m/z 826.6 → 616.4 and m/z 814.6 → 604.0, respectively. An assay method to quantify

tacrolimus from 1 to 250 ng/mL in whole blood was qualified by ensuring that linearity, selectivity,

intra- and inter-batch reproducibility, and stability were within the acceptance criteria. Consistent and

high extraction recovery of tacrolimus was ensured from blood with low- (20%), mid- (45%), and

high-hematocrit (65%) levels with minimal matrix effects. Apparent instability at ambient temperature

or 4 °C possibly due to reduced recovery suggests that tacrolimus in VAMS should be stored at −25 °C

until assay. Potential reduced recovery over time from VAMS should be taken into consideration in

method optimization.

Fast and efficient zirconia-based reversed phase chromatography for selective determination of

triptans in rat plasma

Sameh Ahmed, Noha N. Atia

ABSTRACT

Selective and fast chromatographic method was essential for the determination of triptans, selective

serotonin receptor (5-HT1) agonists, in biological specimens. However, selective chromatographic

separation of these highly basic drugs is a challenging problem on silica-based stationary phases.

Zirconia-based stationary phases have been introduced as an efficient alternative for silica-based

columns offering unique stability, selectivity, and retention ability for various classes of drugs. Herein,

a new selective, fast and reproducible high performance liquid chromatographic method (HPLC) was

developed and validated for the determination of four triptans in plasma samples namely; Sumatriptan

(SMT), Zolmitriptan (ZLT), Eletriptan (ELT) and Rizatriptan (RZT). Zirconia-based polybutadiene

(PBD) column was used for the separation and quantitation of the studied triptans in rat plasma based

on mixed mode ion exchange and reversed phase chromatography. Zirconia-PBD column (ZirChrom-

PBD) has enhanced chemical and thermal stability, selectivity and provides high resolution for the

investigated triptans in short analysis time compared with the commonly used C18 columns. A simple

isocratic separation mode was used with a mobile phase consisted of acetonitrile and 10 mM

phosphate buffer adjusted to pH 3.0 (20:80; v/v) at flow rate 1.0 mL min−1. The column was

maintained at 50 °C and effluent was monitored by photodiode array detector (PDA). The developed

method was validated in agreement with US-FDA guidelines and was appropriate for analysis of

triptans in plasma samples. The linearity range obtained for the developed HPLC method was 15–2000

ng mL−1 with detection limits of 4.8- 6.2 ng mL−1 for all the studied triptans. The developed

zirchonia-PBD-HPLC method was applied successfully to study the pharmacokinetics of ZLT in rats

after a single oral dose. The method was proved to be valuable for therapeutic drug monitoring and

bioavailability studies of the studied triptans.

196

Monitoring breast cancer treatment using a Fourier transform infrared spectroscopy-based

computational model

J. Depciuch, E. Kaznowska, S. Golowski, A. Koziorowska, J. Cebulski

ABSTRACT

Breast cancer affects one in four women, therefore, the search for new diagnostic technologies and

therapeutic approaches are of critical importance. This involves the development of diagnostic tools to

facilitate the detection of cancer cells, which is useful for assessing the efficacy of cancer therapies.

One of the major challenges for chemotherapy is the lack of tools to monitor efficacy during the course

of treatment. Vibrational spectroscopy appears to be a promising tool for such a purpose, as it yields

Fourier transformation infrared (FTIR) spectra which can be used to provide information on the

chemical composition of the tissue. Previous research by our group has demonstrated significant

differences between the infrared spectra of healthy, cancerous and post-chemotherapy breast tissue.

Furthermore, the results obtained for three extreme patient cases revealed that the infrared spectra of

post-chemotherapy breast tissue closely resembles that of healthy breast tissue when chemotherapy is

effective (i.e., a good therapeutic response is achieved), or that of cancerous breast tissue when

chemotherapy is ineffective. In the current study, we compared the infrared spectra of healthy,

cancerous and post-chemotherapy breast tissue. Characteristic parameters were designated for the

obtained spectra, spreading the function of absorbance using the Kramers–Kronig transformation and

the best fit procedure to obtain Lorentz functions, which represent components of the bands. The

Lorentz function parameters were used to develop a physics-based computational model to verify the

efficacy of a given chemotherapy protocol in a given case. The results obtained using this model

reflected the actual patient data retrieved from medical records (health improvement or no

improvement). Therefore, we propose this model as a useful tool for monitoring the efficacy of

chemotherapy in patients with breast cancer.

Enhancement in recovery of drugs with high protein binding efficiency from human plasma

using magnetic nanoparticles

Aniruddha Bhati, Rucha P. Desai, C.N. Ramchand

ABSTRACT

In this paper, we propose an alternate method for bioanalytical extraction of drugs from human plasma

samples using bare magnetic nanoparticles. The magnetic nanoparticles (MNPs) were used for

deproteination of biological samples that further assist in extraction of plasma bound drugs for

bioanalytical studies. The method uses basic solvents (ethanol, methanol, etc.) rather than the

expensive and toxic solvents. The MNPs provide several advantages like avoiding the use of

centrifuge machine, and making extraction time effective. The average time involved for the sample

preparation is around 30–40 min. The developed method was examined for seven different drugs

having moderate (40–70%) to high (>80%) plasma protein binding efficiency. The present study

focuses on the principle of magnetic nanoparticle based extraction of drug that binds with the plasma

protein. In calcitriol (protein binding efficiency >99%), it was observed that the drug extraction

efficiency could be enhanced by 16% using the present method. However, we assume that still there is

a scope for improving the extraction efficiency by optimizing proper solvent for the specific drug. The

use of magnetic nanoparticles makes the extraction cost effective and quick with improved efficiency.

197

Simultaneous quantification of endothelin receptor antagonists and phosphodiesterase 5

inhibitors currently used in pulmonary arterial hypertension

Yeliz Enderle, Lukas Witt, Heinrike Wilkens, Ekkehard Grünig, Jürgen Burhenne

ABSTRACT

Combination treatment with endothelin receptor antagonists (ERA) and phosphodiesterase 5 inhibitors

(PDE5I) improved efficacy of pulmonary arterial hypertension (PAH) therapy. However, drug–drug

interactions, variable exposure, non-adherence can influence plasma levels. For these reasons, drug

quantification may be advantageous particularly in patients with poor treatment responses. We

developed, validated, and applied an assay for the simultaneous quantification of ambrisentan,

bosentan, macitentan, sildenafil, and tadalafil as well as their main (and partly active) metabolites in

human plasma. This method is based on LC–MS/MS separation for a rapid and sensitive quantification

with stable isotopically labelled analogues as internal standards for each drug and metabolite. Sample

preparation was carried out using a solid phase extraction protocol based on Oasis HLB material. The

separation was achieved on a Kinetex C18 column and multiple reaction monitoring in negative

ionization mode was used for sensitive detection. The calibrations were linear for all analytes with

correlation coefficients >0.99 within the concentration range observed under a therapeutic PAH dosing

scheme with lower limits of quantification between 0.34 ng/mL (OH-ambrisentan) and 10 ng/mL

(despropyl-macitentan). Intra- and inter-day precision at LLOQ and QC levels ranged between 2.03%

and 19.8%, and 0.65% and 14.0%, respectively. The sample turnover time was 12 min. The

applicability of this versatile LC/MS/MS assay was verified by the successful analysis of clinical

routine samples of patients on PAH medication.

Application of 1H NMR spectroscopy to the metabolic phenotyping of rodent brain extracts: A

metabonomic study of gut microbial influence on host brain metabolism

J.R. Swann, I. Garcia-Perez, V. Braniste, I.D. Wilson, E. Holmes

ABSTRACT

1H NMR Spectroscopy has been applied to determine the neurochemical profiles of brain extracts

from the frontal cortex and hippocampal regions of germ free and normal mice and rats. The results

revealed a number of differences between germ free (GF) and conventional (CV) rats or specific

pathogen-free (SPF) mice with microbiome-associated metabolic variation found to be both species-

and region-dependent. In the mouse, the GF frontal cortex contained lower amounts of creatine, N-

acetyl-aspartate (NAA), glycerophosphocholine and lactate, but greater amounts of choline compared

to that of specific pathogen free (SPF) mice. In the hippocampus, the GF mice had greater creatine,

NAA, lactate and taurine content compared to those of the SPF animals, but lower relative quantities

of succinate and an unidentified lipid-related component. The GF rat frontal cortex contained higher

relative quantities of lactate, creatine and NAA compared to the CV animals whilst the GF

hippocampus was characterized by higher taurine and phosphocholine concentrations and lower

quantities of NAA, N-acetylaspartylglutamate and choline compared to the CV animals. Of note is

that, in both rat and mouse brain extracts, concentrations of hippocampal taurine were found to be

greater in the absence of an established microbiome. The results provide further evidence that brain

biochemistry can be influenced by gut microbial status, specifically metabolites involved in energy

metabolism demonstrating biochemical dialogue between the microbiome and brain.

198

A re-investigation of the phytochemical composition of the edible herb Amaranthus retroflexus

L

Serena Fiorito, Francesco Epifano, Roberta Palmisano, Salvatore Genovese, Vito Alessandro Taddeo

ABSTRACT

In this paper the presence of selected prenylated and unprenylated phenylpropanoids of nutraceutical

value, namely umbelliferone, apigenin, 4′-geranyloxyferulic acid, 7-isopentenyloxycoumarin,

auraptene, and umbelliprenin have been determined in all parts of the edible herb Amaranthus

retroflexus extracted with different methodologies. Roots were seen to contain the widest variety of

unprenylated and prenylated phenylpropanoids both in terms of number of secondary metabolites and

their quantitites. Findings described in the present study underline how A. retroflexus can be

considered as a potential nutraceutical for human welfare.

A vote for robustness: Monitoring serum enzyme activity by thin-layer chromatography of

dabsylated bradykinin products

Malte Bayer, Simone König

ABSTRACT

High-end analytical methods provide excellent data but may lack the robustness required in large

analytical studies. In particular complex chemical matrices may cause difficulties and increase the

need for extensive sample preparation. For screening of patients we thus developed a low-tech assay to

monitor bradykinin degradation by serum proteases. The bradykinin concentration mirrors the activity

of angiotensin-converting enzyme (ACE). Dabsylated bradykinin (DBK) and its labeled fragments

DBK1-8 and DBK1-5 were visualized by thin-layer chromatography using only 3 μL of serum. Lower

DBK1-5 levels indicated reduced ACE activity due to medication (ACE-inhibitors) or disease.

Provided that purified DBK is available, the assay protocol itself is very simple and does not require

any expensive high-end equipment.

199

Salting-out assisted liquid–liquid extraction combined with gas chromatography-mass

spectrometry for the determination of pyrethroid insecticides in high salinity and biological

samples

Zongliang Niu, Chunwei Yu, Xiaowen He, Jun Zhang, Yingying Wen

ABSTRACT

A salting-out assisted liquid–liquid extraction (SALLE) combined with gas chromatography-mass

spectrometry (GC–MS) method was developed for the determination of four pyrethroid insecticides

(PYRs) in high salinity and biological samples. Several parameters including sample pH, salting-out

solution volume and salting-out solution pH influencing the extraction efficiency were systematically

investigated with the aid of orthogonal design. The optimal extraction conditions of SALLE were: 4

mL of salting-out solution with pH = 4 and the sample pH = 3. Under the optimum extraction and

determination conditions, good responses for four PYRs were obtained in a range of 5–5000 ng/mL,

with linear coefficients greater than 0.998. The recoveries of the four PYRs ranged from 74% to 110%,

with standard deviations ranging from 1.8% to 9.8%. The limits of detection based on a signal-to-noise

ratio of 3 were between 1.5–60.6 ng/mL. The method was applied to the determination of PYRs in

urine, seawater and wastewater samples with a satisfactory result. The results demonstrated that this

SALLE-GC–MS method was successfully applied to determine PYRs in high salinity and biological

samples. SALLE avoided the need for the elimination of salinity and protein in the sample matrix, as

well as clean-up of the extractant. Most of all, no centrifugation or any special apparatus are required,

make this a promising method for rapid sample preparation procedure.

HPLC–MS/MS method for troventol determination in human plasma and its application to

biological samples

Aleksandra Nikitina, Alexander Grigoriev, Alla Sidorova

ABSTRACT

For the first time, an HPLC–MS/MS method for the determination pmol/l levels of troventol (TRV)

and clenbuterol as an internal standard (IS) in human plasma was developed, validated and tested on

biological samples. The method included solid phase extraction by Waters Oasis WCX cartridges and

chromatographic separation on a YMC-Pack SIL (100 mm × 2.1 mm, 5 μm, 12 nm) analytical column

with acetonitrile–water–formic acid (50:50:0.1, v/v/v) as the mobile phase; the selected ion transitions

were m/z 332.2→138.2 and m/z 277.0→203.1 for TRV and IS, respectively, in positive ionization

mode. The calibration curve for TRV showed good linearity in the concentration range of 35–500

pg/ml. The method was applied to real samples taken from healthy subjects after inhalation of an

aerosol containing 640 μg of TRV.

200

1,4-Anthraquinone: A new useful pre-column reagent for the determination of N-acetylcysteine

and captopril in pharmaceuticals by high performance liquid chromatography

Rita Gatti, Rita Morigi

ABSTRACT

1,4-Anthraquinone (ANQ) is proposed as a novel pre-column reagent for high performance liquid

chromatography (HPLC) determination of N-acetylcysteine (NAC) and captopril (CAP) in

pharmaceutical formulations. The derivatization reactions were carried out at room temperature: NAC

at pH 8 for 1 min, while CAP at pH 7.5 for 20 min. Both reactions reached completeness at a reagent

to thiol molar ratio of about 2.5. The synthesised derivatives were characterized by 1H NMR and IR.

The chromatographic separations were performed on a C18 Phenomenex Synergi Fusion 4 μm (250

mm × 4.6 mm I.D.) stainless steel column with detection at λ = 300 nm. The mobile phase consisted of

methanol/triethylammonium (TEA) phosphate buffer (pH 3; 0.05 mol/L) 75:25 (v/v) at a flow-rate of

0.4 mL/min for NAC and 88:12 (v/v), at a flow-rate of 0.6 mL/min for CAP. The validation

parameters (linearity, sensitivity, accuracy, precision, specificity and stability) were highly

satisfactory. Linear response was observed (determination coefficient ≥0.9996). Detection limits were

about 8 and 18 ng/mL for NAC and CAP, respectively. Intra-day precision (relative standard

deviation, R.S.D.) was ≤1.58%, for thiol to internal standard (IS) peak area ratio and ≤0.33%, for thiol

and IS retention times (tR), without significant differences between intra- and inter-day data. Thiol

recovery studies were satisfactory (99.50%) with R.S.D. ≤0.56%. The results highlight the high

sensitivity of the method and the remarkable reactivity and selectivity of the reagent towards the thiol

function.

Development a validated highly sensitive LC–MS/MS method for simultaneous quantification of

Ledipasvir, sofosbuvir and its major metabolite GS-331007 in human plasma: Application to a

human pharmacokinetic study

Ola M. Abdallah, Ahmed M. Abdel-Megied, Amira S. Gouda

ABSTRACT

A highly sensitive and rapid LC–MS/MS method was developed, fully optimized and validated for the

simultaneous determination of Ledipasvir (LED) and Sofosbuvir (SOF) in the presence of its major

metabolite GS-331007 in human plasma using Daclatasvir as internal standard (IS). The extraction of

analytes and IS from plasma was performed using liquid-liquid extraction with ethyl acetate. The

chromatographic separation of these prepared samples was achieved on Xterra MS C8 column (4.6 ×

50 mm,5 μm) using gradient elution with a mobile phase of ammonium formate buffer (pH 3.5; 10

mM), acetonitrile and methanol pumped at a flow rate 0.7 mL min−1.The detection was performed on

API4000 triple quadrupole tandem mass spectrometer using multiple reaction monitoring (MRM)

positive electrospray ionization interface. The method was validated according to FDA guidelines for

bio-analytical methods with respect to linearity, accuracy, precision, selectivity, carry-over, stability

and dilution integrity. Linearity was obtained over a concentration range of 0.1–1000, 0.3–3000 and

3.0–3000 ng mL−1 for LED, SOF and GS-331007; respectively by applying weighted least-squares

linear regression method (1/x2). The wider range of quantification in a shorter period of separation

time less than 5.0 min allowed monitoring the serum concentration of analytes up to 144 h. The

proposed method can be successfully applied for pharmacokinetic and bioequivalence studies in

healthy human volunteers.

201

Chemometrics and chromatographic fingerprints to classify plant food supplements according to

the content of regulated plants

E. Deconinck, C.A. Sokeng Djiogo, P. Courselle

ABSTRACT

Plant food supplements are gaining popularity, resulting in a broader spectrum of available products

and an increased consumption. Next to the problem of adulteration of these products with synthetic

drugs the presence of regulated or toxic plants is an important issue, especially when the products are

purchased from irregular sources. This paper focusses on this problem by using specific

chromatographic fingerprints for five targeted plants and chemometric classification techniques in

order to extract the important information from the fingerprints and determine the presence of the

targeted plants in plant food supplements in an objective way. Two approaches were followed: (1) a

multiclass model, (2) 2-class model for each of the targeted plants separately. For both approaches

good classification models were obtained, especially when using SIMCA and PLS-DA. For each

model, misclassification rates for the external test set of maximum one sample could be obtained. The

models were applied to five real samples resulting in the identification of the correct plants, confirmed

by mass spectrometry. Therefore chromatographic fingerprinting combined with chemometric

modelling can be considered interesting to make a more objective decision on whether a regulated

plant is present in a plant food supplement or not, especially when no mass spectrometry equipment is

available.

Surrogate CD16-expressing effector cell lines for determining the bioactivity of therapeutic

monoclonal antibodies

Shalom A. Gurjar, Jeremy P. Derrick, Rebecca J. Dearman, Robin Thorpe, Meenu Wadhwa

ABSTRACT

Traditional antibody dependent cellular cytotoxicity (ADCC) assays use donor derived natural killer

(NK) or peripheral blood mononuclear cells, but donor genetic variability and the technically

challenging nature of the assay means that alternative in vitro assay formats are required. We explored

the utility of two reporter gene cell lines, the J2 and J9, as surrogate effector cells for ADCC assays.

Both express the ADCC relevant Fcγ receptor CD16, crosslinking of which leads to firefly luciferase

expression. For anti-CD20 rituximab and anti-HER2 trastuzumab (both IgG1 monoclonal antibodies,

mAbs) a dose dependent firefly luciferase response was observed exclusively in the presence of their

respective targets, representing the molecular interaction which potentiates ADCC activity.

Importantly, both surrogate effector and NK cell based assays gave statistically similar values for

rituximab ADCC activity. Increased engagement with target cell bound mAbs was determined to be

cytotoxic for the J2 and J9 cell lines at the assay end point (at which luciferase expression is

measured). However, use of the J9 cells containing the constitutively expressed renilla luciferase gene

enabled data normalisation and corrected for fluctuations in both cell number and viability providing

an advantage over currently available surrogate effector cell-lines. Abrogated ADCC activity with

IgG4 mAbs, but enhanced activity with an IgG1 non-fucosylated mAb, was seen with the J9 cell line,

as expected. Additionally, two rituximab products (biosimilars in development) with similar binding

by flow cytometry, N-glycan profiles using HPLC and CD16 binding by surface plasmon resonance

showed comparable ADCC activity to Mabthera. The ADCC activity of another anti-CD20 mAb,

ofatumumab, reported only with primary cell based assays to date was also measured.

202

Dual-target screening of bioactive components from traditional Chinese medicines by hollow

fiber-based ligand fishing combined with liquid chromatography–mass spectrometry

Liang Chen, Xin Wang, Youping Liu, Xin Di

ABSTRACT

A novel strategy was developed for dual-target screening of bioactive components from traditional

Chinese medicines (TCMs). This strategy was based on the use of low-cost microporous hollow fibers

filled with target enzymes as baits to ―fish out‖ the ligands in TCM extracts, followed by identification

of the ligands dissociated from the target-ligand complexes by liquid chromatography–mass

spectrometry. Ganjiang Huangqin Huanglian Renshen Decoction (GHHRD), a classical TCM

prescription for diabetes treatment, was chosen as a model sample to evaluate the feasibility of the

proposed strategy. Three bioactive components were successfully screened out from GHHRD.

Coptisine was identified as the ligand of α-glucosidase and baicalin as the ligand of angiotensin-

converting enzyme (ACE). Berberine was found to be a dual inhibitor of α-glucosidase and ACE. The

results were further verified by enzyme inhibitory assay and molecular docking simulation. The study

suggested that our developed strategy would be a powerful tool for screening bioactive components

from multi-component and multi-target TCMs.

Volume 144 September 2017

Circular dichroism analysis of the calicheamicin-DNA interaction revisited

Gloria Proni, Kristi Tami, Nina Berova, George A. Ellestad

ABSTRACT

Calicheamicin, γ1I, is a remarkable DNA binding-cleaving, enediyne-containing, natural product that

exhibits potent antitumor activity. In this study, we used electronic circular dichroism spectroscopy to

monitor potential drug-induced DNA conformational changes and DNA induced conformational

changes in the calicheamicin aglycone. Three DNA dodecamer sequences were examined: one

containing a primary TCCT binding/cleavage site and two dodecamers containing less prominent

CTCT and TCTC sites. The binding was monitored by taking advantage of the drug‘s unique negative

exciton couplet (−313 nm/+275 nm) in phosphate buffer/ethanol 10%. Specifically the CD analysis

focused at the longest wavelength region around 313 nm where there is no interference by the positive

CD contributions of the DNA. Upon binding at a DNA/drug ratio of 1/1.2 and 1/2.7 a slight red shift

from 313 nm to 319 nm was observed. At a ratio of 1/1.2, the CE intensity remained practically

unchanged from that of free drug, which indicates no conformational changes in the bound aglycone

itself. A larger amount of drug, at a molar ratio of DNA/drug of 1/2.7 but especially at 1/6 and up to

1/10, however, caused a surprisingly distinct decrease in the intensity at this negative CD band and a

further small red-shift to 322 nm, evidence for non-specific binding.

203

Induced circularly polarized luminescence for revealing DNA binding with fluorescent dyes

Marcin Górecki, Francesco Zinna, Tarita Biver, Lorenzo Di Bari

ABSTRACT

To the best of our knowledge this is the first report on the application of induced circularly polarized

luminescence (CPL) for sensing the binding of fluorescent dyes to double stranded DNA. Using

Thiazole Orange (TO) and 4′,6-diamidino-2-phenylindole (DAPI) as models, we show utility and

limitations of CPL in DNA binding studies. The results obtained indicate that CPL can be used as a

new chiroptical tool for this purpose, however, special attention while recording CPL data must be

used, in order to exclude measurement artefacts caused by linear polarization components.

Analysis of stereoselective drug interactions with serum proteins by high-performance affinity

chromatography: A historical perspective

Zhao Li, David S. Hage

ABSTRACT

The interactions of drugs with serum proteins are often stereoselective and can affect the distribution,

activity, toxicity and rate of excretion of these drugs in the body. A number of approaches based on

affinity chromatography, and particularly high-performance affinity chromatography (HPAC), have

been used as tools to study these interactions. This review describes the general principles of affinity

chromatography and HPAC as related to their use in drug binding studies. The types of serum agents

that have been examined with these methods are also discussed, including human serum albumin, α1-

acid glycoprotein, and lipoproteins. This is followed by a description of the various formats based on

affinity chromatography and HPAC that have been used to investigate drug interactions with serum

proteins and the historical development for each of these formats. Specific techniques that are

discussed include zonal elution, frontal analysis, and kinetic methods such as those that make use of

band-broadening measurements, peak decay analysis, or ultrafast affinity extraction.

204

An indirect stereoselective analysis of nebivolol glucuronides in plasma by LC–MS/MS:

Application to clinical pharmacokinetics

Carolina Pinto Vieira, Daniel Valente Neves, Evandro José Cesarino, Adriana Rocha, Vera Lucia

Lanchote

ABSTRACT

Nebivolol is a racemate of the d-isomer responsible for β1 adrenergic receptor antagonism and the l-

isomer responsible for the release of nitric oxide from endothelial cells. Nebivolol is mainly

metabolized to nebivolol glucuronide, which also contribute to the nebivolol β1 adrenoreceptor

antagonism. This study reports the development and validation of an indirect stereoselective method of

analysis of nebivolol glucuronides in plasma by LC–MS/MS. The method was applied to the

investigation of stereoselectivity in the glucuronidation of nebivolol in elderly hypertensive patients (n

= 11) CYP2D6 phenotyped as EM and treated with a single oral dose of the racemate. One-milliliter

plasma aliquots spiked with internal standard (S)-(−)-metoprolol were incubated with 25 μL of β-

glucuronidase (final concentration 2500 unit/mL) at pH 5.0 for 16 h at 37 °C. Linearity for total

nebivolol was 0.2–125 ng of each isomer per mL plasma and permitted analysis of nebivolol

glucuronide isomers up to 48 h after administration of a single oral dose of 10 mg racemate. Regarding

to the nebivolol glucuronide isomers, higher plasma concentrations of the d-isomer were observed

compared to the l-isomer (d/l AUC = 5.4), explaining at least in part the plasma accumulation of

unchanged l-nebivolol (l/d AUC = 1.8). This study also showed metabolic glucuronide

nebivolol/unchanged nebivolol ratios of approximately 6.5 for the l-isomer (AUC 65.3 vs 10.1 ng

h/mL) and approximately 62.1 (335.2 vs 5.4 ng h/mL) for the d-isomer. Considering that d-nebivolol

glucuronide also contributes for β1 adrenergic receptor antagonism, future studies regarding PK-PD of

nebivolol should evaluate not only plasma concentrations of unchanged nebivolol isomers but also

glucuronide nebivolol isomers.

N-Decyl-S-trityl-(R)-cysteine, a new chiral selector for ―green‖ ligand-exchange chromatography

applications

Andrea Carotti, Federica Ianni, Emidio Camaioni, Lucia Pucciarini, Benedetto Natalini

ABSTRACT

In search for new enantioselectivity profiles, the N-decyl-S-trityl-(R)-cysteine [C10-(R)-STC] was

synthesized through a one-step procedure and then hydrophobically adsorbed onto an octadecylsilica

surface to generate a stable chiral stationary phase for ligand-exchange chromatography (CLEC-CSP)

applications. The CLEC analysis was carried out on underivatized amino acids, by using a Cu(II)

sulphate (1.0 mM) containing aqueous eluent system. Most of the analysed compounds (34 out of 45)

were enantiodiscriminated by the C10-(R)-STC-based CSP, with resolution factor (RS) values up to

8.86. Conformationally rigid and hydrophobic ligands often experienced the largest enantioselectivity

effects. A high loadability emerged from the analysis of rac-NorVal (selected as prototype test

compound): up to 20 mg/mL were efficiently enantioseparated with the CLEC-CSP. Two in-line hand-

made cartridges filled with a strong cation-exchange resin allowed the effective catching of Cu(II) ions

after the semi-preparative enantioseparation. The quantitative recovery of the rac-NorVal enantiomers

was made possible by flowing through the cartridge a 5% (v) ammonia solution. The CLEC phase

proved successful in the enantioselective analysis of a commercially available (S)-Leu containing

tablet. Furthermore, in order to understand the molecular basis for a successful use of the C10-(R)-

STC-based CLEC system, a descriptive structure-separation relationship study was performed.

205

The role of chirality in a set of key intermediates of pharmaceutical interest, 3-aryl-substituted-

γ-butyrolactones, evidenced by chiral HPLC separation and by chiroptical spectroscopies

Daniela Rossi, Rita Nasti, Simona Collina, Giuseppe Mazzeo, Sergio Abbate

ABSTRACT

The enantiomers of four chiral 3-aryl-substituted-γ-butyrolactones, key intermediates for the

preparation of compounds of pharmaceutical interest, were successfully isolated by enantioselective

chromatography, employing the Chiralpak AD-H chiral stationary phase. For all compounds the same

elution order was observed, as monitored by a full set of chiroptical methods that we employed,

namely ORD (optical rotatory dispersion), ECD (electronic circular dichroism, or CD in the UV

range), and VCD (vibrational circular dichroism, or CD in the IR range). By density functional theory

(DFT) calculations we were able to determine that the first eluted enantiomer has (S) absolute

configuration in all four cases. We were able to justify the elution order by molecular docking

calculations for all four enantiomeric pairs and suitable modeling of the stationary and mobile phases

of the employed columns. The optimal performance of the chiroptical spectroscopies and of the DFT

calculations allows us to formulate a lactone chirality rule out of the CO stretching region of the VCD

spectra.

Determination of the absolute configuration of a novel tetrasubstituted isoindolinone by

vibrational circular dichroism

Antonio Massa, Paola Rizzo, Francesco Scorzelli, Guglielmo Monaco, Riccardo Zanasi

ABSTRACT

The absolute configuration of a recently prepared asymmetric 3,3-disubstituted isoindolinone (ethyl 2-

benzyl-3-oxo-1-(3-oxobutyl)isoindoline-1-carboxylate), possessing highly promising pharmaceutical

activity, has been determined by means of VCD spectroscopy and DFT calculations. The great

flexibility of the molecule reduces to a few relevant conformers, all contributing in the same way to the

shape of the VCD spectrum for the carbonyl stretching region. Two of the three CO groups of the

molecule interact with each other during the stretching vibration, thus providing a non-conservative

VCD couplet whose signature, together with the VCD sign of the third CO stretching mode,

unequivocally determines the absolute configuration of the molecule, which is found to be (S) for the

(–) optical isomer.

206

GC–MS based Gestational Diabetes Mellitus longitudinal study: Identification of 2-and 3-

hydroxybutyrate as potential prognostic biomarkers

Danuta Dudzik, Marcin Zorawski, Mariusz Skotnicki, Wieslaw Zarzycki, M. Pilar Ramos

ABSTRACT

Gestational Diabetes Mellitus (GDM) causes severe short- and long-term complications for the

mother, fetus and neonate, including type 2-diabetes (T2DM) later in life. In this pilot study, GC–

Q/MS analysis was applied for plasma metabolomics fingerprinting of 24 healthy and 24 women with

GDM at different stages of gestation (second and third trimester) and postpartum (one and three

months). Multivariate (unsupervised and supervised) statistical analysis was performed to investigate

variance in the data, identify outliers and for unbiased assessment of data quality. Plasma fingerprints

allowed for the discrimination of GDM pregnant women from controls both in the 2nd and 3rd

trimesters of gestation. However, metabolic profiles tended to be similar after delivery. Follow up of

these women revealed that 4 of them developed T2DM within 2 years postpartum. Multivariate PLS-

DA models limited to women with GDM showed clear separation 3 months postpartum. In the 2nd

trimester of gestation there was also a clear separation between GDM women that were

normoglycemic after pregnancy and those with recognized postpartum T2DM. Metabolites that had

the strongest discriminative power between these groups in the 2nd trimester of gestation were 2-

hydroxybutyrate, 3-hydroxybutyrate, and stearic acid. We have described, that early GDM comprises

metabotypes that are associated with the risk of future complications, including postpartum T2DM. In

this pilot study, we provide evidence that 2-hydroxybutyrate and 3-hydroxybutyrate may be considered

as future prognostic biomarkers to predict the onset of diabetic complications in women with

gestational diabetes after delivery.

Development and validation of a quantification method for cucurbitacins E and I in rat plasma:

Application to population pharmacokinetic studies

Giovana Maria Lanchoti Fiori, Salvatore D‘Agate, Adriana Rocha, Ana Maria Soares Pereira,

Norberto Peporine Lopes

ABSTRACT

Cucurbitacin E is a potential drug candidate due to its anticancer activity, recognition of its molecular

targets, and synergism with other drugs used for cancer treatment. However, the use of cucurbitacin E

in clinical practice is not possible because of important knowledge gaps in its preclinical and clinical

pharmacokinetic characteristics. Cucurbitacin E is hydrolyzed to cucurbitacin I in plasma and in

human liver microsomes. The aim of this study was to evaluate the population pharmacokinetics of

cucurbitacin E and of its metabolite cucurbitacin I in rats. The method for the sequential analysis of

cucurbitacins E and I in rat plasma was developed using LC–MS/MS. Plasma aliquots of 50 μL were

deproteinized with acetonitrile and clobazam was added as internal standard. The extracts were

injected into an RP-18 column and eluted with a mobile phase consisting of a mixture of

acetonitrile:water:methanol (32:35:33, v/v/v). The method was precise and accurate, showing linearity

in the range of 1–100 ng cucurbitacin E/mL plasma and of 0.4–200 ng cucurbitacin I/mL plasma. The

method was applied to the pharmacokinetic evaluation of cucurbitacin E administered intravenously to

male Wistar rats (1 mg/kg). Serial blood samples were collected up to 24 h after administration. The

plasma concentrations of cucurbitacin E were quantified up to 16 h, while the plasma concentrations of

cucurbitacin I remained below the limit of quantification. A population pharmacokinetic model was

developed for cucurbitacin E using the NONMEM program, with adequate goodness of fit and

predictive performance.

207

Ultra-fast quantitation of voriconazole in human plasma by coated blade spray mass

spectrometry

Marcos Tascon, Germán Augusto Gómez-Ríos, Nathaly Reyes-Garcés, Justen Poole, Janusz Pawliszyn

ABSTRACT

Voriconazole is a triazole broad-spectrum antifungal medication often used to treat fungal infections

caused by Aspergillus and Fusarium species. One of the main challenges associated with the

implementation of this medication is its narrow therapeutic concentration range, demonstrating toxicity

at concentrations above 6 μg/mL and limited efficacy at concentrations below 2 μg/mL. As a result,

methodologies which permit the rapid and accurate quantitation of voriconazole in patients are highly

desirable. In this work two different approaches based on coated blade spray directly coupled to mass

spectrometry (CBS-MS) are introduced; each enabling the quantitation of voriconazole in plasma

samples with a simple and fast sample preparation and no chromatographic step. The first approach

involves a rapid extraction (1 min) of the target analyte from 300 μL of human plasma using

conventional laboratory vessels (e.g. vial, 96-well plate). Alternatively, the second strategy consists of

a 2 min extraction from a plasma droplet (10 μL) placed on the coated area of the blade. Both

procedures were successfully validated and good linearity (R2 ≥ 0.998), accuracy (91–122%) and

precision (<8%) were attained in the concentration range evaluated (0.1–50 μg/mL). Moreover, very

good results in terms of relative matrix effects were obtained given that the slopes of the calibration

curves constructed in five different plasma lots exhibited relative standard deviation (RSD) values

below 7%. Herein we demonstrated that CBS-MS is a technology suitable for the ultra-fast

determination of voriconazole in human plasma samples. Indeed, the proposed methodology can be

easily used either for routine drug monitoring or for in vitro pharmacokinetic studies in applications

where very small sample volumes are available and great temporal resolution is needed.

Pharmacokinetic profile of bilberry anthocyanins in rats and the role of glucose transporters:

LC–MS/MS and computational studies

G. Baron, A. Altomare, L. Regazzoni, V. Redaelli, G. Aldini

ABSTRACT

The aim of the present investigation was to better understand the pharmacokinetic profile of bilberry

(Vaccinium Myrtillus) anthocyanins and the role of glucose transporters (sGLT1 and GLUT2) on their

absorption. In particular, the absorption of 15 different anthocyanins contained in a standardized

bilberry extract (Mirtoselect®) was measured in rats by a validated LC-ESI–MS/MS approach. The

plasma concentration peak (Cmax) of 11.1 ng/mL was reached after 30 min and fasting condition

significantly increased the bioavailability of anthocyanins by more than 7 fold in respect to fed rats.

Glucose co-administration did not interfere with the overall anthocyanin uptake. Bioavailability of

each anthocyanin was then estimated by comparing the relative content in plasma vs extract. The 15

anthocyanins behaved differently in term of bioavailability and both the aglycone and the sugar moiety

were found to affect the absorption. For instance, arabinoside moiety was detrimental while cyanidin

enhanced bioavailability. Computational studies permitted to rationalize such results, highlighting the

role of glucose transporters (sGLT1 and GLUT2) in anthocyanins absorption. In particular a

significant correlation was found for the 15 anthocyanins between sGLT1 and GLUT2 recognition and

absorption.

208

Comparative pharmacodynamic analysis of imidazoline compounds using rat model of ocular

mydriasis with a test of quantitative structure–activity relationships

Joanna Raczak-Gutknecht, Antoni Nasal, Teresa Frąckowiak, Anita Kornicka, Roman Kaliszan

ABSTRACT

Imidazol(in)e derivatives, having the chemical structure similar to clonidine, exert diverse

pharmacological activities connected with their interactions with alpha2-adrenergic receptors, e.g.

hypotension, bradycardia, sedation as well as antinociceptive, anxiolytic, antiarrhythmic, muscle

relaxant and mydriatic effects. The mechanism of pupillary dilation observed after systemic

administration of imidazol(in)es to rats, mice and cats depends on the stimulation of postsynaptic

alpha2-adrenoceptors within the brain. It was proved that the central nervous system (CNS)-localized

I1-imidazoline receptors are not engaged in those effects. It appeared interesting to analyze the CNS-

mediated pharmacodynamics of imidazole(in)e agents in terms of their chromatographic and

calculation chemistry-derived parameters. In the present study a systematic determination and

comparative pharmacometric analysis of mydriatic effects in rats were performed on a series of 20

imidazol(in)e agents, composed of the well-known drugs and of the substances used in experimental

pharmacology. The eye pupil dilatory activities of the compounds were assessed in anesthetized Wistar

rats according to the established Koss method. Among twenty imidazol(in)e derivatives studied, 18

produced diverse dose-dependent mydriatic effects. In the quantitative structure–activity relationships

(QSAR) analysis, the pharmacological data (half maximum mydriatic effect – ED50 in μmol/kg) were

considered along with the structural parameters of the agents from molecular modeling. The

theoretically calculated lipophilicity parameters, CLOGP, of imidazol(in)es, as well as their

lipophilicity parameters from HPLC, log kw, were also considered. The attempts to derive statistically

significant QSAR equations for a full series of the agents under study were unsuccessful. However, for

a subgroup of eight apparently structurally related imidazol(in)es a significant relationship between

log(1/ED50) and log kw values was obtained.

Characterization of oxycodone in vitro metabolism by human cytochromes P450 and UDP-

glucuronosyltransferases

Stéphanie Romand, Dany Spaggiari, Niloufar Marsousi, Caroline Samer, Serge Rudaz

ABSTRACT

The hepatic metabolism of oxycodone by cytochromes P450 (CYP) and the UDP-

glucuronosyltransferases (UGT), the main metabolic enzymes of phase I and phase II, respectively,

was assessed in vitro. The N-demethylation by CYP3A4/5 and the O-demethylation by CYP2D6 in

human liver microsomes (HLM) followed Michaelis-Menten kinetics, with intrinsic clearances of 1.46

μL/min/mg and 0.35 μL/min/mg, respectively. Although noroxycodone and oxymorphone mainly

contribute to the elimination of oxycodone, the simulated total in vivo clearance using in vitro phase I

metabolism was underestimated. For the first time, metabolism of oxycodone by UGT was deeply

investigated using HLM, recombinant enzymes and selective inhibitors. Oxycodone-glucuronide was

mainly produced by UGT2B7 (Km = 762 ± 153 μM, Vmax = 344 ± 20 peak area/min/mg) and to a

lesser extent by UGT2B4 (Km = 2454 ± 497 μM, Vmax = 201 ± 19 peak area/min/mg). Finally, the

kinetics of the drug–drug interactions were assessed using two CYP and UGT cocktail approaches.

Incubations of HLM with phase I and phase II drug probes showed that oxycodone mainly decreased

the in vitro activities of CYP2D6, CYP3A4/5, UGT1A3, UGT1A6 and UGT2B subfamily with an

important impact on UGT2B7.

209

Structural and functional integrity of human serum albumin: Analytical approaches and clinical

relevance in patients with liver cirrhosis

Marina Naldi, Maurizio Baldassarre, Marco Domenicali, Manuela Bartolini, Paolo Caraceni

ABSTRACT

Human serum albumin (HSA) is the most abundant circulating plasma protein. Besides a significant

contribution to the osmotic pressure, it is also involved in the fine regulation of many other

physiological processes, including the balance of the redox state, the inflammatory and/or

immunological responses, and the pharmacokinetic and pharmacodynamics of many drugs. Growing

evidence suggests that HSA undergoes structural and functional damage in diseases characterized by

an enhanced systemic inflammatory response and oxidative stress, as it occurs in chronic liver disease.

Based on their clinical relevance, this review provides a summary of the most common post-

translational modifications affecting HSA structural integrity and functions and their clinical relevance

in the field of liver disease. The review also provides a critical description of the analytical approaches

employed for the investigation of conformational alterations and the identification/quantitation of

specific post-translational modifications affecting HSA. Finally, the analytical methods available for

the assessment of two of the most clinically relevant non-oncotic properties of HSA, namely the

binding capacity and the antioxidant activity, are critically reviewed. Among the available techniques

particular attention is given to those proposed for the in vitro and in vivo investigation of structurally

modified albumin.

Targeted proteomics of cannabinoid receptor CB1 and the CB1b isoform

Soumita Ghosh, Isabel González-Mariscal, Josephine M. Egan, Ruin Moaddel

ABSTRACT

Cannabinoid receptors (CBR), including CB1 and CB2 have been therapeutic targets for a number of

conditions. Recently, splice variants of the CB1R have been identified in humans. The isoforms differ

in their N-terminus sequence and pharmacological activity relative to the CB1R, as a result, the

differentiation between the CB1 receptor and its isoform is required. As a result, a selected reaction

monitoring mass spectrometry (SRM-MS) method was developed for the quantitation of CB1 and the

CB1b isoform in CHO cells transduced with CB1 and CB1b. The SRM-MS protocol was assessed

with isotopically labeled peptide standards and had high reproducibility of intra-day assay (CVs from

1.9 to 4.3% for CB1 and 0.5 to 5.9% for CB1b) and inter-day assay (CVs from 1.2 to 5.2% for CB1

and 1.2 to 6.1% for CB1b).

210

Application of an ESI-QTOF method for the detailed characterization of GSK-3β inhibitors

Angela De Simone, Jessica Fiori, Marina Naldi, Annalisa D‘Urzo, Vincenza Andrisano

ABSTRACT

The crucial role of Glycogen Synthase Kinase 3 (GSK-3β) as a pivotal player in Alzheimer's Disease

(AD) has recently inspired significant attempts to design and synthesize potent kinase inhibitors. In

fact GSK-3β is considered the main kinase which catalyzes the microtubule-associated protein tau

hyper-phosphorylation and the neurofibrillary tangles (NFT) in vitro and in vivo, The first classes of

GSK-3β inhibitors were classified as ATP-competitive and, therefore, they lack of an efficient degree

of selectivity over other kinases. In light of this consideration, many efforts are devoted to characterize

new non ATP-competitive GSK-3β inhibitors, endowed with high selectivity. In parallel, there is an

urgent need to develop new analytical methodologies for the hit selection (highthroughput screening)

and ligand binding characterization in terms of potency, affinity and mechanism of action. The new

methodology for GSK-3β enzymatic activity determination can be adopted as a realistic alternative to

the currently used radioactive, luminescence and fluorescence detection methods, each showing

limitations in terms of safety and interferences. Herein, we propose an alternative and selective

electrospray ionization quadrupole time-of-flight (ESI-QTOF) method, based on the direct

quantification of phosphorylated substrate muscle glycogen synthase GSM, a peptide resembling the

high affinity sequence of natural substrate muscle glycogen synthase 1, for the detailed

characterization of GSK-3β inhibitors. The method was validated in terms of accuracy and

reproducibility of GSM signal intensity with a relative standard deviation RSD% value of 3.55%;

Limit of Detection (LOD): 0.006 μM; Lower Limit of Quantification (LLOQ): 0.02 μM; linearity r2

0.9951. The kinetic constants (KM and vmax) of the GSK-3β catalyzed kinase reaction and the

inhibitory potency of known ligands (IC50), were determined. All the obtained results were in

agreement with those reported in literature or obtained in house by the standard reference

luminometric approach. The proposed method was applied to the elucidation of well known inhibitors

mechanism of action by the construction of a Lineweaver–Burk plot and the Ki determination.

Furthermore, the potency, affinity and mechanism of action of a new non ATP-competitive compound

were established.

Quantitative estimation of cholinesterase-specific drug metabolism of carbamate inhibitors

provided by the analysis of the area under the inhibition-time curve

Huimin Zhou, Qiaoling Xiao, Wen Tan, Yiyi Zhan, Marco Pistolozzi

ABSTRACT

Several molecules containing carbamate groups are metabolized by cholinesterases. This metabolism

includes a time-dependent catalytic step which temporary inhibits the enzymes. In this paper we

demonstrate that the analysis of the area under the inhibition versus time curve (AUIC) can be used to

obtain a quantitative estimation of the amount of carbamate metabolized by the enzyme. (R)-

bambuterol monocarbamate and plasma butyrylcholinesterase were used as model carbamate-

cholinesterase system. The inhibition of different concentrations of the enzyme was monitored for 5 h

upon incubation with different concentrations of carbamate and the resulting AUICs were analyzed.

The amount of carbamate metabolized could be estimated with <15% accuracy (RE%) and ≤23%

precision (RSD%). Since the knowledge of the inhibition kinetics is not required for the analysis, this

approach could be used to determine the amount of drug metabolized by cholinesterases in a selected

compartment in which the cholinesterase is confined (e.g. in vitro solutions, tissues or body fluids),

either in vitro or in vivo.

211

A new method to characterize the kinetics of cholinesterases inhibited by carbamates

Qiaoling Xiao, Huimin Zhou, Hong Wei, Huaqiao Du, Marco Pistolozzi

ABSTRACT

The inhibition of cholinesterases (ChEs) by carbamates includes a carbamylation (inhibition) step, in

which the drug transfers its carbamate moiety to the active site of the enzyme and a decarbamylation

(activity recovery) step, in which the carbamyl group is hydrolyzed from the enzyme. The

carbamylation and decarbamylation kinetics decide the extent and the duration of the inhibition, thus

the full characterization of candidate carbamate inhibitors requires the measurement of the kinetic

constants describing both steps. Carbamylation and decarbamylation rate constants are traditionally

measured by two separate set of experiments, thus making the full characterization of candidate

inhibitors time-consuming. In this communication we show that by the analysis of the area under the

inhibition-time curve of cholinesterases inhibited by carbamates it is possible to calculate the

decarbamylation rate constant from the same data traditionally used to characterize only the

carbamylation kinetics, therefore it is possible to obtain a full characterization of the inhibition with a

single set of experiments. The characterization of the inhibition kinetics of human and dog plasma

butyrylcholinesterase and of human acetylcholinesterase by bambuterol and bambuterol

monocarbamate enantiomers was used to demonstrate the validity of the approach. The results showed

that the proposed method provides reliable estimations of carbamylation and decarbamylation rate

constants thus representing a simple and useful approach to reduce the time required for the

characterization of carbamate inhibitors.

Cyclodextrins as inhibitors of the precipitation of riboflavin-5’-phosphate due to presence of zinc

chloride: A NMR investigation

Federica Aiello, Gloria Uccello-Barretta, Niccolò Falugiani, Francesca Nardelli, Federica Balzano

ABSTRACT

Several cyclodextrins (CDs) were probed in order to counteract the precipitation of riboflavin-5‘-

phosphate (or flavin mononucleotide, FMN-P) due to the presence of divalent cations, by exploiting

Nuclear Magnetic Resonance (NMR) spectroscopy both for quantitative analyses and stereochemical

characterizations. Among CDs, β-cyclodextrin (β-CD) showed the best solubilizing power in virtue of

the formation of a 1–2 FMN-P/β-CD complex, the stereochemistry of which was ascertained by

ROESY (Rotating-frame Overhauser Enhanced SpectroscopY) measurements.

212

Monitoring drug–serum protein interactions for early ADME prediction through Surface

Plasmon Resonance technology

Edoardo Fabini, U. Helena Danielson

ABSTRACT

Many molecules fail to reach the market due to poor pharmacokinetic (PK) properties, rendering the

potential drug virtually unavailable for the primary target despite efficient administration to the body.

PK properties of endogenous and exogenous compounds in mammals are dependent, among other

factors, on their ability to interact with serum proteins. The extent of binding can greatly influence

their ADME (adsorption, distribution, metabolism and execration) profile. Reliable and cost-effective

bioavailability studies, early in the drug discovery process, can lead to an improvement of the success

rate for compounds entering clinical trials. Optical biosensors based on surface plasmon resonance

(SPR) detection emerged as an efficient approach to obtain large amounts of information about the

binding of small molecules to serum proteins. Simple, automated and fast assays provide a good

throughput, versatility and highly informative data output, rendering the methodology particularly

suited for early screening. The ability to provide basic information on PK can be easily coupled to

structure–activity relationship analysis. In this review, features of the technology and its employment

for the study of serum protein–small molecule interactions are presented and discussed.

Capillary electrophoresis in the context of drug discovery

Elena Farcaş, Lionel Pochet, Jacques Crommen, Anne-Catherine Servais, Marianne Fillet

ABSTRACT

Capillary Electrophoresis is a very efficient and resolutive separation technique used for many years in

the analytical field. Despite all its assets, CE remains poorly used in drug discovery. This can be

explained by the relatively low number of experienced CE practitioners, the maturity of HPLC in the

pharmaceutical industry and some intrinsic limitations of the technique. The objective of this review is

to focus our attention on recent developments of this technique in three different drug discovery areas:

bioassays, drug-plasma interactions and drug metabolism studies. These developments were based on

two important abilities of CE: the capacity to measure non-covalent interactions in solution and the

ability to use a portion of the capillary as a reactor while the rest of the capillary is used for the

separation of the product of the reaction.

213

Simultaneous analysis of nucleobases, nucleosides and ginsenosides in ginseng extracts using

supercritical fluid chromatography coupled with single quadrupole mass spectrometry

Yang Huang, Tingting Zhang, Yumei Zhao, Haibo Zhou, Zhengjin Jiang

ABSTRACT

Nucleobases, nucleosides and ginsenosides, which have a significant impact on the physiological

activity of organisms, are reported to be the active components of ginseng, while they are less present

in ginseng extracts. Few analytical methods have been developed so far to simultaneously analyze

these three classes of compounds with different polarities present in ginseng extracts. In the present

study, a simple and efficient analytical method was successfully developed for the simultaneous

separation of 17 nucleobases, nucleosides and ginsenosides in ginseng extracts using supercritical fluid

chromatography coupled with single quadrupole mass spectrometry (SFC-MS). The effect of various

experimental factors on the separation performance, such as the column type, temperature and

backpressure, the type of modifier and additive, and the concentration of make-up solvent were

systematically investigated. Under the selected conditions, the developed method was successfully

applied to the quality evaluation of 14 batches of ginseng extracts from different origins. The results

obtained for the different batches indicate that this method could be employed for the quality

assessment of ginseng extracts.

Combined approach using capillary electrophoresis, NMR and molecular modeling for

ambrisentan related substances analysis: Investigation of intermolecular affinities, complexation

and separation mechanism

Benedetta Pasquini, Fabrizio Melani, Claudia Caprini, Massimo Del Bubba, Sandra Furlanetto

ABSTRACT

A comprehensive investigation on the CE separation mechanisms and on the inclusion complexation

with CyDs of the chiral drug S-ambrisentan (S-AMB), its R-enantiomer and other impurities was

performed by different techniques. A CE method was previously set up allowing the simultaneous

determination of the enantiomeric purity and of impurities of S-AMB, based on the addition of SDS

micelles and γ-cyclodextrin (γCyD) to borate buffer. In this study, the electrophoretic behavior of the

analytes in terms of selectivity and mobility with respect to the addition of different CyDs was first

investigated, evidencing the presence of interactions for all the CyDs, but the unique ability of γCyD

for obtaining the separation of all the compounds. By molecular modeling, aggregates between SDS

micelles and analytes, and inclusion complexes between CyDs, SDS and/or analytes of different

stoichiometries were simulated. The potential and the gain energy of complexes were calculated on the

minimized conformations, showing the great tendency of γCyD of forming mixed complexes with one

or two SDS molecules and with the analyte, even if with different affinities among the analytes. For

1:1:1 mixed complexes with different CyDs, the highest difference of potential energy between the

enantiomers‘ complexes was observed for γCyD. Two-dimensional NOE spectroscopy experiments

were performed for S-AMB and I1 and pointed out the interactions of the aromatic moiety of the

analytes and of SDS aliphatic chain with γCyD protons, confirming the existence of γCyD mixed

complexes. The high affinity of SDS for the γCyD cavity was suggested to justify the fundamental role

of SDS in modulating and achieving the CE separation, due to its influence both on the stability and on

the type of complexes between γCyD and the analytes.

214

Molecularly imprinted polymer for glutathione by modified precipitation polymerization and its

application to determination of glutathione in supplements

Yukari Nakamura, Shizuka Masumoto, Hisami Matsunaga, Jun Haginaka

ABSTRACT

Molecularly imprinted polymers (MIP) particles for glutathione (GSH) with a narrow particle size

distribution were prepared by modified precipitation polymerization using methacrylic acid as a

functional monomer, divinylbenzene as a crosslinker and water as a co-solvent. The particle diameters

of the MIP and non-imprinted polymer (NIP) prepared under the optimum conditions were 3.81 ± 0.95

(average ± standard deviation) and 3.39 ± 1.22 μm, respectively. The retention and molecular-

recognition properties of the prepared MIP were evaluated using a mixture of acetonitrile and water as

a mobile phase in hydrophilic interaction chromatography. With an increase of acetonitrile content, the

retention factor of GSH was increased on the MIP. In addition to shape recognition, hydrophilic

interactions seem to work for the recognition of GSH on the MIP. The MIP had a specific molecular-

recognition ability for GSH, while glutathione disulfide, l-Glu, l-Cys, Gly-Gly and l-Cys-Gly could not

be retained or recognized on the MIP. The effect of column temperature revealed that the separation of

GSH on the MIP was entropically driven. Binding experiments and Scatchard analyses revealed that

one binding sites were formed on both the MIP and NIP, while the MIP gave higher affinity and

capacity for GSH than the NIP. Furthermore, the MIP was successfully applied for determination of

GSH in the supplements.

Efficacy of a titanium dioxide nanoparticles − based indoor anti-odor product as assessed by

electronic nose and gaschromatography–mass spectrometry

Mara Mirasoli, Roberto Gotti, Massimo Di Fusco, Giulia Basaglia, Aldo Roda

ABSTRACT

Indoor air pollutants and odorants may have psychological and physical impact on exposed individuals

and the unpleasant room air is considered as one of the factors associated with sick building syndrome

comprising general symptoms such as headache and lethargy. Approaches for improving the quality of

indoor air are thus important as support for human health and well-being. Photo-oxidation catalyzed by

titanium dioxide (TiO2), is one of the methods used for elimination of volatile organic compounds,

which are the cause of odor nuisance in indoor and outdoor air. In the present investigation, the

efficacy of an experimental anti-odor air freshener based on TiO2 nanoparticles was estimated by

testing its ability in removing from a small air chamber (200 mL) the odor of triethylamine solutions

(50 μL at concentrations between 0.700 to 700 mM), used as a model volatile molecule for simulating

fish-like unpleasant indoor environment. The evaluation was performed by electronic nose which

provided a holistic and objective data on the efficacy of the product, demonstrating that the effects of

triethylamine even at the highest tested concentrations can be completely removed by application of

3.0 g of the product at 25% TiO2 nanoparticles concentration. The obtained results were confirmed by

gaschromatography-mass spectrometry (GC–MS) analysis addressed to the quantitative determination

of residual triethylamine in the environment after treatment by the anti-odor product.

215

Comprehensive study on the effects of sodium and potassium additives in size exclusion

chromatographic separations of protein biopharmaceuticals

Alexandre Goyon, Alain Beck, Jean-Luc Veuthey, Davy Guillarme, Szabolcs Fekete

ABSTRACT

To separate proteins solely based on their difference in hydrodynamic volume in size exclusion

chromatography (SEC), the ionic strength of the mobile phase has to be increased in order to avoid

secondary ionic interactions between proteins and the stationary phase. However, adding salts to the

mobile phase can have a serious effect on protein aggregation and can lead to artifacts. In the present

study, several monoclonal antibodies (mAbs) and the antibody-drug conjugate (ADC), trastuzumab

emtansine were selected to study the effect of mobile phase salt additive on aggregation

measurements. In a first instance, the same aggregation ratios between the dimeric and monomeric

forms of ten mAbs approved by the Food and Drug Administration (FDA) and the European Medicine

Agency (EMA) were obtained with three UHP-SEC columns. However, SEC analysis using various

amounts of NaCl provided surprising results for rituximab, e.g. presence of 0.8% aggregates with a

mobile phase containing 0.2 M NaCl, while no aggregates were observed without NaCl in the mobile

phase. Despite the absence of monomeric protein adsorption at the surface of the SEC resin, the

comparison of sodium- and potassium-based salts demonstrated the superiority of potassium-based

salts to reduce possible secondary electrostatic interactions, mainly between protein dimers and the

SEC support as well as to lower protein-salts interaction. To investigate the effect of mobile phase salt

additives on SEC measurements, fluorescence spectroscopy provided insights related to the possible

contribution of protein tertiary structure. Indeed, biopharmaceuticals could be classified depending on

the exposure of their tryptophan residues to the solvent in order to understand their propensity to

interact with the stationary phase or/and to undergo self-association.

Application of a rapid HILIC-UV method for synthesis optimization and stability studies of

immunogenic neo-glycoconjugates

F. Rinaldi, S. Tengattini, E. Calleri, T. Bavaro, C. Temporini

ABSTRACT

Proteins and glycoproteins with therapeutic activity are susceptible to environmental factors, which

can cause their degradation and the loss of their activity. Thus, the maintenance of their stability during

the production process is a critical factor. In this work, a simple and rapid hydrophilic interaction

liquid chromatography HILIC-UV method was validated in terms of accuracy, precision, linearity,

LOD, LOQ and specificity and applied to the investigation of the stability of intact proteins and their

neo-glycoconjugates with antigenic activity against tuberculosis. The method proved to be suitable for

the estimation of the degradation of the proteins under critical conditions (i.e. freeze-thaw cycles) and

for the monitoring of their coupling reaction with saccharidic moieties, without the need of sample

preparation. In addition, the chromatographic analysis allowed calculating the yields of the protein

glycosylation reaction.

216

Simple and rapid LC–MS method for the determination of circulating albumin

microheterogeneity in veal calves exposed to heat stress

Maurizio Baldassarre, Marina Naldi, Marco Domenicali, Sabrina Volo, Angelo Peli

ABSTRACT

Heat stress has a major impact on veal calves welfare and productivity. Prolonged exposure to warm

temperature is associated with several alterations of physiologic processes and increased systemic

inflammation and oxidative stress. Bovine serum albumin (BSA) is the most abundant plasma protein

and, besides the regulation of osmotic pressure, carries several additional functions, including

antioxidant, immunomodulatory, binding and transport activities. Such non-oncotic properties are

closely related to structural integrity of the circulating molecule and may be compromised in stressful

microenvironments as it occurs in heat stressed animals. Thus, in the present study we developed and

validated an LC–MS analytical technique for the characterization of circulating BSA

microheterogeneity in veal calves exposed to heat stress. The method was specifically tailored to the

structural characteristics of the BSA molecule as well as to the complexity of the biological samples,

allowing the identification of several BSA isoforms, each characterized by a specific structural defect.

The mass spectrometry based approach enabled the identification of BSA isoforms with reversible and

irreversible oxidation and/or glycation and the native BSA, the only isoform endowed with structural

and functional integrity. We found that, in veal calves, heat stress is associated to a significant

reduction of the native BSA and to a significant increment of the reversibly and irreversibly oxidized

BSA.

Molecular fingerprinting of principal neurons in the rodent hippocampus: A neuroinformatics

approach

D.J. Hamilton, C.M. White, C.L. Rees, D.W. Wheeler, G.A. Ascoli

ABSTRACT

Neurons are often classified by their morphological and molecular properties. The online knowledge

base Hippocampome.org primarily defines neuron types from the rodent hippocampal formation based

on their main neurotransmitter (glutamate or GABA) and the spatial distributions of their axons and

dendrites. For each neuron type, this open-access resource reports any and all published information

regarding the presence or absence of known molecular markers, including calcium-binding proteins,

neuropeptides, receptors, channels, transcription factors, and other molecules of biomedical relevance.

The resulting chemical profile is relatively sparse: even for the best studied neuron types, the

expression or lack thereof of fewer than 70 molecules has been firmly established to date. The mouse

genome-wide in situ hybridization mapping of the Allen Brain Atlas provides a wealth of data that,

when appropriately analyzed, can substantially augment the molecular marker knowledge in

Hippocampome.org. Here we focus on the principal cell layers of dentate gyrus (DG), CA3, CA2, and

CA1, which together contain approximately 90% of hippocampal neurons. These four anatomical

parcels are densely packed with somata of mostly excitatory projection neurons. Thus, gene expression

data for those layers can be justifiably linked to the respective principal neuron types: granule cells in

DG and pyramidal cells in CA3, CA2, and CA1. In order to enable consistent interpretation across

genes and regions, we screened the whole-genome dataset against known molecular markers of those

neuron types. The resulting threshold values allow over 6000 very-high confidence (>99.5%)

expressed/not-expressed assignments, expanding the biochemical information content of

Hippocampome.org more than five-fold. Many of these newly identified molecular markers are

potential pharmacological targets for major neurological and psychiatric conditions.

217

Volume 145 October 2017

Two-dimensional liquid chromatography in pharmaceutical analysis Instrumental aspects,

trends and applications

Marion Iguiniz, Sabine Heinisch

ABSTRACT

The interest in two-dimensional liquid chromatography (2D-LC) has been growing up since the last

decades. This promising technique appears as a relevant solution for various analytical challenges

encountered in pharmaceutical analysis. The objective of this review is to give an overview of past,

current and emerging trends in 2D-LC techniques applied to pharmaceutical compounds. The

referenced studies cover the late 1980s to the present. Information regarding the different aspects of

this analytical technique, including chromatographic conditions, instrumental setup and compounds of

interest, was compiled and summarized into a synoptic table. Particular attention is paid to key features

including (i) the interfaces used for coupling the two dimensions, (ii) the application fields, and (iii)

the chromatographic modes that can be combined together. Finally an attempt is made to predict future

advances in two-dimensional separation techniqes for pharmaceutical analysis.

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A

generic method development approach

Balázs Bobály, Valentina D‘Atri, Alain Beck, Davy Guillarme, Szabolcs Fekete

ABSTRACT

Hydrophilic interaction liquid chromatography (HILIC) is a well-established technique for the

separation and analysis of small polar compounds. A recently introduced widepore stationary phase

expanded HILIC applications to larger molecules, such as therapeutic proteins. In this paper, we

present some generic HILIC conditions adapted for a wide range of FDA and EMA approved

recombinant monoclonal antibody (mAb) species and for an antibody-drug conjugate (ADC). Seven

approved mAbs possessing various isoelectric point (pI) and hydrophobicity as well as a cysteine

conjugated ADC were used in this study. Samples were digested by IdeS enzyme and digests were

further fragmented by chemical reduction. The resulting fragments were separated by HILIC. The

main benefit of HILIC was the separation of polar variants (glycovariants) in a reasonable analysis

time at the protein level, which is not feasible with other chromatographic modes. Three samples were

selected and chromatographic conditions were further optimized to maximize resolution. A

commercial software was used to build up retention models. Experimental and predicted

chromatograms showed good agreement and the average error of retention time prediction was less

than 2%. Recovery of various species and sample stability under the applied conditions were also

discussed.

218

A new platform for serological analysis based on porous 3-dimensional polyethylene sinter

bodies

Mohammed Alasel, Michael Keusgen

ABSTRACT

A new sensitive and selective platform, three-dimensional immunosensor, has been developed for a

rapid serological diagnosis; detection of a Borrelia infection was considered as a model assay. The

immunosensor is based on a 3-dimensional (3D) porous solid surface (sinter body) with dimensions of

2 × 2.5 mm where a recombinant variable lipoprotein surface-exposed protein (VlsE; Borrelia-antigen)

is immobilized by different techniques. The sinter body served as a robust and inexpensive carrier,

which facilitated a successful hydrophobic adsorption as well as covalent immobilization of the

antigen with sufficient amounts of on the surface. Because of sinter body‘s porosity, the detection

could be performed in an immune affinity flow system based on a little disposable plastic column. The

flow of reagents through the column is advantageous in terms of reducing the non-specific interaction

and shortening the test time. Furthermore, three labels were tested for a colorimetric detection: i) a

horseradish peroxidase (HRP) labeled secondary antibody, ii) nanoparticles based on Sudan IV, and

iii) gold nanoparticles modified with protein A. HRP secondary labeled antibody provides the most

sensitive test, 1000 fold dilution of serum sample can be clearly detected in only 20 min. Gold

nanoparticles modified with protein A were used as a direct label or as a catalyst for reduction of silver

ions. Direct detection with gold nanoparticles provides short time of analysis (5 min) while detection

of metallic silver required longer time (12 min) but with improved sensitivity. Nanoparticles based on

Sudan IV showed high background and were less favorable.

Designing a calibration set in spectral space for efficient development of an NIR method for

tablet analysis

Md Anik Alam, James Drennen, Carl Anderson

ABSTRACT

Designing a calibration set is the first step in developing a multivariate spectroscopic calibration

method for quantitative analysis of pharmaceutical tablets. This step is critical because successful

model development depends on the suitability of the calibration data. For spectroscopic-based

methods, traditional concentration based techniques for designing calibration sets are prone to have

redundant information while simultaneously lacking necessary information for a successful calibration

model. A method for designing a calibration set in spectral space was developed. The pure component

spectra of a tablet formulation were used to define the spectral space of that formulation. This method

maximizes the information content of measurements and minimizes sample requirements to provide an

efficient means for developing multivariate spectroscopic calibration. A comparative study was

conducted between a commonly employed full factorial approach to calibration development and the

newly developed technique. The comparison was based on a system to quantify a model drug,

acetaminophen, in pharmaceutical compacts using near infrared spectroscopy. A 2-factor full factorial

design (acetaminophen with 5 levels and MCC: Lactose with 3 levels) was used for calibration

development. Three replicates at each design point resulted in a total of 45 tablets for the calibration

set. Using the newly developed spectral based method, 11 tablets were prepared for the calibration set.

Partial least square (PLS) models were developed from respective calibration sets.

219

On-line prediction of the glucose concentration of CHO cell cultivations by NIR and Raman

spectroscopy: Comparative scalability test with a shake flask model system

Bence Kozma, Edit Hirsch, Szilveszter Gergely, László Párta, András Salgó

ABSTRACT

In this study, near-infrared (NIR) and Raman spectroscopy were compared in parallel to predict the

glucose concentration of Chinese hamster ovary cell cultivations. A shake flask model system was

used to quickly generate spectra similar to bioreactor cultivations therefore accelerating the

development of a working model prior to actual cultivations. Automated variable selection and several

pre-processing methods were tested iteratively during model development using spectra from six shake

flask cultivations. The target was to achieve the lowest error of prediction for the glucose

concentration in two independent shake flasks. The best model was then used to test the scalability of

the two techniques by predicting spectra of a 10 l and a 100 l scale bioreactor cultivation. The NIR

spectroscopy based model could follow the trend of the glucose concentration but it was not

sufficiently accurate for bioreactor monitoring. On the other hand, the Raman spectroscopy based

model predicted the concentration of glucose in both cultivation scales sufficiently accurately with an

error around 4 mM (0.72 g/l) that is satisfactory for the on-line bioreactor monitoring purposes of the

biopharma industry. Therefore, the shake flask model system was proven to be suitable for scalable

spectroscopic model development.

Conventional and accelerated-solvent extractions of green tea (camellia sinensis) for

metabolomics-based chemometrics

Joshua J. Kellogg, Emily D. Wallace, Tyler N. Graf, Nicholas H. Oberlies, Nadja B. Cech

ABSTRACT

Metabolomics has emerged as an important analytical technique for multiple applications. The value of

information obtained from metabolomics analysis depends on the degree to which the entire

metabolome is present and the reliability of sample treatment to ensure reproducibility across the

study. The purpose of this study was to compare methods of preparing complex botanical extract

samples prior to metabolomics profiling. Two extraction methodologies, accelerated solvent extraction

and a conventional solvent maceration, were compared using commercial green tea [Camellia sinensis

(L.) Kuntze (Theaceae)] products as test cases. The accelerated solvent protocol was first evaluated to

ascertain critical factors influencing extraction using a D-optimal experimental design study. The

accelerated solvent and conventional extraction methods yielded similar metabolite profiles for the

green tea samples studied. The accelerated solvent extraction yielded higher total amounts of extracted

catechins, was more reproducible, and required less active bench time to prepare the samples. This

study demonstrates the effectiveness of accelerated solvent as an efficient methodology for

metabolomics studies.

220

Phase separation of in situ forming poly (lactide-co-glycolide acid) implants investigated using a

hydrogel-based subcutaneous tissue surrogate and UV–vis imaging

Yu Sun, Henrik Jensen, Nickolaj J. Petersen, Susan W. Larsen, Jesper Østergaard

ABSTRACT

Phase separation of in situ forming poly (lactide-co-glycolide acid) (PLGA) implants with agarose

hydrogels as the provider of nonsolvent (water) mimicking subcutaneous tissue was investigated using

a novel UV–vis imaging-based analytical platform. In situ forming implants of PLGA-1-methyl-2-

pyrrolidinone and PLGA-triacetin representing fast and slow phase separating systems, respectively,

were evaluated using this platform. Upon contact with the agarose hydrogel, the phase separation of

the systems was followed by the study of changes in light transmission and absorbance as a function of

time and position. For the PLGA-1-methyl-2-pyrrolidinone system, the rate of spatial phase separation

was determined and found to decrease with increasing the PLGA concentration from 20% to 40%

(w/w). Hydrogels with different agarose concentrations (1% and 10% (w/v)) were prepared for

providing the nonsolvent, water, to the in situ forming PLGA implants simulating the injection site

environment. The resulting implant morphology depended on the stiffness of hydrogel matrix,

indicating that the matrix in which implants are formed is of importance. Overall, the work showed

that the UV–vis imaging-based platform with an agarose hydrogel mimicking the subcutaneous tissue

holds potential in providing bio-relevant and mechanistic information on the phase separation

processes of in situ forming implants.

Mid-infrared and near-infrared spectroscopy for rapid detection of Gardeniae Fructus by a

liquid-liquid extraction process

Lingyan Tao, Zhonglin Lin, Jiashan Chen, Yongjiang Wu, Xuesong Liu

ABSTRACT

Gardeniae Fructus is widely used in the pharmaceutical industry, and many studies have confirmed its

medical and economic value. In this study, samples collected from different liquid-liquid extraction

batches of Gardeniae Fructus were detected by mid-infrared (MIR) and near-infrared (NIR)

spectroscopy. Seven analytes, neochlorogenic acid (5-CQA), cryptochlorogenic acid (4-CQA),

chlorogenic acid (3-CQA), geniposidic acid (GEA), deacetyl-asperulosidic acid methyl ester

(DAAME), genipin-gentiobioside (GGB), and gardenoside (GA), were chosen as quality property

indexes of Gardeniae Fructus. The two kinds of spectra were each used to build models by single

partial least squares (PLS). Additionally, both spectral data were combined and modeled by multiblock

PLS. For single spectroscopy modeling results, NIR had a better prediction for high-concentration

analytes (3-CQA, DAAME, GGB, and GA) whereas MIR performed better for low-concentration

analytes (5-CQA, 4-CQA, and GEA). The multiblock methodology was found to be better compared

to single spectroscopy models for all seven analytes. Specifically, the coefficients of determination

(R2) of the NIR, MIR, and multiblock PLS calibration models of all seven components were higher

than 0.95. Relative standard errors of prediction (RSEP) were all less than 7%, except for models of

GGB, which were 10.36%, 13.24%, and 8.15% for the NIR-PLS, MIR-PLS, and multiblock models,

respectively. These results indicate that MIR and NIR spectrographic techniques could provide a new

choice for quality control in industrial production of Gardeniae Fructus.

221

Quantitative analysis of a biopharmaceutical protein in cell culture samples using automated

capillary electrophoresis (CE) western blot

Dong Xu, Kentaro Marchionni, Yunli Hu, Wei Zhang, Zoran Sosic

ABSTRACT

An effective control strategy is critical to ensure the safety, purity and potency of biopharmaceuticals.

Appropriate analytical tools are needed to realize such goals by providing information on product

quality at an early stage to help understanding and control of the manufacturing process. In this work,

a fully automated, multi-capillary instrument is utilized for size-based separation and western blot

analysis to provide an early readout on product quality in order to enable a more consistent

manufacturing process. This approach aims at measuring two important qualities of a

biopharmaceutical protein, titer and isoform distribution, in cell culture harvest samples. The acquired

data for isoform distribution can then be used to predict the corresponding values of the final drug

substance, and potentially provide information for remedy through timely adjustment of the

downstream purification process, should the expected values fall out of the accepted range.

Synchronous characterization of carbohydrates and ginsenosides yields deeper insights into the

processing chemistry of ginseng

Shan-Shan Zhou, Jun Xu, Ming Kong, Ka-Man Yip, Hu-Biao Chen

ABSTRACT

Carbohydrates and ginsenosides in ginseng are biologically interrelated. Their synchronous analysis is

therefore essential in chemical research on ginseng to characterize its ―holistic‖ quality. Here we

investigated the processing chemistry of red ginseng (RG), a ginseng product processed by water-

steaming, for which both carbohydrates and ginsenosides were qualitatively and quantitatively

determined through multiple analytical techniques. Results revealed that the steam-processing not only

qualitatively and quantitatively altered the ginsenosides but also affected the polymeric carbohydrates

via changing their physiochemical parameters, i.e. water-solubility, molecular size, types and ratios of

constituent monosaccharides. Potential mechanisms involved in the transformation of ginseng

chemicals are proposed and discussed, including hydrolysis (deglycosylation, demalonylation,

deacetylation), dehydration, polymerization, volatilization, reduction and the Maillard reaction. The

study strengthens the research on the processing chemistry of RG, and therefore should be helpful for

elucidating the scientific basis of RG preparation and application.

222

Development and validation of an ICP-MS method for the determination of elemental impurities

in TP-6076 active pharmaceutical ingredient (API) according to USP 〈232〉/〈233〉

Osama Chahrour, John Malone, Mark Collins, Vrushali Salmon, Nick Dunwoody

ABSTRACT

The new guidelines of the United States pharmacopeia (USP), European pharmacopeia (EP) and

international conference on harmonization (ICH) regulating elemental impurities limits in

pharmaceuticals signify the end of unspecific analysis of metals as outlined in USP 〈231〉. The new

guidelines specify both daily doses and concentration/limits of elemental impurities in pharmaceutical

final products, active pharmaceutical ingredients (API) and excipients. In chapter USP 〈233〉

method implementation, validation and quality control during the analytical process are described. We

herein report the use of a stabilising matrix that overcomes low spike recovery problem encountered

with Os and allows the determination of all USP required elemental impurities (As, Cd, Hg, Pb, V, Cr,

Ni, Mo, Cu, Pt, Pd, Ru, Rh, Os and Ir) in a single analysis. The matrix was used in the validation of a

method to determine elemental impurities in TP-6076 active pharmaceutical ingredient (API) by ICP-

MS according to the procedures defined in USP〈233〉 and to GMP requirements. This validation

will support the regulatory submission of TP-6076 which is a novel tetracycline analogue effective

against the most urgent multidrug-resistant gram-negative bacteria. Evaluation of TP-6076 in IND-

enabling toxicology studies has led to the initiation of a phase 1 clinical trial.

Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1


Oluwatosin O. Dada, Romesh Rao, Natalie Jones, Nomalie Jaya, Oscar Salas-Solano

ABSTRACT

Fragmentation of monoclonal antibodies is a critical quality attribute routinely monitored to assess the

purity and integrity of the product from development to commercialization. Cleavage in the upper

hinge region of IgG1 monoclonal antibodies is a common fragmentation pattern widely studied by size

exclusion chromatography (SEC). Capillary electrophoresis with sodium dodecylsulfate (CE-SDS) is a

well-established technique commonly used for monitoring antibody fragments as well, but its

comparability to SEC in monitoring hinge fragments has not been established until now. We report a

characterization strategy that establishes the correlation between hinge region fragments analyzed by

SEC and CE-SDS. Monoclonal antibodies with elevated hinge fragments were generated under low pH

stress conditions and analyzed by SEC and CE-SDS. The masses of the fragments generated were

determined by LC-MS. Electrophoretic migration of the hinge fragmentation products in CE-SDS

were determined based on their mass values. Comparative assessment of fragments by SEC, and CE-

SDS showed similar correlation with incubation time. This study demonstrates that CE-SDS can be

employed as a surrogate technique to SEC for monitoring hinge region fragments. Most importantly,

combination of these techniques can be used to obtain comprehensive understanding of fragment

related characteristics of therapeutic protein products.

223

Revisiting blood-brain barrier: A chromatographic approach

Xavier Subirats, Laura Muñoz-Pascual, Michael H. Abraham, Martí Rosés

ABSTRACT

Drugs designed to reach a pharmacological CNS target must be effectively transported across the

blood-brain barrier (BBB), a thin monolayer of endothelial cells tightly attached together between the

blood and the brain parenchyma. Because of the lipidic nature of the BBB, several physicochemical

partition models have been studied as surrogates for the passive permeation of potential drug

candidates across the BBB (octanol-water, alkane-water, PAMPA...). In the last years, biopartition

chromatography is gaining importance as a noncellular system for the estimation of biological

properties in early stages of drug development. Microemulsions (ME) are suitable mobile phases,

because of their ease of formulation, stability and adjustability to a large number of compositions

mimicking biological structures. In the present work, several microemulsion liquid chromatographic

(MELC) systems have been characterized by means of the Abraham‘s solvation parameter model, in

order to assess their suitability as BBB distribution or permeability surrogates. In terms of similarity

between BBB and MELC systems (dispersion forces arising from solute non-bonded electrons,

dipolarity/polarizability, hydrogen-bond acidity and basicity, and molecular volume), the passive

permeability surface area product (log PS) for neutral (including zwitterions), fully and partially

ionized drugs was found to be well correlated with the ME made of 3.3% SDS (w/v; surfactant) 0.8%

heptane (w/v; oil phase) and 6.6% 1-butanol (w/v; co-surfactant) in 50 mM aqueous phosphate buffer,

pH 7.4.

Liquid chromatographic enantioseparation of carbocyclic β-amino acids possessing limonene

skeleton on macrocyclic glycopeptide-based chiral stationary phases

Tímea Orosz, Nóra Grecsó, Gyula Lajkó, Zsolt Szakonyi, Antal Péter

ABSTRACT

Polar-ionic and reversed-phase high-performance liquid chromatographic separations of limonene-

based cyclic β-amino acid enantiomers were carried out by using macrocyclic glycopeptide-based

chiral selectors applying Chirobiotic T, TAG and R columns. The effects of additives, concentration of

the co- and counter-ions and the temperature in polar-ionic mobile phase systems were studied. The

influence of pH, MeOH content and alcohol additives were investigated in the reversed-phase mode.

The difference in the change in standard enthalpy Δ(ΔH°), entropy Δ(ΔS°), and free energy Δ(ΔG°)

was calculated from the linear van't Hoff plots derived from the ln α vs 1/T curves in the temperature

range 5–40 °C. Unusual temperature behavior was observed on Chirobiotic TAG for most of the

analytes: decreased retention times were accompanied with increased separation factors with

increasing temperature, and separation was entropically-driven. For two of the studied analytes

enthalpically-driven enantioseparations were observed. The elution sequence was determined in all

cases, but no general rule could be established.

224

On-line coupling of molecularly imprinted solid phase extraction with liquid chromatography

for the fast determination of coumarins from complex samples

Andrea Machyňáková, Ivona Lhotská, Katarína Hroboňová, Dalibor Ńatínský

ABSTRACT

In this work, an on-line SPE-HPLC method with spectrophotometric detection was developed for the

determination of coumarins in complex samples. For the on-line cleanup of samples, a molecularly

imprinted polymer was packed into the column cartridge and coupled directly with HPLC (MISPE-

HPLC) using a column switching system. The separation of coumarins was performed on a C18 core-

shell column (100 × 4.6 mm, 5 μm) with a mobile phase consisting of 0.3% acetic acid/acetonitrile

with gradient elution at a flow-rate of 1 mL min−1. The total time of the whole analytical run,

including the extraction step, was 13.25 min. The on-line MISPE-HPLC method was optimized and

validated. The results showed good linearity (0.10–100 μg mL−1) with correlation coefficients higher

than 0.99. The LOD values were from 0.03 to 0.15 μg mL−1. The proposed method was successfully

applied for analysis of real samples (Cassia cinnamon, chamomile tea, and Tokaj specialty wines) and

obtained recoveries varied from 78.7% to 112.2% with an RSD less than 9%.

Metabolic profiling analysis of Siraitia grosvenorii revealed different characteristics of green

fruit and saccharified yellow fruit

Chengnan Fang, Qingqing Wang, Xinyu Liu, Guowang Xu

ABSTRACT

Siraitia grosvenorii is an economic and medicinal plant, its fruit is considered to be good to health for

its diverse bioactive ingredients. However, the clarification of chemical composition and their changes

after saccharification procedure are not well performed. In present study, a metabolomics method

based on ultra-high-performance liquid chromatography tandem quadrupole time-of-flight mass

spectrometry was developed for metabolic profiling acquisition of Siraitia grosvenorii extract.

Furthermore, information dependent analysis (IDA) combined with self-constructed LC–MS/MS

identification system for metabolites were employed to identify primary and secondary metabolites in

Siraitia grosvenorii. A total of 126 metabolites were identified or tentatively identified. The obvious

differences of metabolic profiling between green fruit and saccharified yellow fruit were observed, and

metabolites showed their own distribution characteristics in peel, flesh and seed. The majority of the

nutrients and effective components were more distributed in flesh and peel, and saccharification was

conducive to accumulation of sweet glycosides. This study not only expanded metabolite composition

information of Siraitia grosvenorii, but also specified distribution characteristics of identified

metabolites.

225

Comparison of α-glucosidase inhibitory effect and bioactive constituents of Anemarrhenae

Rhizoma and Fibrous Roots

Si-Hui Nian, Hui-Jun Li, E-Hu Liu, Ping Li

ABSTRACT

Comprehensive utilization of medicinal plant resources is of great significance for sustainable

development of traditional Chinese medicines. In the present study, the α-glucosidase inhibitory

activities of the rhizome and fibrous root of Anemarrhena Asphodeloides Bunge, were compared

detailedly, and a high performance liquid chromatography coupled with electrospray ionization tandem

triple quadrupole mass spectrometry (HPLC-QQQ/MS) method was developed for simultaneous

quantification of seven bioactive constituents including neomangiferin, mangiferin, isomangiferin,

timosaponin BII, timosaponin B, timosaponin AIII, and timosaponin N in 40 batches of samples. The

results demonstrated that fibrous root extracts had more potent α-glucosidase inhibitory activity than

rhizome extracts. Mangiferin and isomangiferin were abundant in fibrous root, while the analyzed

saponins were rich in rhizome. Based on the chemometrics methods including principal component

analysis (PCA), orthogonal partial least square discriminant analysis (OPLS-DA), and partial least

square (PLS), mangiferin and isomangiferin might be mainly responsible for α-glucosidase inhibitory

activity of the genus. These findings indicate that the established HPLC-QQQ/MS method was proven

to be useful and efficient for quality control of Anemarrhena materials, and fibrous root had the

potential to be utilized as anti-diabetic medicinal resource.

Characterization of forced degradation products of torasemide through MS tools and

explanation of unusual losses observed during mass fragmentation of drug and degradation

products through density functional theory

Moolchand Kurmi, Neha Patel, Shalu Jhajra, Prasad V. Bharatam, Saranjit Singh

ABSTRACT

Mass spectrometry tools (HRMS/LC-HRMS, MSn, and/or on-line H/D exchange) were employed to

establish mass fragmentation pattern of torasemide and to characterize its degradation products.

During collision-induced dissociation, multiple rearrangement processes and unusual losses of sulfur

(S), sulfanyl (HS), sulfur dioxide (SO2), sulphinic acid radical (HSO2), sulfur monoxide (SO), carbon

monoxide (CO), formyl radical (CHO) and C5H3NOS were observed. The same were successfully

explained by study of energy profiles, established by application of density functional theory (DFT).

226

Metabolic profiling of the traditional Chinese medicine formulation Yu Ping Feng San for the


in U937 cells

Stefanie Nikles, Marlene Monschein, Huiqin Zou, Yong Liu, Rudolf Bauer

ABSTRACT

Yu Ping Feng San (YPFS) is a classical TCM formulation which has been traditionally used for

treatment of immune system related diseases such as chronic bronchitis, allergic rhinitis and asthma.

The formula is a mixture of Radix Saposhnikoviae (Fangfeng), Radix Astragali (Huangqi), and

Rhizoma Atractylodis macrocephalae (Baizhu). TLC- and LC-DAD-ESI-MS/MS methods have been

developed for the analysis of the metabolic profiles of the single herbs and of the formula. Decoctions

and ASE extracts were analyzed in order to trace components of the individual herbs in YPFS. Nine

constituents of Radix Saposhnikoviae, ten constituents of Radix Astragali and five constituents of

Rhizoma Atractylodis macrocephalae have been assigned in the chemical profiles of the formula,

which now allow the standardisation of YPFS. The pharmacological testing showed that all extracts

significantly inhibited expression of TNF-α, IFN-γ, and IL-1β in U937 cells, while the inhibition of IL-

4 was consistently low. Compared to conventional analyses which are focused on a limited set of

compounds, metabolomics approaches, together with novel data processing tools, enable a more

holistic comparison of the herbal extracts. In order to identify the constituents which are relevant for

the immunomodulatory effects of the formula, metabolomics studies (PCA, OPLS-DA) have been

performed using UPLC/QTOF MS data.

A comprehensive stability-indicating HPLC method for determination of chloroquine in active

pharmaceutical ingredient and tablets: Identification of oxidation impurities

Ana Silva Coelho, Clara Elisa Pontes Chagas, Rodrigo Maia de Pádua, Gerson Antônio Pianetti,

Christian Fernandes

ABSTRACT

Malaria is the most common parasitic disease in humans. It is estimated that 3 billion people live under

the risk of contracting this disease in the world. Chloroquine (CQ) is the drug of choice to treat cases

of non-complicated malaria. Forced degradation studies are important to know the drug‘s potentials

degradation products and to develop a stability indicating method. Thus, chloroquine active

pharmaceutical ingredient (API), chloroquine tablets and placebo were submitted to a detailed forced

degradation study, using several stressing agents. The results were used on the development of a

stability indicating method, using high performance liquid chromatography. The method was validated

showing selectivity, precision, accuracy, robustness and linearity in the range of 30–360 μg/mL of

chloroquine. Chloroquine API and tablets were susceptible to alkaline hydrolysis with NaOH 1 mol/L,

and to oxidation with H2O2 3.0%. Two degradation products were formed in oxidative test. Kinetics

of chloroquine degradation in alkaline hydrolysis was performed for both API and tablets. The

calculated decay constant (k1) was 0.223 days−1 for API and 0.182 days−1 for tablets, while the half-

life (t1/2) was 3.1 days for API and 3.8 days for tablets. Chemical structures have been proposed for

the two degradation products formed in the presence of H2O2, using an UHPLC-UV-MS/MS

approach.

227

Identification of impurities in macrolides by liquid chromatography–mass spectrometric


retention relationship (QSRR)

Xia Zhang, Jin Li, Chen Wang, Danqing Song, Changqin Hu

ABSTRACT

Macrolides are multicomponent drugs whose impurity control is always a challenge demanding

analysis method with good sensitivity and selectivity. Three separate, sensitive, accurate liquid

chromatography tandem mass spectrometry methods (LC–MS) were developed for the measurement

of three 16-membered ring macrolides (josamycin, josamycin propionate and midecamycin acetate)

and related substances in commercial samples. The characteristics of impurities in macrolides were

summarized as useful guidance for the impurity analysis of this class of drugs. For each drug, a large

number of unknown components have been detected with the high-sensitive MS detector and possible

structures of the majority of them were postulated based on the summarized fragmentation rules of 16-

membered ring macrolides. A QSRR model was constructed by multilinear regression to predict the

retention times of identified impurities which were not detected by the LC–MS methods, without

obtaining their reference standards. Satisfactory performance was obtained during leave-one-out cross-

validation with a predictive ability (Q2) of 0.95. The generalisation ability of the model was further

confirmed by an average error of 2.3% in external prediction. The best QSRR model, based on eight

molecular descriptors, exhibited a promising predictive performance and robustness.

The impact of ZnO and TiO2 on the stability of clotrimazole under UVA irradiation:

Identification of photocatalytic degradation products and in vitro cytotoxicity assessment

Agata Kryczyk, Paweł Żmudzki, Paulina Koczurkiewicz, Joanna Piotrowska, Urszula Hubicka

ABSTRACT

In order to ensure the safe and effective use of pharmaceutical products especially for topical

administration photostability testing is necessary. The current paper presents an in-depth analysis of

the stability of one of the most common antifungal agents, namely clotrimazole. Clotrimazole has

proven to be stable under UVA irradiation in applied experimental conditions, but the presence of

catalysts such as ZnO and TiO2 has contributed significantly to the degradation of this compound. The

findings indicate that its photocatalytic degradation reactions followed the pseudo first-order kinetics

with rate constant depending on the pH and the used solvent. Using LC–MS/MS, 14 presumable

degradation products of clotrimazole were identified and the plausible transformation pathways were

proposed. The in vitro cytotoxicity risk evaluation based on photostability of clotrimazole was also

performed using the Human skin fibroblast cell line (BJ) ATCC™ CRL-2522. There was no

statistically significant difference between cells viability in all analyzed combinations of clotrimazole,

TiO2/ZnO, and UVA irradiation (p < 0.05).

228

Chemometrically assisted development and validation of LC–MS/MS method for the analysis of

potential genotoxic impurities in meropenem active pharmaceutical ingredient

Katerina Grigori, Yannis L. Loukas, AnĎelija Malenović, Vicky Samara, Yannis Dotsikas

ABSTRACT

A sensitive Liquid Chromatography tandem mass spectrometry (LC–MS/MS) method was developed

and validated for the quantitative analysis of three potential genotoxic impurities (318BP, M9, S5) in

meropenem Active Pharmaceutical Ingredient (API). Due to the requirement for LOD values in ppb

range, a high concentration of meropenem API (30 mg/mL) had to be injected. Therefore, efficient

determination of meropenem from its impurities became a critical aim of this study, in order to divert

meropenem to waste, via a switching valve. ‎ After the selection of the important factors affecting

analytes‘ elution, a Box-Behnken design was utilized to set the plan of experiments conducted with

UV detector. As responses, the separation factor s between the last eluting impurity and meropenem,

as well as meropenem retention factor k were used. Grid point search methodology was implemented

aiming to obtain the optimal conditions that simultaneously comply to the conflicted criteria. Optimal

mobile phase consisted of ACN, methanol and 0.09% HCOOH at a ratio 71/3.5/15.5 v/v. All

impurities and internal standard omeprazole were eluted before 7.5 min and at 8.0 min the eluents were

directed to waste. The protocol was transferred to LC–MS/MS and validated according to ICH

guidelines.

Identification and interconversion of isomeric 4,5-functionalized 1,2,3-thiadiazoles and 1,2,3-

triazoles in conditions of electrospray ionization

D.M. Mazur, M.E. Zimens, V.A. Bakulev, A.T. Lebedev

ABSTRACT

1,2,3-Triazoles and 1,2,3-thiadiazoles have been receiving permanent interest due to their exciting

chemical reactivity and interesting biological properties including antibacterial, anticancer and

antiviral activities. There are four compounds bearing 1H-1,2,3-triazole core in clinical studies which

may appear in the market of drugs in nearest future. Definitely reliable methods of their identification

and quantification should be developed by that time. Mass spectrometry showed itself as the most

reliable method of analysis when dealing with trace levels of organic compounds in the mixtures and

in the most complex matrices, including biological ones. In the present study tandem mass

spectrometry was used to study fragmentation pathways of protonated and deprotonated molecules of

isomeric 4,5-functionalized 1,2,3-thiadiazoles and 1,2,3-triazoles in conditions of electrospray

ionization (ESI). A group of marker ions allowing differentiation between the targeted isomeric

compounds was established. Besides, interconversion of these isomers into one another was studied in

the gas phase in conditions mimicking these processes in solution.

229

Dissolution assessment of allopurinol immediate release tablets by near infrared spectroscopy

Jelena Smetińko, Sneņana Miljanić

ABSTRACT

The purpose of this study was to develop a NIR spectroscopic method for assessment of drug

dissolution from allopurinol immediate release tablets. Thirty three different batches of allopurinol

immediate release tablets containing constant amount of the active ingredient, but varying in excipients

content and physical properties were introduced in a PLS calibration model. Correlating allopurinol

dissolution reference values measured by the routinely used UV/Vis method, with the data extracted

from the NIR spectra, values of correlation coefficient, bias, slope, residual prediction determination

and root mean square error of prediction (0.9632, 0.328%, 1.001, 3.58, 3.75%) were evaluated. The

obtained values implied that the NIR diffuse reflectance spectroscopy could serve as a faster and

simpler alternative to the conventional dissolution procedure, even for the tablets with a very fast

dissolution rate (>85% in 15 minutes). Apart from the possibility of prediction of the allopurinol

dissolution rate, the other multivariate technique, PCA, provided additional data on the non-chemical

characteristics of the product, which could not be obtained from the reference dissolution values.

Analysis on an independent set of samples confirmed that a difference between the UV/Vis reference

method and the proposed NIR method was not significant. According to the presented results, the

proposed NIR method may be suitable for practical application in routine analysis and for continuously

monitoring the product's chemical and physical properties responsible for expected quality.

Enhanced and green extraction polyphenols and furanocoumarins from Fig (Ficus carica L.)

leave using deep eutectic solvents

Tong Wang, Jiao Jiao, Qing-Yan Gai, Peng Wang, Yu-Jie Fu

ABSTRACT

Nowadays, green extraction of bioactive compounds from medicinal plants has gained increasing

attention. As green solvent, deep eutectic solvent (DES) have been highly rated to replace toxic

organic solvents in extraction process. In present study, to simultaneous extraction five main bioactive

compounds from fig leaves, DES was tailor-made. The tailor-made DES composed of a 3:3:3 molar

ratio of glycerol, xylitol and D-(−)-Fructose showed enhanced extraction yields for five target

compounds simultaneously compared with traditional methanol and non-tailor DESs. Then, the tailor-

made DES based extraction methods have compared and microwave-assisted extraction was selected

and optimized due to its high extraction yields with lower time consumption. The influencing

parameters including extraction temperature, liquid-solid ratio, and extraction time were optimized

using response surface methodology (RSM). Under optimal conditions the extraction yield of

caffeoylmalic acid, psoralic acid-glucoside, rutin, psoralen and bergapten was 6.482 mg/g, 16.34 mg/g,

5.207 mg/g, 15.22 mg/g and 2.475 mg/g, respectively. Macroporous resin D101 has been used to

recovery target compounds with recovery yields of 79.2%, 83.4%, 85.5%, 81.2% and 75.3% for

caffeoylmalic acid, psoralic acid-glucoside, rutin, psoralen and bergapten, respectively. The present

study suggests that DESs are truly designer and efficient solvents and the method we developed was

efficient and sustainable for extraction main compounds from Fig leaves.mg/g

230

Site- and species-specific hydrolysis rates of cocaine

Levente Szöcs, Gergely Völgyi, Ákos Urai, Sándor Hosztafi, Béla Noszál

ABSTRACT

The hydroxide-catalyzed non-enzymatic hydrolysis of cocaine is quantified in terms of ten site- and

species-specific rate constants in connection with also ten site- and species-specific acid-base

equilibrium constants, comprising all the twelve coexisting species in solution. This characterization

involves the major and minor decomposition pathways via benzoylecgonine and ecgonine methyl

ester, respectively, leading to ecgonine, the final product. Hydrolysis has been found to be 10–330

times faster at site 2 than at site 3, depending on the ionization status of the amino moiety and the rest

of the molecule. Nitrogen protonation accelerates the hydrolyses approximately ten times both at site 2

and site 3.

Comparative stability-indicating chromatographic methods for determination of 4-

hexylresorcinol in pharmaceutical formulation and shrimps

Rafeek F. Shokry, Lories I. Bebawy, Mohamed R. Elghobashy, Samah S. Abbas

ABSTRACT

Three chromatographic stability-indicating methods were developed for determination of 4-

hexylresorcinol in pure form and in a pharmaceutical formulation. Method A was based on a gradient

elution liquid chromatographic HPLC determination of 4-hexylresorcinol, its related impurities and in

presence of its degradation products. UPLC–MS/MS (Method B) was described for determination of

the cited drug in presence of its degradation products. Method C was a thin- layer chromatography

(TLC)-densitometry method for the separation and determination of the active ingredient, one of its

related impurities and in presence of its degradation products. The mechanism of alkali, oxidative and

photodegradation of 4-hexylresorcinol was studied according to ICH guidelines. The degradation

products were characterized by the LC–MS/MS method. Methods A and B were applicable for

determination of 4-hexylresorcinol residues in shrimp meat. The studied drug was easily degraded in

alkali medium giving toxic compounds. The results obtained by the proposed methods were

statistically analyzed and compared with those obtained by applying a reported method.

231

Enantiomeric separation of seven β-agonists by NACE—Study of chiral selectivity with

diacetone-d-mannitol–boric acid complex

Lili Lv, Lijuan Wang, Jun Li, Yajun Jiao, Hongyuan Yan

ABSTRACT

A rapid and effective nonaqueous capillary electrophoresis (NACE)–ultraviolet (UV) method was

developed for the enantiomeric separation of seven β-agonists. Diacetone-d-mannitol–boric acid

complex was used as a new chiral selector. It was in situ synthesized by the reaction of diacetone-d-

mannitol and boric acid in methanol medium containing triethylamine. The effects of diacetone-d-

mannitol, boric acid, and triethylamine concentrations on the enantioseparation were carefully

investigated. Under the optimized conditions, baseline enantioseparation could be obtained for six of

the tested β-agonists within 12 min. These results were better than that obtained with d-mannitol–boric

acid complex in previous work. 11B nuclear magnetic resonance (11B NMR) was applied to determine

the fraction of boron species and confirm the formation of diacetone-d-mannitol–boric acid complex.

Validation of the established NACE method was also carried out according to ICH guidelines.

Calibration curves showed good linearity with correlation coefficients (r) ≥ 0.9992 over a certain

concentration range for each enantiomer of the tested five β-agonists. The relative standard deviations

(RSDs) of intra-day precisions and inter-day precisions of migration times were ≤1.4% (n = 6), and

≤6.3% (n = 10), respectively. That of peak areas were ≤3.7% (n = 6), and ≤5.6% (n = 10), respectively.

The limits of detection (LODs) and the limits of quantitation (LOQs) based on the signal-to-noise

ratios of 3 and 10 were found below 1.25 μg mL−1 and 5.00 μg mL−1, respectively.

An optimized HPLC-UV method for quantitatively determining sesquiterpenes in

Nardostachyos Radix et Rhizoma

Vu Ngoc Han Le, Trong Quan Khong, Min Kyun Na, Kyung Tae Kim, Jong Seong Kang

ABSTRACT

Nardostachyos Radix et Rhizoma (NR), the root and rhizome from either Nardostachys jatamansi

Batal or Nardostachys jatamansi DC, is known to have biological functions including neuro-protective

and anti-pancreatitis activity. The main bioactive compounds within NR are all classified as

sesquiterpenes, and include desoxo-narchinol A, nardosinonediol, and nardosinone. Although NR is a

valuable herb that is widely used in many Asian countries, robust quality control protocols for NR are

still in question, especially those that can analyze the three main active compounds. Current

quantitative methods within the Chinese Pharmacopoeic use nardosinone as a marker compounds. One

compound cannot represent a complicated matrix, and is thus insufficient to control the quality of this

herbal medicine. Moreover, there are no high-performance liquid chromatography (HPLC) methods

that can simultaneously analyze desoxo-narchinol A (DA), nardosinonediol (NE), and nardosinone

(ND) within NR. This study aimed to establish an efficient quality control protocol by developing an

analytical method that simultaneously detects the three sesquiterpenes with HPLC using response

surface methodology (RSM) to optimize sample preparation. Optimized HPLC conditions included a

mobile phase of 0.1% formic acid in water (A), and a 0.1% formic acid in acetonitrile (B) under an

elution program of 20% B–80% B for 30 min at 254 nm. The method was well validated,

demonstrating satisfactory linearity, accuracy, precision, recovery, repeatability, and stability.

Optimized conditions for creating the analytical sample were predicted by RSM using a Box-Behnken

design.

232

Photostability testing using online reactor HPLC hyphenation and mass spectrometric

compound identification illustrated by ketoprofen as model compound

Jaber Assaf, Diego Zulkiewicz Gomes, Bernhard Wuest, Maria Kristina Parr

ABSTRACT

Investigations on the photochemical stability of pharmaceutical substances are mandatory in drug

development and licensing as photo-induced degradation of an active pharmaceutical ingredient (API)

may not only lead to decreased API concentrations but also to toxic or reactive products. Thus, the US

Food and Drug Administration (FDA) and the International Council for Harmonisation of Technical

Requirements for Pharmaceuticals for Human Use (ICH) issued Guidance for Industry Q1B

―Photostability Testing of New Drug Substances and Products‖ for testing of pure but also packed

drugs. However, photoproducts are also known to be generated in vivo under sunlight exposure of the

skin and lead to considerable amounts of adverse drug effects. Herein we present an alternative system

that may be used for photostability testing mimicking both situations. It combines a tailored

photoreactor with an exchangeable pen light source and a modified HPLC system with online-SPE.

Identification of photoproducts may be performed using mass spectrometry. The potential of accurate

mass spectrometry as a tool for identification of photoproducts was demonstrated as well. A

comparison of the online photoreactor system and the traditional photochamber irradiation was

performed using ketoprofen for proof of concept. In both designs acetylbenzophenone and

ethylbenzophenone were detected as main photoproducts. The new device allows for fast and easy

photostability studies that may help to reduce time consuming in vitro experiments and animal trials.

Using state of the art instruments kinetic studies could also easily be performed with qualitative and

quantitative perspectives combined into one experimental design with only very low amounts of API

needed. This may be useful in early drug development, where only small amounts of API are available.

Scale-up may also be easily realized for the generation of reference material for quantification and

quality control (QC) processes as well as for toxicity testing.

Optoelectronic iron detectors for pharmaceutical flow analysis

Natalia Rybkowska, Robert Koncki, Kamil Strzelak

ABSTRACT

Compact flow-through optoelectronic detectors fabricated by pairing of light emitting diodes have

been applied for development of economic flow analysis systems dedicated for iron ions

determination. Three analytical methods with different chromogens selectively recognizing iron ions

have been compared. Ferrozine and ferene S based methods offer higher sensitivity and slightly lower

detection limits than method with 1,10‐phenantroline, but narrower ranges of linear response. Each

system allows detection of iron in micromolar range of concentration with comparable sample

throughput (20 injections per hour). The developed flow analysis systems have been successfully

applied for determination of iron in diet supplements. The utility of developed analytical systems for

iron release studies from drug formulations has also been demonstrated.

233

Isoconversional approach for non-isothermal decomposition of un-irradiated and photon-

irradiated 5-fluorouracil

Hala Sh. Mohamed, AbdelRahman A. Dahy, Refaat M. Mahfouz

ABSTRACT

Kinetic analysis for the non-isothermal decomposition of un-irradiated and photon-beam-irradiated 5-

fluorouracil (5-FU) as anti-cancer drug, was carried out in static air. Thermal decomposition of 5-FU

proceed in two steps. One minor step in the temperature range of (270–283 °C) followed by the major

step in the temperature range of (285–360 °C). The non-isothermal data for un-irradiated and photon-

irradiated 5-FU was analyzed using linear (Tang) and non-linear (Vyazovkin) isoconversional

methods. The results of the application of these free models on the present kinetic data showed quite a

dependence of the activation energy on the extent of conversion. For un-irradiated 5-FU, the non-

isothermal data analysis indicates that the decomposition is generally described by A3 and A4 modeles

for the minor and major decomposition steps, respectively. For a photon-irradiated sample of 5-FU

with total absorbed dose of 10 Gy, the decomposition is controlled by A2 model throughout the

coversion range. The activation energies calculated in case of photon-irradiated 5-FU were found to be

lower compared to the values obtained from the thermal decomposition of the un-irradiated sample

probably due to the formation of additional nucleation sites created by a photon-irradiation. The

decomposition path was investigated by intrinsic reaction coordinate (IRC) at the B3LYP/6-

311++G(d,p) level of DFT. Two transition states were involved in the process by homolytic rupture of

NH bond and ring secession, respectively.

Characterization of impurities in sodium cromoglycate drug substance and eye drops using LC-

ESI-ion trap MS and LC-ESI-QTOF MS

Peixi Zhu, Jingxian Lu, Zhijian Wang, Weike Su, Erwin Adams

ABSTRACT

As requested by regulatory authorities, impurity profiling is an important issue of quality control. In

this work, a simple and sensitive liquid chromatographic (LC) method compatible with mass

spectrometry (MS) was developed to study related substances and degradation products in sodium

cromoglycate drug substance and eye drops. The method used a Sunfire column (4.6 mm × 150 mm,

3.5 μm). Mobile phase A consisted of 10 mM ammonium formate and mobile phase B were

acetonitrile. Linear gradient elution with a post-run time of 8 min was performed as follows: 0–30 min,

3% B to 50% B; 30–35 min, 50% B. The flow rate was set at 1.0 mL/min. Degradation experiments

were performed to check the stability indicating properties of the developed method. Based on MSn

spectral data and exact mass measurements, the chemical structures of 2 unknown impurities and 6

unknown degradation products were characterized, including impurity C listed in the European

Pharmacopoeia as unknown structure. In addition, a plausible mechanism for the formation of the

degradation products was also proposed.

234

Method validation and nanoparticle characterization assays for an innovative amphothericin B

formulation to reach increased stability and safety in infectious diseases

Maraine Catarina Tadini, Ana Maria de Freitas Pinheiro, Daniel Blascke Carrão, Ana Luiza Scarano

Aguillera Forte, Franciane Marquele-Oliveira

ABSTRACT

Drug Delivery Systems (DDS) of known drugs are prominent candidates towards new and more-

effective treatments of various infectious diseases as they may increase drug bioavailability, control

drug delivery and target the site of action. In this sense, the encapsulation of Amphotericin B (AmB) in

Nanostructured Lipid Carriers (NLCs) designed with pH-sensible phospholipids to target infectious

tissues was proposed and suitable analytical methods were validated, as well as, proper nanoparticle

characterization were conducted. Characterization assays by Dinamic Light Scattering (DLS) and

Atomic Force Microscopy demonstrated spherical particles with nanometric size 268.0 ± 11.8 nm and

Zeta Potential −42.5 ± 1.5 mV suggestive of important stability. DSC/TGA and FT-IR assessments

suggested mechanical encapsulation of AmB. The AmB aggregation study indicated that the

encapsulation provided AmB at the lowest cytotoxic form, polyaggregate. Analytical methods were

developed and validated according to regulatory agencies in order to fast and assertively determine

AmB in nanoparticle suspension and, in Drug Encapsulation Efficiency (EE%), release and stability

studies. The quantification method for AmB in NLC suspension presented linearity in 5.05–60.60 μg

mL−1 range (y = 0.07659x + 0.05344) and for AmB in receptor solution presented linearity in 0.15–

10.00 μg mL−1 range (y = 54609x + 263.1), both with r ≥ 0.999. EE% was approximately 100% and

according to the release results, at pH 7.4, a sustained controlled profile was observed for up 46 h.

Surfactants enhance recovery of poorly soluble drugs during microdialysis sampling:

Implications for in vitro dissolution-/permeation-studies

Sebastian Koplin, Mont Kumpugdee-Vollrath, Annette Bauer-Brandl, Martin Brandl

ABSTRACT

Aim of this project was to investigate the applicability of a recently developed in vitro microdialysis-

sampling approach in connection with a dissolution-/permeation (D/P) system, especially the impact of

surfactants within the perfusion fluid. The D/P-system is based on side-by-side chambers, separated by

a barrier that simulates the intestinal barrier. Here, in contrast to conventional D/P-systems, the

dissolution of the drug (donor chamber concentration) is followed by microdialysis sampling. This

approach appears promising, because it is expected not to disturb the dynamic interplay between drug-

dissolution (-release) and drug permeation. Furthermore, it should allow quantification of the unbound

(free) drug concentration. In the first step, it was assessed, if the addition of the anionic surfactant

sodium dodecyl sulphate (SDS) to the perfusate of the microdialysis system affects the recovery of the

(slightly) water-soluble model drug acyclovir and the poorly water soluble model drug celecoxib

(CXB). SDS had no influence on acyclovir-recovery, but substantially enhanced CXB-recovery, partly

due to improved extraction efficiency, partly due to inhibition of loss of CXB due to non-specific

binding to surfaces and the probe. The fraction of CXB recovered from aqueous CXB-solutions by

microdialysis sampling using SDS-containing perfusates correlated well with the celecoxib

concentration in the samples, but was found independent of the SDS-concentrations (above critical

micelle concentration).

235

Simultaneous identification and quantification of polymethoxyflavones, coumarin and phenolic

acids in Ageratum conyzoides by UPLC-ESI-QToF-MS and UPLC-PDA

Larissa G. Faqueti, Louis P. Sandjo, Maique W. Biavatti

ABSTRACT

Ageratum conyzoides L. is a plant widely used in traditional medicine of tropical and subtropical

regions for its anti-inflammatory and antinociceptive properties. Nevertheless, the chemical

composition of its medicinal preparation has not been yet accurately established. In this study,

chromatographic methods were developed for the simultaneous identification and quantification of the

main compounds in A. conyzoides aqueous extract, using UPLC-PDA-ESI-QToF-MS. The qualitative

analyses defined by MS/MS analysis allowed the identification of 27 compounds in the aqueous

extract, including the toxic pyrrolizidine alkaloids, phenolic acids, coumarin and

polymethoxyflavones. Among the metabolites, twelve were detected for the first time in the species.

The quantitative method was validated according to the official guidelines, demonstrating to be

specific, linear, precise, accurate and sensitive for the quantification of chlorogenic acid, coumaric

acid, coumarin (1,2-benzopyranone), 5,6,7,3′,4′,5′-hexamethoxyflavone, nobiletin, 5′-methoxynobiletin

and eupalestin.

Degradation and metabolite formation of estrogen conjugates in an agricultural soil

Li Ma, Scott R. Yates

ABSTRACT

Estrogen conjugates are precursors of free estrogens such as 17ß-estradiol (E2) and estrone (E1),

which cause potent endocrine disrupting effects on aquatic organisms. In this study, microcosm

laboratory experiments were conducted at 25 °C in an agricultural soil to investigate the aerobic

degradation and metabolite formation kinetics of 17ß-estradiol-3-glucuronide (E2-3G) and 17ß-

estradiol-3-sulfate (E2-3S). The aerobic degradation of E2-3G and E2-3S followed first-order kinetics

and the degradation rates were inversely related to their initial concentrations. The degradation of E2-

3G and E2-3S was extraordinarily rapid with half of mass lost within hours. Considerable quantities of

E2-3G (7.68 ng/g) and E2-3S (4.84 ng/g) were detected at the end of the 20-d experiment, particularly

for high initial concentrations. The major degradation pathway of E2-3G and E2-3S was oxidation,

yielding the primary metabolites 17ß-estrone-3-glucuronide and 17ß-estrone-3-sulfate, respectively.

Common metabolites were E2, the second primary metabolite, and E1, the secondary metabolite.

Additionally, ring B unsaturated estrogens and their sulfate conjugates were tentatively proposed as

minor metabolites. The persistence of E2-3G and E2-3S (up to 20 d) suggests that the high rate of

application of conjugated estrogen-containing substances could be responsible for the frequent

detection of free estrogens in surface and subsurface water.

236

Chemical analysis and potential endocrine activities of aluminium coatings intended to be in

contact with cosmetic water

Elias Bou-Maroun, Laurence Dahbi, Marie-Pierre Gomez-Berrada, Philippine Pierre, Marie-Christine

Chagnon

ABSTRACT

The objective of the work was to check the presence of Non-Intended Added Substances (NIAS) with

hormonal activities in aluminium coatings extracts coded: AA, BBF, MC and RR, furnished by four

different suppliers. Water samples were prepared at room temperature or at 40 °C for three months to

verify the storage effect on the coatings. Solid phase extraction was used to concentrate and to extract

coating substances. Hormonal activities were checked in vitro using reporter gene bioassays. Except

BBF, all extracts induced a weak but significant estrogenic agonist activity in the human cell line.

Using an estrogenic antagonist (ICI-182, 780), the answer was demonstrated specific in the bioassay.

RR was the only extract to induce a concentration dependent anti-androgenic response in the MDA-

KB2 cell line. Analysis performed using GC–MS and HPLC–MS detected 12 substances in most of the

extracts. 8 NIAS were present. Among them, 4 were identified with certainty: HMBT, BGA, DCU and

BPA. Estrogenic potency was BPA > DCU > BGA > HMBT. HMBT was also anti-androgenic at high

concentration. Combining chemical analysis and bioassays data, we demonstrated that in the RR and

the RR40 extracts, the observed estrogenic response was mainly due to BPA, the anti-androgenic

activity of RR could be due to a synergism between HMBT and BPA. For MC and AA, estrogenic

responses appear to be due to the presence of DCU. Except BBF, storage conditions tended to increase

estrogenic activities in all extracts.

Identification and determination of related substances of ceftaroline fosamil in medicinal

product by high performance liquid chromatography with diode array detection and tandem

mass spectrometry

Izabela Karpiuk, Katarzyna Michalska, Katarzyna Bus, Monika Kiljan, Stefan Tyski

ABSTRACT

Ceftaroline fosamil, the prodrug of ceftaroline, is an advanced-generation cephalosporin antibacterial

agent approved for treatment in the European Union in 2012. The drug is dedicated to curing

complicated skin and soft tissue infections and community-acquired pneumonia. The developed

analytical method of high performance liquid chromatography (HPLC) followed by diode array

detector (DAD) set at 243 nm was used to identify and determine degradation products of ceftaroline

fosamil. The elaborated method demonstrated good selectivity and linearity [r > 0.998 for the range

0.8–1.2 mg/mL (80–120%) and r > 0.997 for the range 0.005–0.015 mg/mL (0.5–1.5%)]. Limit of

detection (LOD) and limit of quantification (LOQ) values were equal to 0.15 μg/mL and 0.5 μg/mL,

respectively. The forced decomposition of ceftaroline fosamil carried out under acidic, basic,

oxidative, photolytic and thermal conditions revealed the high sensitivity of ceftaroline on

photodegradation and thermolysis processes. Representative Ceftaroline samples were selected and

analysed by LC coupled with electrospray ionisation time-of-flight mass spectrometry (LC-ESI-Q-

TOF-MS) to identify the related substances that appeared under stress conditions. The LC-DAD

method was transferred without any modification to LC–MS/MS system, allowing it to correlate

results back to the LC-DAD method.

237

Quantification of concentrated Chinese medicine granules by quantitative polymerase chain

reaction

Yat-Tung Lo, Pang-Chui Shaw

ABSTRACT

Determination of the amount of constituent in a multi-herb product is important for quality control. In

concentrated Chinese medicine granules (CCMG), no dregs are left after dissolution of the CCMG.

This study is the first to examine the feasibility of using quantitative polymerase chain reaction

(qPCR) to find the amount of CCMG in solution form. DNA was extracted from Hirudo and Zaocys

CCMG mixed at different ratios and amplified in qPCR using species-specific primers. The threshold

cycle (CT) obtained was compared with the respective standard curves. Results showed that

reproducible quantification results could be obtained (1) for 5–50 mg CCMG using a modified DNA

extraction protocol, (2) amongst DNA extracted from the same batch of CCMG and (3) amongst

different batches of CCMG from the same company. This study demonstrated the constitute amount of

CCMG in a mixture could be determined using qPCR. This work has extended the application of DNA

techniques for the quantification of herbal products and this approach may be developed for quality

assurance in the CCMG industry.

Chiral separation of terbutaline and non-steroidal anti-inflammatory drugs by using a new

lysine–bridged hemispherodextrin in capillary electrophoresis

V. Cucinotta, M. Messina, A. Contino, G. Maccarrone, A. Giuffrida

ABSTRACT

A method for the separation of a mixture of terbutaline and non-steroidal anti-inflammatory drugs was

developed using capillary electrophoresis with a new hemispherodextrin, ad hoc designed, the lysine −

bridged hemispherodextrin (THLYSH). The use of lysine residues to bridge the trehalose capping unit

moiety to the cyclodextrin cavity gives rise to a receptor with two long chains with amine nitrogen

atoms, whose charge can be easily tuned as a function of the solution pH. The new hemispherodextrin

was accurately characterised by ESI–MS and NMR spectroscopy, also highlighting its protonation

behaviour. Circular dichroism and ESR spectroscopy measurements were also carried out to test its

inclusion ability towards anthraquinone-3-sulfonate and its metal coordination ability towards

copper(II) ion, respectively. Analogously to the other hemispherodextrins, the main skill of this new

derivative lies in its chiral selector properties, as shown by the separation of the enantiomeric pairs of

terbutaline and ibuprofen, flurbiprofen, suprofen and tiaprofenic acid by capillary electrophoresis. The

focused use of the solution equilibria involved in the separations made it possible to understand the

phenomena occurring in solution, and to finely tune the charge status of the receptor. In this way the

chiral separation of the racemic mixture was successfully obtained, even if the receptor was

individually used, differently by the other hemispherodextrins previously studied whose chiral

separation capabilities are present only if used as binary mixtures.

238

Separation and characterization of allergic polymerized impurities in cephalosporins by 2D-

HPSEC × LC-IT-TOF MS

Yu Xu, DanDan Wang, Lan Tang, Jian Wang

ABSTRACT

Eleven unknown allergic impurities in cefodizime, cefmenoxime and cefonicid were separated and

characterized by a trap-free two-dimensional high performance size exclusion chromatography

(HPSEC) and reversed phase liquid chromatography (RP-HPLC) coupled to high resolution ion

trap/time-of-flight mass spectrometry (2D-HPSEC × LC-IT-TOF MS) with positive and negative

modes of electrospray ionization method. Separation and characterization the allergic polymerized

impurities in β-lactam antibiotics were on the basis of column-switching technique which effectively

combined the advantages of HPSEC and the ability of RP-HPLC to identify the special impurities. In

the first dimension HPSEC, the column was Xtimate SEC-120 analytical column (7.8 mm × 30 cm, 5

μm), and the gradient elution used pH 7.0 buffer-acetonitrile as mobile phase And the second

dimension analytical column was ZORBAX SB-C18 (4.6 × 150 mm, 3.5 μm) with ammonium formate

solution (10 mM) and ammonium formate (8 mM) in [acetonitrile-water (4:1, v/v)] solution as mobile

phase. Structures of eleven unknown impurities were deduced based on the high resolution MSn data

with both positive and negative modes, in which nine impurities were polymerized impurities. The

forming mechanism of β-lactam antibiotic polymerization in cephalosporins was also studied. The

question on incompatibility between non-volatile salt mobile phase and mass spectrometry was solved

completely by multidimensional heart-cutting approaches and online demineralization technique,

which was worthy of widespread use and application for the advantages of stability and repeatability.

Phytochemical analysis and anti-inflammatory evaluation of compounds from an aqueous

extract of Croton cajucara Benth

Adamara M. Nascimento, Daniele Maria-Ferreira, Fernando T. Dal Lin, Alexandre Kimura, Lauro M.

de Souza

ABSTRACT

Croton cajucara Benth is a medicinal plant popularly used in the Brazilian Amazonia, where it is

known as sacaca, being consumed as tea, decoction or infusion of the leaves and stem bark. From a

decoction of the leaves, a comprehensive phytochemical analysis was developed by liquid

chromatography-mass spectrometry. Many compounds were identified for the first time in C. cajucara,

such as O-glycosides of kaempferol and quercetin, flavonoid-C-glycosides, tannins and cinnamic acid

derivatives. These compounds were fractionated by polarity and assayed for their anti-inflammatory

activity, using a model of mice edema, induced by an intraplantar injection of carrageenan. All

fractions exhibited anti-inflammatory properties.

239

Supercritical fluid chromatography approach for a sustainable manufacture of new

stereoisomeric anticancer agent

Alina Ghinet, Yasmine Zehani, Emmanuelle Lipka

ABSTRACT

Two routes aimed at the manufacture of unprecedented stereoisomeric combretastatin A-4 analogue

were described: flash chromatography vs supercritical fluid chromatography. The latter has many

advantages over liquid chromatography and was therefore chosen for the small scale separation of

methyl 1-[(3-hydroxy-4-methoxyphenyl) (3,4,5-trimethoxyphenyl)methyl]-5-oxo-l-prolinate 5, with

potential antitumoral activity. After a screening of six different polysaccharide based chiral stationary

phases and four co-solvents, the percentage of co-solvent, the flow-rate and the outlet pressure were

optimized through a design of experiments (DoE). The preparation of 50 mg of each stereoisomer was

achieved successfully on a Chiralpak AD-H with isopropanol as a co-solvent. Productivity (kkd),

solvent usage and environmental factor (E Factor) were calculated. Flash chromatography and

supercritical fluid chromatography approaches were compared in terms of yield and purity of each

stereoisomer manufactured.

Use of ionic liquids as headspace gas chromatography diluents for the analysis of residual

solvents in pharmaceuticals

Omprakash Nacham, Tien D. Ho, Jared L. Anderson, Gregory K. Webster

ABSTRACT

In this study, two ionic liquids (ILs), 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide

([BMIM][NTf2]) and trihexyltetradecylphosphonium bis[(trifluoromethyl)sulfonyl]imide ([P66614]

[NTf2]) were examined as contemporary diluents for residual solvent analysis using static headspace

gas chromatography (SHS-GC) coupled with flame ionization detection (FID). ILs are a class of non-

molecular solvents featuring negligible vapor pressure and high thermal stabilities. Owing to these

favorable properties, ILs have potential to enable superior sensitivity and reduced interference,

compared to conventional organic diluents, at high headspace incubation temperatures. By employing

the [BMIM][NTf2] IL as a diluent, a 25-fold improvement in limit of detection (LOD) was observed

with respect to traditional HS-GC diluents, such as N-methylpyrrolidone (NMP). The established IL-

based method demonstrated LODs ranging from 5.8 parts-per-million (ppm) to 20 ppm of residual

solvents in drug substances. The optimization of headspace extraction conditions was performed prior

to method validation. An incubation temperature of 140 °C and a 15 min incubation time provided the

best sensitivity for the analysis. Under optimized experimental conditions, the mass of residual

solvents partitioned in the headspace was higher when using [BMIM][NTf2] than NMP as a diluent.

The analytical performance was demonstrated by determining the repeatability, accuracy, and linearity

of the method. Linear ranges of up to two orders of magnitude were obtained for class 3 solvents.

Excellent analyte recoveries were obtained in the presence of three different active pharmaceutical

ingredients. Owing to its robustness, high throughput, and superior sensitivity, the HS-GC IL-based

method can be used as an alternative to existing residual solvent methods.

240

Adduct ion-targeted qualitative and quantitative analysis of polyoxypregnanes by ultra-high

pressure liquid chromatography coupled with triple quadrupole mass spectrometry

Xu Wu, Lin Zhu, Jiang Ma, Yang Ye, Ge Lin

ABSTRACT

Polyoxypregnane and its glycosides (POPs) are frequently present in plants of Asclepiadaceae family,

and have a variety of biological activities. There is a great need to comprehensively profile these

phytochemicals and to quantify them for monitoring their contents in the herbs and the biological

samples. However, POPs undergo extensive adduct ion formation in ESI-MS, which has posed a

challenge for qualitative and quantitative analysis of POPs. In the present study, we took the advantage

of such extensive adduct ion formation to investigate the suitability of adduct ion-targeted analysis of

POPs. For the qualitative analysis, we firstly demonstrated that the sodium and ammonium adduct ion-

targeted product ion scans (PIS) provided adequate MS/MS fragmentations for structural

characterization of POPs. Aided with precursor ion (PI) scans, which showed high selectivity and

sensitivity and improved peak assignment confidence in conjunction with full scan (FS), the

informative adduct ion-targeted PIS enabled rapid POPs profiling. For the quantification, we used

formic acid rather than ammonium acetate as an additive in the mobile phase to avoid simultaneous

formation of sodium and ammonium adduct ions, and greatly improved reproducibility of MS response

of POPs. By monitoring the solely formed sodium adduct ions [M+Na]+, a method for simultaneous

quantification of 25 POPs in the dynamic multiple reaction monitoring mode was then developed and

validated. Finally, the aforementioned methods were applied to qualitative and quantitative analysis of

POPs in the extract of a traditional Chinses medicinal herb, Marsdenia tenacissima (Roxb.) Wight et

Arn., and in the plasma obtained from the rats treated with this herb.

Quantitative determination of dobutamine in newborn pig plasma samples by HPLC–MS/MS

O.E. Albóniga, M.L. Alonso, M.E. Blanco, O. González, R.M. Alonso

ABSTRACT

A novel gradient reverse phase high performance liquid chromatography tandem mass spectrometry

(HPLC/MS-MS) was performed as a method for the determination of dobutamine hydrochloride

(DOB) in newborn pig plasma samples. It was developed and validated after optimization of sample

treatment and various chromatographic and mass spectrometric conditions. Trimethoxydobutamine

(TMD) was used as internal standard. Heptafluorobutyric acid (HFBA) and ethyl acetate were used for

the treatment of plasma samples. The separation of dobutamine and internal standard was done using a

Kinetex F5 (50 × 2.1 mm, 2.6 μm, 100 Å) analytical column. The mobile phase was a mixture of

acetonitrile and HCOOH 0.01%. The column oven temperature was optimized at 40° C and the flow

rate was 0.25 mL/min. DOB and TMD were detected by multiple reaction monitoring (MRM) mode in

ESI+, using a cone voltage (CV) of 25 V and a collision energy (CE) of 25 eV. The weighted

calibration curve (1/x2) was found to be linear over the concentration range of 1–100 ng/mL (r2 >

0.999). The limit of quantification (LLOQ) of the method was 1 ng/mL. The values of selectivity,

carryover, LLOQ, linearity, accuracy, precision, matrix effect, stability and recovery obtained meet the

acceptable range according to European Medicines Agency (EMA) and Food and Drug Administration

(FDA) guidelines. The method was efficiently applied to quantify DOB in plasma samples from a

pharmacokinetic/pharmacodynamic study in a disease model of newborn piglet.

241

An integrative investigation of the toxicity of Aconiti kusnezoffii radix and the attenuation effect

of its processed drug using a UHPLC-Q-TOF based rat serum and urine metabolomics strategy

Zhenyu Sui, Qing Li, Lin Zhu, Zhenru Wang, Kaishun Bi

ABSTRACT

Aconiti kusnezoffii radix (AKR), the root of Aconitum kusnezoffii Reichb., is commonly used in the

treatment of the rheumatoid arthritis. However, the clinical application is limited due to its potential

toxicity. Therefore, to investigate the mechanism of its potential neurotoxicity and nephrotoxicity, a

comprehensive metabolomics study combined with serum biochemistry and histopathology

measurements was carried out. A UHPLC-Q-TOF mass spectrometry based metabolomics approach

was applied to characterize the AKR toxicity, while the toxicity attenuation effects of Aconiti

kusnezoffii radix cocta (AKRC) on Wistar rats were also investigated. Two chromatographic

techniques involving reversed-phase chromatography and hydrophilic interaction chromatography

were combined for the serum and urine detection, which balanced the integrity and selectivity of the

two matrices. Principal component analysis was used to determine the groups, and principal

component analysis discriminant analysis was carried out to confirm the important variables. Then, the

developed integrative toxicity evaluation method was applied to assess the toxicity of AKR and the

attenuation effect of AKRC. The highly sensitive and specific toxic biomarkers, which can provide

practical bases were identified for the diagnosis of the neurotoxicity and nephrotoxicity induced by

AKR. In all, a total of 19 putative biomarkers were characterized, and related metabolic pathways were

identified. The study demonstrated that the established metabolomics strategy is a powerful approach

for investigating the mechanisms of herbal toxicity and the attenuation effect of a processing method

and would provide medical solutions for other toxic herbal medications and further clinical evidence

on how AKR improves symptoms of rheumatoid arthritis patients.

Calibration and validation of a MCC/IMS prototype for exhaled propofol online measurement

Felix Maurer, Larissa Walter, Martin Geiger, Jörg Ingo Baumbach, Sascha Kreuer

ABSTRACT

Propofol is a commonly used intravenous general anesthetic. Multi-capillary column (MCC) coupled

Ion-mobility spectrometry (IMS) can be used to quantify exhaled propofol, and thus estimate plasma

drug concentration. Here, we present results of the calibration and analytical validation of a MCC/IMS

pre-market prototype for propofol quantification in exhaled air. Calibration with a reference gas

generator yielded an R2 ≥ 0.99 with a linear array for the calibration curve from 0 to 20 ppbv. The

limit of quantification was 0.3 ppbv and the limit of detection was 0.1 ppbv. The device is able to

distinguish concentration differences > 0.5 ppbv for the concentration range between 2 and 4 ppbv and

> 0.9 ppbv for the range between 28 and 30 ppbv. The imprecision at 20 ppbv is 11.3% whereas it is

3.5% at a concentration of 40 ppbv. The carry-over duration is 3 min. The MCC/IMS we tested

provided online quantification of gaseous propofol over the clinically relevant range at measurement

frequencies of one measurement each minute.

242

Biochemical and functional analysis of corticotropin releasing factor purified from an aqueous

extract of human placenta used as wound healer

Namrata Singh, Debasish Bhattacharyya

ABSTRACT

Human placental extract constitutes of innumerable therapeutically important components mostly used

in wound healing arising from the skin and burn injuries. However, there is still some bioactive present

in the placental extracts yet to be characterized to better under the complex process of wound healing

mediated by the placental extract. In this study, the presence of corticotropin releasing factor (CRF) in

an aqueous extract of human placenta was detected and quantified by dot blot and CRF-ELISA

immunoassay kit respectively. Subsequently, it was purified by immuno-affinity chromatography and

quantified as 0.45 ± 0.05 μg of CRF per ml of placental extract where its molecular weight found to be

4.78 kDa by MALDI-TOF. To study functional analysis of CRF, an in vitro WI-38 lung fibroblast cell

scratch wound model was used which indicated proliferation, motility of cells after treatment with

purified CRF. Moreover, reduction in apoptosis rate of cells during closure of wound was observed

from microscopy studies and FACS analysis. Also, Antalarmin, an antagonist of CRF type 1 receptor

inhibited the wound closure potency of the purified component. Faster healing of wound with an

elevation of IL-6 and TGF-β during early stages of repair by placental CRF was observed on excision

rat model. The process of healing was accompanied by the decrease in the level of TNF-α and IFN-γ.

Toxic compounds from tobacco in placenta samples analyzed by UPLC-QTOF-MS

Somayeh Mohammadi, Celia Domeno, Isabel Nerin, Margarita Aznar, Cristina Nerin

ABSTRACT

Polycyclic aromatic hydrocarbons (PAHs), tobacco-specific nitrosamines (TSNAs) and aromatic

amines are carcinogens present in cigarette smoke. These compounds are distributed in the human

body and they could be transferred to the foetus during the pregnancy. Placenta is the main barrier to

these toxic compounds and its study is the objective of this work. A method based on solid-phase

extraction (SPE) with ultra-performance liquid chromatography−tandem quadrupole-time-of-flight

mass spectrometry (UPLC-QTOF-MS) has been examined and optimized for the analysis of 9 target

analytes (4 tobacco-specific nitrosamines and some of their metabolites, 3 aromatic amines, nicotine

and cotinine) in 26 placenta samples from smoking and non-smoking women. Limits of detection

(LODs) were in the range of 3–27 ng/g of placenta. Nicotine, cotinine, N-nitrosoanatabine (NAT) and

4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK) metabolite, 4-(methylnitrosamino)-1-(3-

pyridyl)-1-butanol (NNAL) were detected in the placenta samples of smoking woman. Nicotine was

detected in 3 out of 8 placentas from smoking women, always below the limit of quantification (88

ng/g). This could be expected, as the half-life of nicotine in the body is limited to about 0.5–3 h.

Cotinine, the main metabolite from nicotine, was detected in all placentas from smoking women at

concentrations between 17.2 and 61.8 ng/g, reaching the highest values for those women that smoked

the highest number of cigarettes. NAT and NNAL were detected in all placentas from smoking

women, always below the limit of quantification (40 ng/g and 33 ng/g respectively).

243

HPLC-HG-AFS determination of arsenic species in acute promyelocytic leukemia (APL) plasma

and blood cells

Meihua Guo, Wenjing Wang, Xin Hai, Jin Zhou

ABSTRACT

Arsenic trioxide (ATO) has been successfully used in the treatment of acute promyelocytic leukemia

(APL). To clarify the arsenic species in APL patients, high performance liquid chromatography-

hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) and HG-AFS methods were

developed and validated to quantify the plasma concentrations of inorganic arsenic (As(III) and As(V))

and methylated metabolites (MMA and DMA), and the total amounts of arsenic in blood cells and

plasma. Blood cells and plasma were digested with mixtures of HNO3H2O2 and analyzed by HG-

AFS. For arsenic speciation, plasma samples were prepared with perchloric acid to precipitate protein.

The supernatant was separated on an anion-exchange column within 6 min with isocratic elution using

13 mM CH3COONa, 3 mM NaH2PO4, 4 mM KNO3 and 0.2 mM EDTA-2Na. The methods provided

linearity range of 0.2–20 ng/mL for total arsenic and 2.0–50 ng/mL for four arsenic species. The

developed methods for total arsenic and arsenic species determination were precise and accurate. The

spiked recoveries ranged from 81.2%-108.6% and the coefficients of variation for intra- and inter-

batch precision were less than 9.3% and 12.5%, respectively. The developed methods were applied

successfully for the assay of total arsenic and arsenic species in 5 APL patients.

Quantification of IDP-73152, a novel antibiotic, in plasma from mice, rats and humans using an


pharmacokinetic studies

Myongjae Lee, Dohee Kim, Jeongcheol Shin, Hee-Yeol Lee, Suk-Jae Chung

ABSTRACT

IDP-73152, a novel inhibitor of a bacterial peptide deformylase, was recently approved as a new,

investigational drug in Korea for the clinical management of infections caused by Gram positive

bacteria. The objective of this study was to develop/validate a simple and robust analytical method for

the determination of IDP-73152 in plasma samples from rodents and humans, and to assess the

feasibility of the assay for use in pharmacokinetic studies using animal models. Plasma samples were

processed using a standard method for protein precipitation and an aliquot of the extract then injected

onto an UHPLC–MS/MS system. The drug and IDP-117293, an internal standard, were analyzed in

the positive ion-mode by electrospray ionization and quantified by monitoring the transition at m/z

555.2 → 245.2 for IDP-73152 and 563.3 → 253.1 for the internal standard, respectively. The lower

and upper limit of the assay was determined to be 5 and 10000 ng/ml, respectively, with an acceptable

linearity (R > 0.999) in the response-concentration relationship. Validation parameters, including

accuracy, precision, dilution, recovery, matrix effect and stability were found to be within the

acceptable ranges recommended by the assay validation guidelines of the United States FDA. The

method was successfully applied to the quantification of IDP-73152 in plasma from mice/rats that had

received a single oral administration of 80 mg/kg IDP-73152, in the form of the mesylate salt. These

findings suggest that the validated assay can be used in preclinical and clinical pharmacokinetic studies

of IDP-73152.

244

New approach for the diagnosis of histamine intolerance based on the determination of

histamine and methylhistamine in urine

Oriol Comas-Basté, M.Luz Latorre-Moratalla, Roberta Bernacchia, M.Teresa Veciana-Nogués,

M.Carmen Vidal-Carou

ABSTRACT

Histamine intolerance is a disorder in the homeostasis of histamine due to a reduced intestinal

degradation of this amine, mainly caused by diamine oxidase (DAO) enzyme deficiency, which

provokes its accumulation in plasma and the appearance of adverse health affects. A new approach for

the diagnosis of this intolerance could be the determination of histamine and its metabolites in urine.

The aim of this work was to develop and validate a rapid method to determine histamine and

methylhistamine in human urine by Ultra High Performance Liquid Chromatography and Fluorimetric

detection (UHPLC-FL). The proposed method is a consistent procedure to determine histamine and

methylhistamine in less than 11 min with adequate linearity and sensitivity. Relative standard

deviation was always lower than 5.5%, ensuring method precision; and mean recovery was greater

than 99% for both analytes. The structure of histamine and methylhistamine conjugated with OPA

were confirmed by UHPLC-ITD-FTMS which enabled to unequivocally identify both analytes in

standards and also in urine samples. The analysis of histamine and methylhistamine in urine samples

could be a potential new approach for the routine diagnosis of histamine intolerance, more patient-

friendly and with clear advantages in terms of equipment and personnel demand for sample collection

in comparison with current plasmatic DAO activity determination.

Development and validation of sensitive LC–MS/MS method for the quantification of SUVN-502

and its metabolite and its application for first in human pharmacokinetic study

Ramakrishna Nirogi, Devender Reddy Ajjala, Raghupathi Aleti, Lakshmiprasanna Rayapati, Naga

Surya Prakash Padala

ABSTRACT

A sensitive and rapid LC–MS/MS method was developed and validated for the quantification of

SUVN-502 and M1 of SUVN-502, a 5-HT6 receptor antagonist for the treatment of dementia

associated with Alzheimer‘s disease. Following solid-phase extraction, SUVN-502 and M1 of SUVN-

502 and IS were eluted with 10 mM ammonium acetate (pH 4.0) and acetonitrile using a rapid gradient

program on reverse phase column. Multiple reaction monitoring mode was used to monitor the

respective transitions of m/z 478.2 → 377.7 for SUVN-502 and m/z 464.1 → 377.7 for M1 of SUVN-

502. The assay exhibited a linear dynamic range of 10–10000 pg/mL for SUVN-502 and 20–20000

pg/mL for M1 of SUVN-502 in human plasma. Acceptable precision and accuracy were obtained for

concentrations over the standard curve range. The within batch accuracy and precision were within

acceptable limits. All the other validation parameters were within the acceptable limits. The validated

method was applied to analyze human plasma samples obtained from a human pharmacokinetic study

consisting single and multiple ascending doses.

245

Glycosylation patterns of selected proteins in individual serum and cerebrospinal fluid samples

Isabella Karlsson, Lorena Ndreu, Alessandro Quaranta, Gunnar Thorsén

ABSTRACT

A method we previously developed has been applied to the determination of the glycosylation pattern

of specific proteins in biological samples. Six proteins (alpha-1-antitrypsin, transferrin, haptoglobin,

C1 inhibitor, alpha-1 acid glycoprotein, and immunoglobulin G) were studied in serum samples from

five individuals and cerebrospinal fluid (CSF) samples from three individuals, to investigate the

expected normal distribution of glycosylation patterns and to assess whether this methodology can be

used to discriminate between samples from different individuals. For serum samples, the differences

were shown to be small, while much larger differences were found for the CSF samples, with a greater

number of glycoforms present. This can be linked to the occurrence of differential glycosylation in

proteins expressed in the brain compared with proteins expressed elsewhere in the body. The

developed method could distinguish differences in the glycosylation pattern of specific proteins in the

individual samples, which was not reflected in the glycan content of total CSF. This is the first time

that the glycoforms of several of these proteins have been investigated in CSF.

MIL-101(Cr)@GO for dispersive micro-solid-phase extraction of pharmaceutical residue in

chicken breast used in microwave-assisted coupling with HPLC–MS/MS detection

Yudan Wang, Xinpeng Dai, Xi He, Lin Chen, Xiaohong Hou

ABSTRACT

In this work, MIL-101(Cr)@GO (Graphite Oxide) was synthesized using a hydrothermal synthesis

method and was applied as a dispersive micro-solid-phase extraction (D-μ-SPE) sorbent for the

efficient concentration of four residual drugs (metronidazole, MNZ; tinidazole, TNZ;

chloramphenicol, CAP; sulfamethoxazole, SMX). Meanwhile, the extraction process was optimized by

combining it with microwave-assisted extraction. Factors affecting the D-μ-SPE efficiency, such as

selection of sorbent materials, pH of the sample solution, salting-out effect, amount of used material,

extraction time, desorption solvent and desorption time, were studied. Under the optimal extraction

conditions, the linearity ranged from 10 to 1000 ng kg−1 and 1–100 ng kg−1 (r2 ≥ 0.9928) for the

target analytes. The limits of detection were between 0.08 and 1.02 ng kg−1, and the limits of

quantitation were between 0.26 and 3.40 ng kg−1. Additionally, the developed method also exhibited

good precision (RSD ≤ 2.5%), repeatability (RSD ≤ 4.3%), high recoveries (88.9%–102.3%) and low

matrix effects (78.2%–95.1%). The proposed method proved to be an efficient and reliable approach

for the determination of the analytes. Finally, we successfully detected the four drugs in chicken

breast.

246

Development of a robust reporter gene assay to measure the bioactivity of anti-PD-1/anti-PD-L1

therapeutic antibodies

Lan Wang, Chuanfei Yu, Yalan Yang, Kai Gao, Junzhi Wang

ABSTRACT

Being regarded as the ‗cancer panacea‘, the anti-PD-1/anti-PD-L1 monoclonal antibodies (mAbs) have

become the R&D focus of biopharmaceutical industries. Several marketed such mAbs have been

proved particularly effective in treating various cancers. However, the cell-based bioassay to measure

the biological activities of the anti-PD-1/anti-PD-L1 mAbs as the lot release or stability test has been a

great challenge to quality control laboratories due to the immunomodulating nature of the mAbs. Here,

we describe the development and validation of a reporter gene assay consisting of two-cell systems to

measure the bioactivity of the anti-PD-1/anti-PD-L1 mAbs. We have generated two cell lines, the

CHO-PD-L1-CD3L cell line that stably expresses PD-L1 and the membrane-anchored anti-CD3 single

chain antibody fragment (scFv) named CD3L and the Jurkat-PD-1-NFAT cell line that stably

expresses PD-1 and the luciferase gene under the control of the NFAT response elements from the IL-

2 promoter. The results show good dose-dependent responsiveness to the mAbs and excellent

performance characteristics including specificity, accuracy and precision. The biological relevance of

the assay, the passage stability of the two cell lines, and the capability of measuring various anti-PD-

1/anti-PD-L1 mAbs render this assay applicable not only in lot release and stability test but also in

characterization and development of new anti-PD1/anti-PD-L1 mAbs.

LC–MS/MS-ESI method for simultaneous quantification of darolutamide and its active

metabolite, ORM-15341 in mice plasma and its application to a pharmacokinetic study

Sreekanth Dittakavi, Pavan Kumar V.S.P. Nagasuri, Suresh P. Sulochana, Syed Mohd Saim, Ramesh

Mullangi

ABSTRACT

A sensitive and rapid LC–MS/MS method was developed and validated for the simultaneous

quantitation of darolutamide and its active metabolite i.e. ORM-15341 in 50 μL mice plasma using

bicalutamide as an internal standard (I.S.) as per regulatory guidelines. Sample processing was

accomplished through liquid-liquid extraction. Chromatographic separation was achieved using an

Atlantis C18 column with an isocratic mobile phase comprising 0.2% formic acid:acetonitrile (35:65,

v/v) at a flow rate of 0.8 mL/min within 2.5 min. Detection and quantitation were done by multiple

reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 397 →

202, 395 → 202 and 429 → 255 for darolutamide, ORM-15341 and I.S, respectively in the negative

ionization mode. The calibration curve was linear from 0.61–1097 ng/mL for both darolutamide and

ORM-15341. The intra- and inter-day precisions were in the range of 1.34–13.8 and 4.85–12.9 and

3.91–13.7 and 6.54–14.2%, for darolutamide and ORM-15341, respectively. Darolutamide and ORM-

15341 were found to be stable under different stability conditions. The validated method was applied

to a pharmacokinetic study in mice.

247

Metabolite identification of AZD8055 in Sprague-Dawley rats after a single oral administration

using ultra-performance liquid chromatography and mass spectrometry

Md Mamunur Rashid, Hyun-A Oh, Hyunbeom Lee, Byung Hwa Jung

ABSTRACT

AZD8055 is an ATP-competitive specific dual mTOR inhibitor and exhibited potent antitumor activity

on several types of solid tumors. However, the metabolism of AZD8055 in the body still remains

unknown. In this study, metabolite identification of AZD8055 was performed using ultra high-

performance liquid chromatography-ion trap mass spectrometry (UHPLC-IT-MS) through both in

vitro and in vivo approaches using rat liver microsomes (RLMs) and rat plasma, urine and feces,

respectively. A total of eight putative metabolites (five phase I and three phase II) were identified, and

a tentative metabolic pathway was suggested for the first time. Considering the accurate mass and

mass fragmentations of the detected metabolites, their plausible structures were suggested.

Demethylation, hydroxylation, oxidation and morpholine ring opening were the major

biotransformation processes for the phase-I metabolism, while phase-II metabolites were merely

generated by the glucuronide conjugation reaction. The cumulative excretion of AZD8055 in urine and

feces was 0.13% and 1.11% of the dose, respectively. When the semi-quantitative analysis of the

metabolites was performed using UHPLC–MS/MS (ultra-performance liquid chromatography tandem

mass spectrometry) to evaluate the overall trend of metabolites formation and excretion, AZD8055

was excreted more in the form of the metabolites than itself and their formation was very fast.

Therefore it was presumed that biotransformation was playing a crucial role in its elimination.

Ultimately, this study provides novel insights regarding the in vitro and in vivo biotransformations of

AZD8055. Further investigations of metabolites of this potent anti-cancer compound could be

beneficial for the antitumor drug design and development process.

Quantification of 16 β-lactams in chicken muscle by QuEChERS extraction and UPLC-Q-

Orbitrap-MS with parallel reaction monitoring

Qing Chen, Xiao-Dong Pan, Bai-Fen Huang, Jian-Long Han

ABSTRACT

A method is described for the analysis of 16 β-lactams in chicken muscle by UPLC-quadrupole(Q)-

Orbitrap-MS with parallel reaction monitoring (PRM). QuEChERS approach includes clean-up step by

sorbent of primary-secondary amine (PSA) and C18 was adopted for sample preparation. Q-Orbitrap

with PRM showed high sensitivity with limits of detection (LODs) ranged from 0.01 μg kg−1 to 0.35

μg kg−1. The method was further validated by intra- and inter-day test with spiking levels less than

MRLs (maximum residue limits, the European Union). Recovery (83–112%) and precision values

(RSDs <15%) for all studied analytes were obtained. The result indicates that UPLC-Q-Orbitrap

coupled with QuEChERS preparation can serve as a routine quantification method for β-lactam

residues in chicken muscles.

248

Characterization of the phase I and phase II metabolic profile of tolvaptan by in vitro studies

and liquid chromatography–mass spectrometry profiling: Relevance to doping control analysis

Monica Mazzarino, Valeria Buccilli, Xavier de la Torre, Ilaria Fiacco, Francesco Botrè

ABSTRACT

Phase I and phase II biochemical reactions involved in the biotransformation pathways of tolvaptan

were characterized by LC–MS-based techniques and in vitro models to identify the most appropriate

marker(s) of intake. The effects of physiological and non-physiological factors on the metabolic profile

of tolvaptan were also evaluated. In vitro approaches were based on the use of pooled human liver

microsomes and recombinant isoforms of cytochrome P450 and uridine diphospho glucuronosyl-

transferase. Sample preparation included liquid/liquid extraction at neutral pH with tert-butyl methyl-

ether. In the case of the study of phase II metabolism an additional enzymatic hydrolysis step was

performed. The chromatographic separation was carried out using reversed-phase chromatography,

whereas detection was performed by either triple-quadrupole or time-of-flight analyzers in positive

electrospray ionization and different acquisition modes. Our data show that tolvaptan is metabolized to

at least 20 phase I metabolites, the biotransformation reactions being catalyzed mainly by CYP3A4

and CYP3A5 isoforms. The phase-I reactions include hydroxylation (in different positions),

carboxylation, oxidation, hydrogenation, dealkylation, isomerization and a combination of the above.

Most of the phase I metabolites undergo glucuronidation, carried out mostly by UGT2B7 and

UGT2B17 isoforms. Dealkylated, mono-hydroxylated and carboxylated metabolites both in the free

and in the glucuronidated form appear to be the most suitable urinary diagnostic markers for the

detection of tolvaptan intake in doping control.

An LC–MS/MS method for simultaneous determination of nine steroidal saponins from Paris

polyphylla var. in rat plasma and its application to pharmacokinetic study

Guangyi Yang, Wei Lu, Meng Pan, Chenning Zhang, Gao Song

ABSTRACT

Paris polyphylla var is an herbal plant herb widely used in Traditional Chinese Medicine. The purpose

of this study is to develop an Ultra Performance Liquid Chromatography-tandem mass spectrometer

(UPLC–MS) method to quantify the major components (i.e., nine saponins) from P. polyphylla in

plasma samples. A UItra BiPh column (100 × 2.1 mm, 5 μm) was used with acetonitrile/0.1% formic

acid in water as mobile phases. The analytes were quantified using a Waters XEVO TQ mass

spectrometer via multiple reaction monitoring (MRM) with positive scan mode. A protein precipitation

method was used to extract the analytes from rat plasma. The inter/intra-day precision, accuracy,

recovery, matrix effect, and stability were evaluated per the FDA guidance. The method showed

linearity in the concentration ranges of 2.4–1250 ng/mL. The intra-day and inter-day precisions (RSD)

of these analytes at three different levels were less than 15.0%. The extraction recoveries of these

analytes were from 83.8% to 109.4% and the matrix effects ranged from 87.4% to 105.4%. The

stabilities of these compounds in plasma were evaluated by analyzing three different concentrations

following storage at 25 °C for 6 h, and −80 °C for 30 days. All the samples displayed less than 15.0%

variations. The validated method was successfully used to a pharmacokinetic (PK) study using

Sprague Dawley (SD) rats with intravenous (i.v.) and oral (p.o.) administration of P. polyphylla

extract.

249

N-glucuronidation catalyzed by UGT1A4 and UGT2B10 in human liver microsomes: Assay

optimization and substrate identification

Danyi Lu, Qian Xie, Baojian Wu

ABSTRACT

N-glucuronidation is an important pathway for metabolism and disposition of tertiary amines in

humans. This reaction is mainly catalyzed by the enzymes UGT1A4 and UGT2B10. However, the

metabolic patterns of UGT1A4- and UGT2B10-mediated N-glucuronidation are not fully clear. In this

study, we first optimized in vitro reaction conditions for N-glucuronidation by using specific substrates

(i.e., trifluoperazine for UGT1A4, cotinine and amitriptyline for UGT2B10). Furthermore, we found

that hepatic N-glucuronidation showed significant species differences. In addition, UGT1A4 and

UGT2B10 were primarily responsible for N-glucuronidation of many tertiary amines, including

asenapine, loxapine, clozapine, chlorpromazine, dothiepin, doxepin, mirtazapine, mianserin,

chlorcyclizine, cyclizine, promethazine, cyclobenzaprine, imatinib, retrorsine, strychnine and brucine.

In conclusion, this study provides an in vitro assay system for evaluating N-glucuronidation of amines.

Also, UGT1A4- and UGT2B10-mediated N-glucuronidation might play significant roles in

metabolism and detoxification of tertiary amines in humans.

A simple high performance liquid chromatography–mass spectrometry method for Therapeutic

Drug Monitoring of isavuconazole and four other antifungal drugs in human plasma samples

Giovanna Fatiguso, Fabio Favata, Ilaria Zedda, Amedeo De Nicolò, Antonio D‘Avolio

ABSTRACT

Triazoles chanced the prevention and treatment of invasive fungal infections, but their

pharmacokinetic properties are still unclear. In particular, isavuconazole (ISC) is a new broad-

spectrum antifungal triazole approved in 2015 as first-line treatment for intravenous and oral use

against invasive aspergillosis and for mucormycosis. Nowadays, the optimal management of the

treatments with triazoles requires the use of Therapeutic Drug Monitoring (TDM), in order to prevent

sub-therapeutic or toxic concentrations. In turn, the routine use of TDM requires reliable quantification

methods The aim of this work was the development and full validation of a HPLC-mass spectrometry

assay for the simultaneous quantification of fluconazole, itraconazole, isavuconazole, posaconazole

and voriconazole in human samples. Both standards and quality controls were prepared in human

plasma. After the addition of internal standard (6,7-dimethyl-2,3-di(2-pyridyl)quinaxoline for

voriconazole, posaconazole and itraconazole; stable isotope labeled compounds for fluconazole and

isavuconazole), protein precipitation with acetonitrile and dilution with water were performed.

Chromatographic separation was performed on Atlantis® T3 5 μm 4.6 × 150 mm column, with a

gradient of water and acetonitrile, both added with 0.05% formic acid. Accuracy, intra-day and inter-

day imprecision fitted FDA and EMA guidelines, while matrix effects and recoveries resulted stable

between samples for each analyte. Stability results were in accordance with previously published data.

Finally, we tested this method by monitoring plasma concentrations in real patients and using external

quality controls with good results. This method resulted very simple, fast, cheap and very useful for

TDM application, to improve clinical management of antifungal therapy in critically ill patients.

250

Metabolic profiling of dehydrodiisoeugenol using xenobiotic metabolomics

Qian-Qian Lv, Xiao-Nan Yang, Dong-Mei Yan, Wei-Qing Liang, Fei Li

ABSTRACT

Dehydrodiisoeugenol (DDIE), a representative and major benzofuran-type neolignan in Myristica

fragrans Houtt., shows anti-inflammatory and anti-bacterial actions. In order to better understand its

pharmacological properties, xenobiotic metabolomics was used to determine the metabolic map of

DDIE and its influence on endogenous metabolites. Total thirteen metabolites of DDIE were identified

through in vivo and in vitro metabolism, and seven of them were reported for the first time in the

present study. The identity of DDIE metabolites was achieved by comparison of the MS/MS

fragmentation pattern with DDIE using ultra-performance chromatography electrospray ionization

quadrupole time-of-flight mass spectrometry (UPLC-ESI- QTOFMS). Demethylation and ring-

opening reaction were the major metabolic pathways for in vivo metabolism of DDIE. Recombinant

cytochrome P450 s (CYPs) screening revealed that CYP1A1 is a primary enzyme contributing to the

formation of metabolites D1-D4. More importantly, the levels of two endogenous metabolites 2,8-

dihydroxyquinoline and its glucuronide were significantly elevated in mouse urine after DDIE

exposure, which explains in part its modulatory effects on gut microbiota. Taken together, these data

contribute to the understanding of the disposition and pharmacological activities of DDIE in vivo.

Development and validation of a reliable method for thiopurine methyltransferase (TPMT)


genotypic correlations

Supaporn Wiwattanakul, Santirhat Prommas, Nuttawut Jenjirattithigarn, Siwalee Santon, Chonlaphat

Sukasem

ABSTRACT

A liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for the

determination of thiopurine methyltransferase (TPMT) activity in human whole blood lysate, based on

conversion of 6-mercaptopurine (6-MP) by TPMT to 6-methylmercaptopurine (6-MMP) using S-

adenosyl-l-methionine (SAM) as the methyl donor. This method was improved from the previous

laborious method for washing of red cell lysate preparation to develop whole blood EDTA lysate. In

addition, the TPMT incubation was optimized and the chromatography was performed in a short

runtime of 7 min on a C18-column by detection via triple quadrupole mass spectrometry. The MS/MS

was optimally tuned to monitor mass to charge a ratio (m/z) for 6-MMP 167.2 → 151.9 and the isotope

6-MMP-d3 with m/z of 170.5 → 152.2 were applied as an internal standard. The calibration curve

covered the range of 2.5–360 ng/ml and the correlation coefficient was greater than 0.999. The

accuracy of this method was determined in four concentrations of control of quality that ranged

between 99.33 and 106.33%. The intra-assay coefficient of variation (CV) was less than 4.41% and the

inter-assay was less than 5.43%. This method developed for measuring TPMT by LC–MS/MS is a

reliable, safe, and simple with a small volume requirement (100 μl of whole blood EDTA). The assay

was used to study TPMT activity in 132 Thai children with a range from 29.0 to 89.1 nmol 6-MMP/g

Hb/h with means and median values of TPMT activity 55.9 ± 12.47 nmol 6-MMP/g Hb/h and 54.2

nmol 6-MMP/g Hb/h.

251

Development and validation of a GC–MS method for the determination of hydroxyzine and its

active metabolite, cetirizine, in whole blood

Maria Katselou, Sotiris Athanaselis, Panagiota Nikolaou, Artemisia Dona, Ioannis Papoutsis

ABSTRACT

A simple, rapid, sensitive and accurate gas chromatography–mass spectrometric method was

developed and validated for the simultaneous determination of hydroxyzine and cetirizine in whole

blood. Solid-phase extraction procedure using Bond Elut LRC Certify II columns was used for the

isolation of hydroxyzine and cetirizine from 1 mL whole blood followed by derivatization with a

mixture of acetic anhydride:n-propanol (1:1, v/v). Limits of detection and quantification were 1.50 and

5.00 ng/mL, respectively. The assay was linear within the concentration range of 5.00–1000.0 ng/mL

and the correlation coefficient was R2 ≥ 0.993 for both analytes. Absolute recovery was determined at

three quality control concentration levels and was found to be at least 87.2% for both substances. Intra-

day and inter-day accuracy values for both hydroxyzine and cetirizine were ranged from −1.2 to 3.8%

and −2.7 to 2.0%, respectively, at the three concentration levels studied, whereas their respective intra-

day and inter-day precision values were less than 9.9 and 6.5%, respectively, in terms of relative

standard deviation (%RSD). The developed method was successfully applied for the quantification of

hydroxyzine and cetirizine concentrations in whole blood, during the investigation of clinical cases

where these two antihistamines were detected.

An on-spot internal standard addition approach for accurately determining colistin A and

colistin B in dried blood spots using ultra high-performance liquid chromatography–tandem

mass spectrometry

I-Lin Tsai, Ching-Hua Kuo, Hsin-Yun Sun, Yu-Chung Chuang, Yun-Jung Tsai

ABSTRACT

Outbreaks of multidrug-resistant Gram-negative bacterial infections have been reported worldwide.

Colistin, an antibiotic with known nephrotoxicity and neurotoxicity, is now being used to treat

multidrug-resistant Gram-negative strains. In this study, we applied an on-spot internal standard

addition approach coupled with an ultra high-performance liquid chromatography-tandem mass

spectrometry (LC–MS/MS) method to quantify colistin A and B from dried blood spots (DBSs). Only

15 μL of whole blood was required for each sample. An internal standard with the same yield of

extraction recoveries as colistin was added to the spot before sample extraction for accurate

quantification. Formic acid in water (0.15%) with an equal volume of acetonitrile (50:50 v/v) was used

as the extraction solution. With the optimized extraction process and LC–MS/MS conditions, colistin

A and B could be quantified from a DBS with respective limits of quantification of 0.13 and 0.27 μg

mL−1, and the retention times were < 2 min. The relative standard deviations of within-run and

between-run precisions for peak area ratios were all < 17.3%. Accuracies were 91.5-111.2% for lower

limit of quantification, low, medium, and high QC samples. The stability of the easily hydrolyzed

prodrug, colistin methanesulfonate, was investigated in DBSs. Less than 4% of the prodrug was found

to be hydrolyzed in DBSs at room temperature after 48 h. The developed method applied an on-spot

internal standard addition approach which benefited the precision and accuracy.

252

Comparative study of single/combination use of Huang-Lian-Jie-Du decoction and berberine on

their protection on sepsis induced acute liver injury by NMR metabolic profiling

Yan Lv, Junsong Wang, Dingqiao Xu, Shanting Liao, Lingyi Kong

ABSTRACT

Sepsis is a serious clinical disease with a high mortality rate all around the world. Liver organ

dysfunction is an important sign for the severity and outcome of sepsis in patients. In this study, 1H

NMR-based metabolomics approach and biochemical assays were applied to investigate the metabolic

profiling for cecal ligation and puncture (CLP) induced acute liver injury, the therapeutical effect of

single/combination use of Huang-Lian-Jie-Du decoction (HLJDD) and berberine, and the interaction

of them. Metabolomics analysis revealed significant perturbations in livers of septic rats, which could

be ameliorated by HLJDD, berberine and their combination treatment. Berberine could better rectified

glycolysis and nucleic acid metabolism in the liver. HLJDD had exceptional better anti-inflammatory,

antibacterial and antioxidative effects than berberine. The interaction of berberine and HLJDD could

further strengthen the anti-inflammation and anti-oxidation, but with poor effect on amino acids

metabolism. These findings highlighted the feasibility of the integrated NMR based metabolomics

approach to understand the pathogenesis of diseases, the action mechanisms of therapy and the herb-

drug interaction.

Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices:


separations

Ádám Tölgyesi, Enikő Barta, Andrea Simon, Thomas J. McDonald, Virender K. Sharma

ABSTRACT

Veterinary drugs containing synthetic anabolic steroid and nitroimidazole active agents are not allowed

for their applications in livestock of the European Union (EU). This paper presents analyses of twelve

selected steroids and six nitroimidazole antibiotics at low levels (1.56 μg/L–4.95 μg/L and 0.17 μg/kg–

2.14 μg/kg, respectively) in body fluids and egg incurred samples. Analyses involved clean-up

procedures, high performance liquid chromatography (HPLC) separation, and tandem mass

spectrometric screening and confirmatory methods. Target steroids and nitroimidazoles in samples

were cleaned by two independent supported liquid extraction and solid phase extraction procedures.

Separation of the selected compounds was conducted on Kinetex XB C-18 HPLC column using

gradient elution. The screening methods utilised supported liquid extraction that enabled fast and cost

effective clean-up. The confirmatory methods were improved by extending the number of matrices and

compounds, and by introducing an isotope dilution mass spectrometry for nitroimidazoles. The new

methods were validated according to the recommendation of the European Union Reference

Laboratories and the performance characteristics evaluated met fully the criteria. The methods were

applied to incurred samples in the proficiency tests. The obtained results of Z-scores demonstrated the

applicability of developed protocols of the methods to real samples. The confirmatory methods were

applied to the national monitoring program and natural contamination of prednisolone could be

detected in urine at low concentration in few samples.

253

Validation of an HPLC-UV method for analysis of Kaempferol-loaded nanoemulsion and its

application to in vitro and in vivo tests

Mariana Colombo, Gabriela de Lima Melchiades, Fabrício Figueiró, Ana Maria Oliveira Battastini,

Letícia Scherer Koester

ABSTRACT

A simple and reliable HPLC-UV method for Kaempferol (KPF) determination in a Kaempferol-loaded

nanoemulsion (KPF-NE), samples from mucosa permeation/retention studies, and murine brain was

developed and validated according to international guidelines. The analyses were performed on a

reversed-phase C18 column at 35 °C and under UV detection at 368 nm. The mobile phase was

composed of methanol:formic acid 0.1% (75:25, v/v) and was eluted at an isocratic flow rate of 1.0

mL/min. The method was selective and sensitive for KPF analysis in matrix extracts, and linear in the

range of 0.25–7.5 μg/mL. The method was also considered precise, accurate, and robust. The recovery

rates of KPF from the porcine nasal mucosa and murine brain were higher than 85%. Low matrix

effect was observed to determine KPF, including biological matrices. The applicability of the method

was confirmed in all different approaches, i.e., quantification of KPF in nanoemulsion, in vitro

permeation/retention of KPF across porcine nasal mucosa, and in vivo quantification of KPF in brain

samples after nasal administration in rats. Thus, the method is effective and reliable to determine KPF

in different real samples. The proposed method, therefore, provides a useful quantification approach to

routine processes, to the development of drug delivery systems, and to KPF quantification in different

biological matrices. Furthermore, the method is applicable in bioavailability studies and the developed

formulation (KPF-NE) is suitable for preclinical trials in different brain disorders.

Development of direct assays for Toxoplasma gondii and its use in genomic DNA sample

Lívia M. Alves, Vinícius R. Rodovalho, Ana C.H. Castro, Márcia A.R. Freitas, Ana G. Brito-Madurro

ABSTRACT

This work describes an approach for the selection and detection of specific DNA probes related to

Toxoplasma gondii, a protozoan parasite responsible for toxoplasmosis. The detection system was

developed on graphite carbon electrode modified with poly(3-hydroxybenzoic acid) sensitized with

ToxG1 probe. The hybridization of the specific genomic DNA related to T. gondii showed good

response by direct detection of guanine residue oxidation using differential pulse voltammetry (DPV).

The biosensor was able to distinguish both the complementary and non-complementary targets and

detect up to 100 ng μL−1 of the T. gondii genomic DNA. The hybridization (ToxG1: T. gondii

genomic DNA) was confirmed by optical measurement. Optical assays using gold

nanoparticles:ToxG1 probe showed a significant change in the absorbance peak in the presence of the

T. gondii genomic DNA according to the electrochemical results. This novel biosensor shows potential

as electrochemical transducer and was successfully applied in the biological sample.

254

Investigation and structural elucidation of a new impurity in bulk drug of cilostazol by

LC/MS/MS, FT-IR and NMR

Zhaoxia Hu, Sanguo Gao, Jue Gao

ABSTRACT

A new impurity was detected in bulk cilostazol (CIL) crude during routine analysis. The impurity

(∼4%, the specification for unknown impurity in crude is not more than 0.20%) has a relative retention

time of 1.46. Based on MS, NMR and IR spectral data, the impurity was identified as 6,6′-bis(4-(1-

cyclohexyl-1H-tetrazol-5-yl) butoxy)-3,3′,4,4′-tetrahydro-[7,7′-biquinoline]-2,2′(1H,1′H)-dione(CIL-

dimer). The precursor of CIL-dimer is an oxidative product of starting material 6-Hydroxy-3,4-

dyhydro-1H-quinolin–2-one(6-HQ), CIL-dimer was formed in the following reaction with 5-(3-

Chloro-propyl)-1-cyclohexyl-1H-tetrazol(CHCBT).

Selective screening of glutaric acid acidurias by capillary electrophoresis-mass spectrometry

Jaime Fernández-Bravo, Fernando de Andrés, Mohammed Zougagh, Ángel Ríos

ABSTRACT

A sensitive and selective method for the separation and quantification of the three organic acids 3-

hydroxy-3-methylglutaric acid, 3-methylglutaric acid, and glutaric acid in human urine samples by CE

with mass spectrometry detection has been developed. This methodology is faster, simpler and less

time-consuming, than other methodologies previously described, and requires of reduced amounts of

reagents as well. Samples are first filtered and then diluted in water. For the electrophoretic separation,

a 20 mM ammonium acetate and 10% methanol solution at pH 9.1 was selected as the running

electrolyte. With 5-s hydrodynamic injection, detection limits ranging from 15.5 to 39.3 μM and linear

responses ranging from the LOQ calculated for each analyte to more than 400 μM were obtained for

the analysis of the different organic acids in less than 13 min. Remarkable selectivity is achieved by

mass spectrometry detection using 0.25% of formic acid in 50% v/v 2-propanol-water solution as

sheath liquid, and enough sensitivity without interferences from the matrices was obtained as well.

This methodology has revealed as an efficient approach to help the 3-hydroxy-3-methylglutaric

aciduria diagnoses in order to discard or confirm the occurrence of the disease as of the presence or

absence of the expected increased levels of these analytes in samples of potential patients.

255

Online turbulent flow extraction coupled with liquid chromatography–tandem mass

spectrometry for high throughput screening of anabolic steroids in horse urine

Hyun Du Shin, Joon Hyuk Suh, Junghyun Kim, Hyun-Deok Cho, Sang Beom Han

ABSTRACT

A high throughput method for simultaneous screening of anabolic steroids and their metabolites (4-

esterendione, trenbolone, boldenone, oxandrolone, nandrolone, methandrostenolone, testosterone, 1-

androstendione, ethisterone, normethandrolone, methyltestosterone, 16β-Hydroxystanozolol,

epitestosterone, bolasterone, norethandrolone, danazol, stanozolol and androstadienone) in equine

urine by online turbulent flow extraction coupled with liquid chromatography-tandem mass

spectrometry was developed. The use of turbulent flow chromatography could simplify pretreatment of

horse urine, which has complex matrices as well as high viscosity. The urine was extracted by mixed-

mode cation exchange solid phase extraction, and hydrolyzed using β-glucuronidase/arylsulfatase.

Then, the sample was automatically loaded on the TurboFlow Cyclone extraction column for removal

of further matrix, followed by separation on a fused core C18 column before MS/MS detection.

Optimization and validation of the method were discussed in detail. All analytes were rapidly detected

within 10 min with high sensitivity (picogram to nanogram per milliliter level), and no interference

was observed. The linearity range was from 0.1–10 ng/mL for nine steroids and 1.0–50 ng/mL for the

others, with correlation of coefficient values over 0.995. Precision and accuracy ranged from 0.1 to

14.5% and 1.7 to 12.4%, respectively. The developed method was successfully applied to the analysis

of anabolic steroids in horse urine after administration of a model drug.

Development of a mixed-mode chromatography with tandem mass spectrometry method for the

quantitative analysis of 23 underivatized amino acids in human serum

Min Sun Choi, Shaheed Ur Rehman, In Sook Kim, Hi-Joon Park, Hye Hyun Yoo

ABSTRACT

In this study, a robust, selective and simplified method was developed and validated for the

simultaneous quantitative analysis of 23 underivatized amino acids in human serum using mixed-mode

chromatography with tandem mass spectrometry (LC–MS/MS). Serum samples were deproteinized

with acetonitrile and subjected to LC–MS/MS analysis. The chromatographic separation of amino

acids was achieved using a mixed-mode column (150 × 3 mm, 3 μm) with a gradient elution system;

the mobile phase consisted of 50 mM ammonium formate and 0.1% formic acid in acetonitrile. The

total run time was 22 min. Eluted compounds were detected in the electrospray ionization-positive

mode with multiple reaction monitoring. The validation study evaluated linearity, repeatability, intra

and inter-day accuracy and precision, and matrix effect. The validation results were satisfactory in all

the tested parameters. This method was successfully applied to the analysis of amino acids in the

clinical sample of human serum.

256

Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance

sensor

Atsushi Shoji, Yumiko Suenaga, Atsushi Hosaka, Yuuki Ishida, Masao Sugawara

ABSTRACT

We describe a simple method for evaluating the inhibition of collagen IV degradation by cathepsin B

with a surface plasmon resonance (SPR) biosensor. The change in the SPR signal decreased with an

increase in the concentration of cathepsin B inhibitors. The order of the inhibitory constant (Ki)

obtained by the SPR method was CA074Me ≈ Z-Phe-Phe-FMK < leupeptin. This order was different

from that obtained by benzyloxycarbonyl-Phe-Phe-Fluoromethylketone (Z-Phe-Phe-FMK) as a peptide

substrate. The comparison of Ki suggested that CA074 and Z-Phe-Phe-FMK inhibited exopeptidase

activity, and leupeptin inhibited the endopeptidase activity of cathepsin B more strongly.

Liquid chromatography-tandem mass spectrometry assay to quantify plitidepsin in human

plasma, whole blood and urine

L. van Andel, H. Rosing, S. Fudio, P. Avilés, J.H. Beijnen

ABSTRACT

Plitidepsin is an anti-cancer drug currently evaluated in phase I/II/III clinical trials. This article

describes the development and validation of a bioanalytical assay to quantify plitidepsin in human

plasma, urine and whole blood using HPLC–MS/MS. The analyte was extracted from the matrix by

liquid–liquid extraction using tert-butyl methyl ether. Final extracts were injected onto a C18 column,

gradient elution was applied for chromatographic separation and detection was performed on a triple

quadrupole mass spectrometer operating in the positive ion mode. The assay was linear over the range

0.1–100 ng/mL, with acceptable accuracy and precision values. This is the first reported bioanalytical

assay quantifying plitidepsin using a stable isotopically labelled standard, achieving a lower limit of

quantification of 0.1 ng/mL in all three matrices, allowing the quantification of trace level of

plitidepsin, and accomplishing this in an analysis time of two minutes only. The presented method was

successfully applied in a mass balance study with plitidepsin in patients with advanced cancer.

257

Rapid analysis of benzalkonium chloride using paper spray mass spectrometry

Jingjing Liu, Wenjie Deng, Muqian Yu, Ruizhi Wen, Bo Chen

ABSTRACT

A paper spray mass spectrometry (PS-MS) method for rapid and reliable analysis of benzalkonium

chloride (BAC) in compound eye drops and body surface disinfectant was developed. The sample was

dropped onto triangular filter paper, and high voltage (3.5 kV) was applied to form an electrospray.

This method can provide the composition of benzalkonium chloride in samples without pretreatment,

solvent or chromatographic separation, and the analysis time is only 10 s. The primary homologues

C12-BAC, C14-BAC and C16-BAC of benzalkonium chloride were quantitatively analyzed using PS-

MS. Samples were subjected to simple dilution and quantified using the internal standard method. Ion

trap mass spectrometry was scanned using SIM mode. The linear ranges of C12-BAC, C14-BAC and

C16-BAC were 1–100 μg mL−1; the linear regression coefficients were 0.998–0.999; the detection

limits (LODs) were 0.1 μg mL−1; the limit of quantifications (LOQs) was <1 μg mL−1, and the

method validation indicated that the method precision and accuracy were good. Compared with HPLC-

UV methods, there was no significant difference in the quantitative determination of the actual

samples, but the analysis time for PS-MS is shorter (2 min). In addition, reagent consumption in PS-

MS is small, and no chromatographic separation is needed, suggesting that PS-MS is especially

suitable for high-throughput analysis.

Rapid screening of non-steroidal anti-inflammatory drugs illegally added in anti-rheumatic

herbal supplements and herbal remedies by portable ion mobility spectrometry

Mengjiao Li, Haiyan Ma, Jinglin Gao, Lina Zhang, Ye Jiang

ABSTRACT

In this work, for the first time, a high-performance ion mobility spectrometry with electrospray

ionization (ESI-HPIMS) method has been employed as a rapid screening tool for the detection of

acetaminophen, ibuprofen, naproxen, diclofenac sodium and indomethacin illegally added in anti-

rheumatic herbal supplements and herbal remedies. Samples were dissolved and filtered through a 0.45

μm microporous membrane, then the filtrate was directly injected into the high-performance ion

mobility spectrometry for analysis. Using this approach, the screening of illegal additions can be

accomplished in as rapid as two to three minutes with no pretreatment required. The proposed method

provided a LOD of 0.06–0.33 μg mL−1, as well as a good seperation of the five NSAIDs. The

precision of the method was 0.1–0.4% (repeatability, n = 6) and 0.9–3.3% (reproducibility, n = 3). The

proposed method appeared to be simple, rapid and highly specific, thus could be effective for the in-

situ screening of NSAIDs in anti-rheumatic herbal supplements and herbal remedies.

258

Simultaneous quantitative analysis of polyethylene glycol (PEG), PEGylated paclitaxel and


spectrometry

Heping Sun, Qi Zhang, Zhi Zhang, Jin Tong, Jingkai Gu

ABSTRACT

PEGylation is practically one of most important modifications of drugs including small molecules,

peptides and proteins, which has been proven to dramatically improve physicochemical properties and

pharmacokinetic behavior of the PEGylated drugs. However, it is a challenge currently to

quantitatively analyze PEG and PEGylated drugs by various analytical methods, even mass

spectrometry because of multiple parent ion distribution of PEG caused by its polydispersity of

molecular weight. Here we developed a robust method with MS/MSALL technique using electrospray

ionization (ESI) source coupled high resolution Quadrupole Time-of-Flight (Q-TOF) mass

spectrometry for the quantification of PEG2K-Paclitaxel (PEG-PTX) and its two metabolites, PEG and

Paclitaxel (PTX). The analysis was performed on a 300SB-C18 column with acetonitrile and 0.1%

formic acid as the mobile phase. Samples were simply prepared by protein precipitation in a small

quantity of plasma (50 μL). Calibration curve was linear within the range of 50.0–4000 ng/mL for

PEG and PEG-PTX and 1.0–1000 ng/mL for PTX. The intra- and inter-day precisions were 3.2–6.9%

and 3.1–6.9% for PEG, 4.1–7.8% and 4.0–9.9% for PEG-PTX, and 3.3–4.8% and 3.1–6.9% for PTX,

respectively. The recoveries were greater than 90% with low matrix effects. Afterwards, the newly

developed method was successfully applied to support a preclinical pharmacokinetic study in six rats

after single intravenous injection of PEG-PTX (51.7 mg/kg).

Lyophilic matrix method for dissolution and release studies of nanoscale particles

Jenni Pessi, Sami Svanbäck, Ilkka Lassila, Edward Hæggström, Jouko Yliruusi

ABSTRACT

We introduce a system with a lyophilic matrix to aid dissolution studies of powders and particulate

systems. This lyophilic matrix method (LM method) is based on the ability to discriminate between

non-dissolved particles and the dissolved species. In the LM method the test substance is embedded in

a thin lyophilic core-shell matrix. This permits rapid contact with the dissolution medium while

minimizing dispersion of non-dissolved particles without presenting a substantial diffusion barrier. The

method produces realistic dissolution and release results for particulate systems, especially those

featuring nanoscale particles. By minimizing method-induced effects on the dissolution profile of

nanopowders, the LM method overcomes shortcomings associated with current dissolution tests.

259

Incorporation of 14C-cholesterol in human adrenal corticocarcinoma H295R cell line and

online-radiodetection of produced 14C-steroid hormone metabolites

Jonas Abdel-Khalik, Erland Björklund, Frederik Knud Nielsen, Martin Hansen

ABSTRACT

This study demonstrates the addition of 14C-cholesterol to the human cell line H295R will in-situ form

radiolabeled steroid hormones allowing for new mechanistic and metabolic insights. The aim of the

present study was to in-situ radiolabel steroid hormones from cell line-incorporated 14C-cholesterol

using the OECD guideline 456, H295R steroidogenesis in-vitro assay. Radiodetection of the steroid

metabolites of the steroidogenic pathway allows for an improved understanding of the various

enzymatic mechanisms involved without necessarily being dependent on quantification. Generated

radiolabeled steroids were analyzed using HPLC hyphenated with a Flow Scintillation Analyzer

(FSA). H295R cells were incubated with radiolabeled cholesterol and cell media were collected and

prepared by solid phase extraction and analyzed with HPLC-FSA. For successful radiolabeling of the

steroids in the steroidogenesis of H295R cells, radioactive cholesterol may potentially only need to be

added just before the cells are incubated for 72 h in well plates. Based on the obtained HPLC-FSA

chromatograms, and confirmation of the observations by studies in the literature, a qualitative time

profile for the production of steroid hormones was estimated. Multiple radiolabeled steroid hormones

were identified by means of analytical standards and UV (ultraviolet) co-chromatography, though the

elucidation of multiple metabolites remains unresolved. Although online radiodetection proved to

suffer from suboptimal sensitivity, the concept of radiolabeling the steroidogenesis in H295R cells

with 14C-cholesterol and detecting the radiolabeled steroid hormones online was proved and may

assist in further toxicological studies.

Verification of the effectiveness of the Fourier transform infrared spectroscopy computational

model for colorectal cancer

J. Depciuch, E. Kaznowska, A. Koziorowska, J. Cebulski

ABSTRACT

Colorectal cancer is one of the most common cancers. Its formation is influenced by genetic and

environmental factors. Despite the continuous development of diagnostic tools and cancer therapies,

there are no methods that allow a real-time estimation of treatment efficiency. This method can be a

vibrational spectroscopy. The resulting infrared spectrum (FTIR) of the tissue gives us information

about the chemical composition and the content of the individual components. We have noticed that

tumor tissues, healthy and after chemotherapy tissues, have different vibrational spectra. It was also

shown that spectra acquired from normal (benign) tissues were similar to those derived from tissues

post-chemotherapy. The similarity was greater, when the effectiveness of chemotherapy, confirmed by

medical documentation, was better. Therefore, we decided to use the physical model proposed in our

earlier paper to verify its correctness and to show whether a particular type of chemotherapy was

effective or not. Comparison of the results obtained from the physical model with patients data have

been found as close to the physical condition.

260

Quantification of paracetamol and 5-oxoproline in serum by capillary electrophoresis:

Implication for clinical toxicology

Tomáń Hloņek, Tomáń Kříņek, Petr Tůma, Miroslava Bursová, Radomír Čabala

ABSTRACT

High anion gap metabolic acidosis frequently complicates acute paracetamol overdose and is generally

attributed to lactic acidosis or compromised hepatic function. However, metabolic acidosis can also be

caused by organic acid 5-oxoproline (pyroglutamic acid). Paracetamol‘s toxic intermediate, N-acetyl-

p-benzoquinoneimine irreversibly binds to glutathione and its depletion leads to subsequent disruption

of the gamma glutamyl cycle and an excessive 5-oxoproline generation. This is undoubtedly an

underdiagnosed condition because measurement of serum 5-oxoproline level is not readily available. A

simple, cost effective, and fast capillary electrophoresis method with diode array detection (DAD) for

simultaneous measurement of both paracetamol (acetaminophen) and 5-oxoproline in serum was

developed and validated. This method is highly suitable for clinical toxicology laboratory diagnostic,

allowing rapid quantification of acidosis inducing organic acid 5-oxoproline present in cases of

paracetamol overdose. The calibration dependence of the method was proved to be linear in the range

of 1.3–250 μg mL−1, with adequate accuracy (96.4–107.8%) and precision (12.3%). LOQ equaled 1.3

μg mL−1 for paracetamol and 4.9 μg mL−1 for 5-oxoproline.

Quantification of cyclocreatine in mouse and rat plasma using hydrophilic-interaction ultra-

performance liquid chromatography-tandem mass spectrometry

Amy Q. Wang, Emma Hughes, Wenwei Huang, Edward H. Kerns, Xin Xu

ABSTRACT

An accurate, rapid and selective method was developed to quantify cyclocreatine in mouse and rat

plasma using hydrophilic interaction (HILIC) ultra-performance liquid chromatography-tandem mass

spectrometry (UPLC–MS/MS). The plasma samples were prepared by protein precipitation with

acetonitrile:methanol (70:30). Chromatographic separation was performed on a HILIC BEH amide

column (2.1 mm × 50 mm, 1.7 μm) with a 3 min gradient elution at a flow rate of 0.5 mL/min. For

mass spectrometric detection, selected reaction monitoring (SRM) was used; the SRM transitions were

m/z 144 → 98 and m/z 144 → 56 for cyclocreatine and m/z 148 → 102 for the internal standard (D4-

cyclocreatine) in the positive ionization mode. No endogenous components interfered with the analysis

of cyclocreatine and the internal standard in mouse and rat plasma. Plasma calibration curves were

constructed in the range of 0.01–25 μM. The correlation coefficient of the calibration curves was

greater than 0.99. The mean intraday assay accuracy for all quality control (QC) replicates was

between 93 and 105%. The mean intraday assay precision (CV%) was 1.9-11% for all QC levels. The

HILIC–UPLC–MS/MS method was successfully applied in pharmacokinetic (PK) studies of

cyclocreatine in mice and rats for the first time. After a single 30 mg/kg oral administration in mice

and rats, the AUC0-∞ (area under the curve) was 84.1 μg h/mL and 91.7 ± 18.0 μg h/mL, respectively.

261

Volumetric adsorptive microsampling-liquid chromatography tandem mass spectrometry assay


validation and comparison with dried blood spot

Sebastiano Barco, Elio Castagnola, Andrea Moscatelli, James Rudge, Giuliana Cangemi

ABSTRACT

Summary: In this paper we show the development and validation of a volumetric absorptive

microsampling (VAMS™)-LC–MS/MS method for the simultaneous quantification of four antibiotics:

piperacillin-tazobactam, meropenem, linezolid and ceftazidime in 10 μL human blood. The novel

VAMS-LC–MS/MS method has been compared with a dried blood spot (DBS)-based method in terms

of impact of hematocrit (HCT) on accuracy, reproducibility, recovery and matrix effect. Antibiotics

were extracted from VAMS and DBS by protein precipitation with methanol after a re-hydration step

at 37 °C for 10 min. LC–MS/MS was carried out on a Thermo Scientific™ TSQ Quantum™ Access

MAX triple quadrupole coupled to an Accela ™UHPLC system. The VAMS-LC–MS/MS method is

selective, precise and reproducible. In contrast to DBS, it allows an accurate quantification without any

HCT influence. It has been applied to samples derived from pediatric patients under therapy. VAMS is

a valid alternative sampling strategy for the quantification of antibiotics and is valuable in support of

clinical PK/PD studies and consequently therapeutic drug monitoring (TDM) in pediatrics.

PLGA Ethionamide Nanoparticles for Pulmonary Delivery: Development and in vivo evaluation

of dry powder inhaler

Sujit Kumar Debnath, Srinivasan Saisivam, Abdelwahab Omri

ABSTRACT

PLGA (50:50) nanoparticles were prepared to sustain the release of Ethionamide in order to decrease

the dose and dosing frequency. It further modified in the form of dry powder inhaler to make suitable

for pulmonary administration and increase drug residency in lungs. Ethionamide loaded PLGA

nanoparticles were prepared by solvent evaporation method. Freeze dried nanoparticles and anhydrous

inhalable grade lactose were mixed manually using geometrical dilution process to modify the

nanoparticles in the form of dry powder inhaler. Animal study was conducted to correlate between in-

vivo and in-vitro. PLGA nanoparticles showed initial burst release followed by zero order release up to

95.17 ± 3.59% in 24 h. Aerodynamic particle size of optimized dry powder inhaler was found as 1.79

μm. There was no significant aggregation of dry powder inhaler during 6 months of stability study.

Area under the concentration-time curve from 0 h to infinity (AUC0−∞) signifies the prolong

residency of ETH in body compartment, revealed from animal study. PLGA 50:50 coated

nanoparticles released Ethionamide for the period of 24 h in simulated lungs fluid. Correlation

between in-vitro dissolution and in-vivo study was established after performing animal study. Prepared

dry powder inhaler maintained Ethionamide concentration above minimum inhibitory concentration

for more than 12 h after single dose administration.

262

Simultaneous determination and pharmacokinetics of danshensu, protocatechuic aldehyde, 4-


MS/MS after oral administration of Compound Danshen Dripping Pills

Wei Li, Hongjie Zhou, Yang Chu, Xiangyang Wang, He Sun

ABSTRACT

Compound Danshen Dripping Pills (CDDP), a herbal patent medicine, is widely used in China for the

prevention and treatment of cardiovascular diseases. A simple, sensitive and reliable method for

simultaneous determination of danshensu (DSS), protocatechuic aldehyde (PCA), and their related

metabolites, 4-hydroxy-3-methyloxyphenyl lactic acid (HMLA) and protocatechuic acid (PAA) in

human plasma was developed and validated based on liquid chromatography tandem mass

spectrometry (LC–MS/MS). The analytes and internal standard (IS), vanillic acid (VAA), were

extracted from plasma with ethyl acetate and separated on a C18 column by using the mobile phase

consisted of methanol-0.1% formic acid via gradient elution. The electrospray ionization (ESI) source

was applied and operated under the multiple reaction monitoring (MRM) mode. The linear calibration

curves were obtained at the concentration ranges of 0.46–1000 ng/mL for DSS and PAA, and 1.38–

1000 ng/mL for PCA and HMLA, respectively. The inter- and intra-day precisions (RSD%) were less

than 13.5%, and the accuracy (±RE%) was within 13.4%. The described method was successfully

applied for the clinical pharmacokinetics of CDDP in Chinese healthy volunteers.

LC–MS bioanalysis of Trastuzumab and released emtansine using nano-surface and molecular-


emtansine-treated breast cancer patients

Noriko Iwamoto, Akihiko Shimomura, Kenji Tamura, Akinobu Hamada, Takashi Shimada

ABSTRACT

Antibody-drug conjugates (ADCs) consist of monoclonal antibody and cytotoxic drugs covalently

attached via stable crosslinkers, and are prospective antibody drugs for cancer therapy. To cover the

overall pharmacokinetic understanding of ADCs, both the antibody and the released drugs are

necessary for practical clinical observation. The nano-surface and molecular-orientation limited

(nSMOL) proteolysis is a universal approach for antibody bioanalysis that enable Fab-selective

proteolysis, which maintains antibody sequence specificity while decreasing excess analyte peptides.

In this study, we describe quantitative assays for ADC in human plasma using nSMOL for the

antibody and polarity-selective liquid–liquid partition with a methanol/ethyl acetate mixed solvent for

the cytotoxic drugs. This approach led to the successful development of LC–MS validated bioanalysis

of the antibody and released drugs within 20% for lower limit of quantitation and 15% for another

concentration setting of Trastuzumab emtansine (T-DM1), Trastuzumab antibody and emtansine

conjugated with crosslinker (DM1-MCC). The validated concentration ranges in human plasma were

0.06–250 μg/mL for T-DM1 and 0.39–200 ng/mL for DM1-MCC. These results indicate that LC–MS

method with a two-sided approach, using nSMOL and liquid–liquid partition, show potential for the

precise pharmacokinetic study for ADC development and treatment.

263

Separation of furostanol saponins by supercritical fluid chromatography

Jie Yang, Lingling Zhu, Yang Zhao, Yongwei Xu, Baiping Ma

ABSTRACT

Supercritical fluid chromatography (SFC) has good separation efficiency and is suitable for separating

weakly polar compounds. Furostanol saponins, as an important kind of steroidal saponins, generally

have two sugar chains, which are polar and hydrophilic. The hydroxyl group at the C-22 position of

furostanol saponins is active and easily reacts with lower alcohols under appropriate conditions. The

separation of hydrophilic furostanol saponins was tested by SFC in this study. The effects of

chromatographic conditions on the separation of the mixed furostanol saponins and their hydroxyl

derivatives at the C-22 position were studied. The conditions for SFC, which included different

column polarity, modifier, additive, and column temperature, were tested. After optimization, the

mixed 10 similar structures of furostanol saponins were separated in 22 min on the Diol column at a

temperature of 40 °C. The mobile phase was CO2 (mobile phase A) and methanol (containing 0.2%

NH3∙H2O and 3% H2O) (mobile phase B). The backpressure was maintained isobarically at 11.03

MPa. SFC was found to be effective in separating the furostanol saponins that shared the same

aglycone but varied in sugar chains. SFC was sensitive to the number and type of sugars. The

resolution of furostanol saponin isomers was not ideal. The extract of Dioscorea zingiberensis C. H.

Wright was profiled by SFC–quadrupole time-of-flight mass spectrometry. The main saponins of the

extract were well separated. Therefore, SFC could be used for separating hydrophilic furostanol

saponins and analyzing traditional Chinese medicines that mainly contained steroidal saponins.

Macroporous monoliths for biodegradation study of polymer particles considered as drug

delivery systems

M.V. Volokitina, V.A. Korzhikov-Vlakh, T.B. Tennikova, E.G. Korzhikova-Vlakh

ABSTRACT

Nanostructures based on biodegradable polymers are often considered as drug delivery systems. The

properties of these nanomaterails towards in vitro biodegradation are very important and usually are

studied using the model physiological conditions. In this work the novel approach based on application

of monolithic immobilized enzyme reactors (IMERs) as the systems for biodegradation study of the

nanoobjects of different nature and morphology was suggested. Rigid nanospheres based on

poly(lactic acid) and self-assembled nanoobjects formed from block-copolymer of glutamic acid and

phenylalanine were applied as model nanomaterials. For that, two enzymes, namely, esterase and

papain were chosen for preparation of the monolithic IMERs. The properties of immobilized enzymes

were compared to those obtained for soluble biocatalysts in the reaction of poly(lactic acid) and

poly(glutamic acid) degradation. The monitoring of substrate destruction process was carried out using

different HPLC modes (anion-exchange, cation-exchange or precipitation-redissolution based process)

also based on application of the same modern stationary phase, namely, macroporous monoliths (CIM

disks and lab-made column). Finally, the applicability of monolithic immobilized enzyme reactors for

degradation of polyester and polypetide-based particles was demonstrated and compared to the process

observed in human blood plasma.

264

A novel enantioseparation approach based on liposome electrokinetic capillary chromatography

Xiaoqi Li, Yingxiang Du, Zijie Feng, Xiaodong Sun, Zhifeng Huang

ABSTRACT

As a novel separation mode of capillary electrophoresis (CE), liposome electrokinetic capillary

chromatography (LEKC) has aroused considerable attention in recent years; however, the

enantioseparation based on this new system has not been previously investigated. In this study, we

proposed a brand-new LEKC chiral separation approach using liposomes comprised of

phosphatidylcholine (PC) and cholesterol as pseudo-stationary phase and sulfobutyl ether-β-

cyclodextrin (SBE-β-CD) as chiral selector. Compared with the single CD system and CD-SDS-

MEKC system, this LEKC method presented an obviously preferable enantioseparation of four model

drugs (naproxen, warfarin, ketoprofen and amlodipine). In this new established system, all the

enantiomers represented baseline separations with the resolution and selectivity respectively achieving

1.584/1.067 (for naproxen), 2.226/1.045 (for warfarin), 1.537/1.038 (for ketoprofen) and 2.592/1.097

(for amlodipine), while other two comparative systems demonstrated no separation or a poor

separation. Several important parameters affecting the enantioseparation, such as buffer pH,

concentration of liposomes, phosphate buffer solution (PBS) and chiral selector (SBE-β-CD), and

applied voltage were systematically investigated. Satisfactory repeatability was achieved through intra-

day, inter-day and batch-to-batch investigations with relative standard deviations less than 3.40%.

Furthermore, the established method was successfully applied to test the chiral impurity of naproxen

sample.

Comparative study on the anticancer activities and binding properties of a hetero metal

binuclear complex [Co(dipic)2Ni(OH2)5]·2H2O (dipic = dipicolinate) with two carrier proteins

Somaye Shahraki, Fereshteh Shiri, Mostafa Heidari Majd, Zohreh Razmara

ABSTRACT

Recognizing of binding mechanisms between drugs and carrier proteins is basic for us to understand

the pharmacokinetics and pharmacodynamics of them. In this research, the anticancer activities of a

binuclear complex [Co(dipic)2Ni(OH2)5]·2H2O (dipic = dipicolinate) against MDA-MB-231 cell

lines were studied. Results of MTT assay and flow cytometry analysis revealed that above complex

can induce the cytotoxicity and the apoptosis in breast cancer cell lines. So, this complex was selected

to investigate its binding to human serum albumin (HSA) and bovine β-lactoglobulin (βLG) by

spectroscopic methods (UV–visible, fluorescence and FT-IR) along with molecular docking technique.

The fluorescence data showed Co-Ni complex quench the fluorescence of both proteins by a static

quenching mechanism and HSA has stronger binding affinity toward Co-Ni complex than βLG. The

binding constant (Kb), number of binding sites (n) and thermodynamic parameters were calculated and

showed that the Co-Ni complex binds to protein (HSA and βLG) through hydrogen bonding and van

der Waals forces with one binding site. The results of UV–visible measurements indicated that the

binding of above complex to HSA and βLG may induce conformational and micro-environmental

changes of studied proteins. Protein–ligand docking analysis confirmed that the Co-Ni complex binds

to residues located in the subdomain IIA of HSA and site II of βLG.

265

Characterization and quantitative analysis of phenolic derivatives in Longxuetongluo Capsule by

HPLC-DAD-IT-TOF-MS

Jing Sun, Yuelin Song, Hui Sun, Wenjing Liu, Jun Li

ABSTRACT

Longxuetongluo Capsule (LTC), which is derived from the total phenolic extract of Chinese dragon‘s

blood, has been proved to be safe as well as effective towards ischemic stroke. However, the effective

material basis remains unclear. The present study thereby focused on the clarification of the qualitative

and quantitative properties for the phenolic derivatives in LTC. Regarding homolog-focused chemical

profiling, the mass fragmentation patterns of the primary subtypes of phenolic compounds such as

homoisoflavanones, flavanes, chalcones, and flavonoid oligomers were summarized by assaying

authentic references with hybrid ion trap time-of-flight mass spectrometry, and the chemical structures

of 124 phenolic compounds, in total, were unambiguously or tentatively annotated in LTC by

matching the accurate mass spectral profiles with the proposed mass cracking rules and those reference

substances. Afterwards, simultaneous determination of 12 primary phenolic compounds was carried

out in different batches of LTC using HPLC-DAD, after that the method was proved to be accurate,

precise, and reproducible according to diverse method validation assays. The obtained findings are

expected to be meaningful for clarifying the effective substances and quality assessment of LTC.

Volume 145 October 2017

Two-dimensional liquid chromatography in pharmaceutical analysis Instrumental aspects,

trends and applications

Marion Iguiniz, Sabine Heinisch

ABSTRACT

The interest in two-dimensional liquid chromatography (2D-LC) has been growing up since the last

decades. This promising technique appears as a relevant solution for various analytical challenges

encountered in pharmaceutical analysis. The objective of this review is to give an overview of past,

current and emerging trends in 2D-LC techniques applied to pharmaceutical compounds. The

referenced studies cover the late 1980s to the present. Information regarding the different aspects of

this analytical technique, including chromatographic conditions, instrumental setup and compounds of

interest, was compiled and summarized into a synoptic table. Particular attention is paid to key features

including (i) the interfaces used for coupling the two dimensions, (ii) the application fields, and (iii)

the chromatographic modes that can be combined together. Finally an attempt is made to predict future

advances in two-dimensional separation techniqes for pharmaceutical analysis.

266

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A

generic method development approach

Balázs Bobály, Valentina D‘Atri, Alain Beck, Davy Guillarme, Szabolcs Fekete

ABSTRACT

Hydrophilic interaction liquid chromatography (HILIC) is a well-established technique for the

separation and analysis of small polar compounds. A recently introduced widepore stationary phase

expanded HILIC applications to larger molecules, such as therapeutic proteins. In this paper, we

present some generic HILIC conditions adapted for a wide range of FDA and EMA approved

recombinant monoclonal antibody (mAb) species and for an antibody-drug conjugate (ADC). Seven

approved mAbs possessing various isoelectric point (pI) and hydrophobicity as well as a cysteine

conjugated ADC were used in this study. Samples were digested by IdeS enzyme and digests were

further fragmented by chemical reduction. The resulting fragments were separated by HILIC. The

main benefit of HILIC was the separation of polar variants (glycovariants) in a reasonable analysis

time at the protein level, which is not feasible with other chromatographic modes. Three samples were

selected and chromatographic conditions were further optimized to maximize resolution. A

commercial software was used to build up retention models. Experimental and predicted

chromatograms showed good agreement and the average error of retention time prediction was less

than 2%. Recovery of various species and sample stability under the applied conditions were also

discussed.

A new platform for serological analysis based on porous 3-dimensional polyethylene sinter

bodies

Mohammed Alasel, Michael Keusgen

ABSTRACT

A new sensitive and selective platform, three-dimensional immunosensor, has been developed for a

rapid serological diagnosis; detection of a Borrelia infection was considered as a model assay. The

immunosensor is based on a 3-dimensional (3D) porous solid surface (sinter body) with dimensions of

2 × 2.5 mm where a recombinant variable lipoprotein surface-exposed protein (VlsE; Borrelia-antigen)

is immobilized by different techniques. The sinter body served as a robust and inexpensive carrier,

which facilitated a successful hydrophobic adsorption as well as covalent immobilization of the

antigen with sufficient amounts of on the surface. Because of sinter body‘s porosity, the detection

could be performed in an immune affinity flow system based on a little disposable plastic column. The

flow of reagents through the column is advantageous in terms of reducing the non-specific interaction

and shortening the test time. Furthermore, three labels were tested for a colorimetric detection: i) a

horseradish peroxidase (HRP) labeled secondary antibody, ii) nanoparticles based on Sudan IV, and

iii) gold nanoparticles modified with protein A. HRP secondary labeled antibody provides the most

sensitive test, 1000 fold dilution of serum sample can be clearly detected in only 20 min. Gold

nanoparticles modified with protein A were used as a direct label or as a catalyst for reduction of silver

ions. Direct detection with gold nanoparticles provides short time of analysis (5 min) while detection

of metallic silver required longer time (12 min) but with improved sensitivity. Nanoparticles based on

Sudan IV showed high background and were less favorable. The assay is distinctive because of the

rapid analysis time with all used labels, longest 20 min. Compared to classical serological methods for

Borrelia diagnosis, the developed method offers a simple, rapid and reliable tool of analysis with

minimal cost and can be easily transferred to other infectious diseases.

267

Designing a calibration set in spectral space for efficient development of an NIR method for

tablet analysis

Md Anik Alam, James Drennen, Carl Anderson

ABSTRACT

Designing a calibration set is the first step in developing a multivariate spectroscopic calibration

method for quantitative analysis of pharmaceutical tablets. This step is critical because successful

model development depends on the suitability of the calibration data. For spectroscopic-based

methods, traditional concentration based techniques for designing calibration sets are prone to have

redundant information while simultaneously lacking necessary information for a successful calibration

model. A method for designing a calibration set in spectral space was developed. The pure component

spectra of a tablet formulation were used to define the spectral space of that formulation. This method

maximizes the information content of measurements and minimizes sample requirements to provide an

efficient means for developing multivariate spectroscopic calibration. A comparative study was

conducted between a commonly employed full factorial approach to calibration development and the

newly developed technique. The comparison was based on a system to quantify a model drug,

acetaminophen, in pharmaceutical compacts using near infrared spectroscopy. A 2-factor full factorial

design (acetaminophen with 5 levels and MCC:Lactose with 3 levels) was used for calibration

development. Three replicates at each design point resulted in a total of 45 tablets for the calibration

set. Using the newly developed spectral based method, 11 tablets were prepared for the calibration set.

Partial least square (PLS) models were developed from respective calibration sets. Model performance

was comprehensively assessed based on the ability to predict acetaminophen concentrations in

multiple prediction sets.

On-line prediction of the glucose concentration of CHO cell cultivations by NIR and Raman

spectroscopy: Comparative scalability test with a shake flask model system

Bence Kozma, Edit Hirsch, Szilveszter Gergely, László Párta, András Salgó

ABSTRACT

In this study, near-infrared (NIR) and Raman spectroscopy were compared in parallel to predict the

glucose concentration of Chinese hamster ovary cell cultivations. A shake flask model system was

used to quickly generate spectra similar to bioreactor cultivations therefore accelerating the

development of a working model prior to actual cultivations. Automated variable selection and several

pre-processing methods were tested iteratively during model development using spectra from six shake

flask cultivations. The target was to achieve the lowest error of prediction for the glucose

concentration in two independent shake flasks. The best model was then used to test the scalability of

the two techniques by predicting spectra of a 10 l and a 100 l scale bioreactor cultivation. The NIR

spectroscopy based model could follow the trend of the glucose concentration but it was not

sufficiently accurate for bioreactor monitoring. On the other hand, the Raman spectroscopy based

model predicted the concentration of glucose in both cultivation scales sufficiently accurately with an

error around 4 mM (0.72 g/l), that is satisfactory for the on-line bioreactor monitoring purposes of the

biopharma industry. Therefore, the shake flask model system was proven to be suitable for scalable

spectroscopic model development.

268

Conventional and accelerated-solvent extractions of green tea (camellia sinensis) for

metabolomics-based chemometrics

Joshua J. Kellogg, Emily D. Wallace, Tyler N. Graf, Nicholas H. Oberlies, Nadja B. Cech

ABSTRACT

Metabolomics has emerged as an important analytical technique for multiple applications. The value of

information obtained from metabolomics analysis depends on the degree to which the entire

metabolome is present and the reliability of sample treatment to ensure reproducibility across the

study. The purpose of this study was to compare methods of preparing complex botanical extract

samples prior to metabolomics profiling. Two extraction methodologies, accelerated solvent extraction

and a conventional solvent maceration, were compared using commercial green tea [Camellia sinensis

(L.) Kuntze (Theaceae)] products as a test case. The accelerated solvent protocol was first evaluated to

ascertain critical factors influencing extraction using a D-optimal experimental design study. The

accelerated solvent and conventional extraction methods yielded similar metabolite profiles for the

green tea samples studied. The accelerated solvent extraction yielded higher total amounts of extracted

catechins, was more reproducible, and required less active bench time to prepare the samples. This

study demonstrates the effectiveness of accelerated solvent as an efficient methodology for

metabolomics studies.

Phase separation of in situ forming poly (lactide-co-glycolide acid) implants investigated using a

hydrogel-based subcutaneous tissue surrogate and UV–vis imaging

Yu Sun, Henrik Jensen, Nickolaj J. Petersen, Susan W. Larsen, Jesper Østergaard

ABSTRACT

Phase separation of in situ forming poly (lactide-co-glycolide acid) (PLGA) implants with agarose

hydrogels as the provider of nonsolvent (water) mimicking subcutaneous tissue was investigated using

a novel UV–vis imaging-based analytical platform. In situ forming implants of PLGA-1-methyl-2-

pyrrolidinone and PLGA-triacetin representing fast and slow phase separating systems, respectively,

were evaluated using this platform. Upon contact with the agarose hydrogel, the phase separation of

the systems was followed by the study of changes in light transmission and absorbance as a function of

time and position. For the PLGA-1-methyl-2-pyrrolidinone system, the rate of spatial phase separation

was determined and found to decrease with increasing the PLGA concentration from 20% to 40%

(w/w). Hydrogels with different agarose concentrations (1% and 10% (w/v)) were prepared for

providing the nonsolvent, water, to the in situ forming PLGA implants simulating the injection site

environment. The resulting implant morphology depended on the stiffness of hydrogel matrix,

indicating that the matrix in which implants are formed is of importance. Overall, the work showed

that the UV–vis imaging-based platform with an agarose hydrogel mimicking the subcutaneous tissue

holds potential in providing bio-relevant and mechanistic information on the phase separation

processes of in situ forming implants.

269

Mid-infrared and near-infrared spectroscopy for rapid detection of Gardeniae Fructus by a

liquid-liquid extraction process

Lingyan Tao, Zhonglin Lin, Jiashan Chen, Yongjiang Wu, Xuesong Liu

ABSTRACT

Gardeniae Fructus is widely used in the pharmaceutical industry, and many studies have confirmed its

medical and economic value. In this study, samples collected from different liquid-liquid extraction

batches of Gardeniae Fructus were detected by mid-infrared (MIR) and near-infrared (NIR)

spectroscopy. Seven analytes, neochlorogenic acid (5-CQA), cryptochlorogenic acid (4-CQA),

chlorogenic acid (3-CQA), geniposidic acid (GEA), deacetyl-asperulosidic acid methyl ester

(DAAME), genipin-gentiobioside (GGB), and gardenoside (GA), were chosen as quality property

indexes of Gardeniae Fructus. The two kinds of spectra were each used to build models by single

partial least squares (PLS). Additionally, both spectral data were combined and modeled by multiblock

PLS. For single spectroscopy modeling results, NIR had a better prediction for high-concentration

analytes (3-CQA, DAAME, GGB, and GA) whereas MIR performed better for low-concentration

analytes (5-CQA, 4-CQA, and GEA). The multiblock methodology was found to be better compared

to single spectroscopy models for all seven analytes. Specifically, the coefficients of determination

(R2) of the NIR, MIR, and multiblock PLS calibration models of all seven components were higher

than 0.95. Relative standard errors of prediction (RSEP) were all less than 7%, except for models of

GGB, which were 10.36%, 13.24%, and 8.15% for the NIR-PLS, MIR-PLS, and multiblock models,

respectively. These results indicate that MIR and NIR spectrographic techniques could provide a new

choice for quality control in industrial production of Gardeniae Fructus.

Quantitative analysis of a biopharmaceutical protein in cell culture samples using automated

capillary electrophoresis (CE) western blot

Dong Xu, Kentaro Marchionni, Yunli Hu, Wei Zhang, Zoran Sosic

ABSTRACT

An effective control strategy is critical to ensure the safety, purity and potency of biopharmaceuticals.

Appropriate analytical tools are needed to realize such goals by providing information on product

quality at an early stage to help understanding and control of the manufacturing process. In this work,

a fully automated, multi-capillary instrument is utilized for size-based separation and western blot

analysis to provide an early readout on product quality in order to enable a more consistent

manufacturing process. This approach aims at measuring two important qualities of a

biopharmaceutical protein, titer and isoform distribution, in cell culture harvest samples. The acquired

data for isoform distribution can then be used to predict the corresponding values of the final drug

substance, and potentially provide information for remedy through timely adjustment of the

downstream purification process, should the expected values fall out of the accepted range.

270

Synchronous characterization of carbohydrates and ginsenosides yields deeper insights into the

processing chemistry of ginseng

Shan-Shan Zhou, Jun Xu, Ming Kong, Ka-Man Yip, Hu-Biao Chen

ABSTRACT

Carbohydrates and ginsenosides in ginseng are biologically interrelated. Their synchronous analysis is

therefore essential in chemical research on ginseng to characterize its ―holistic‖ quality. Here we

investigated the processing chemistry of red ginseng (RG), a ginseng product processed by water-

steaming, for which both carbohydrates and ginsenosides were qualitatively and quantitatively

determined through multiple analytical techniques. Results revealed that the steam-processing not only

qualitatively and quantitatively altered the ginsenosides but also affected the polymeric carbohydrates

via changing their physiochemical parameters, i.e. water-solubility, molecular size, types and ratios of

constituent monosaccharides. Potential mechanisms involved in the transformation of ginseng

chemicals are proposed and discussed, including hydrolysis (deglycosylation, demalonylation,

deacetylation), dehydration, polymerization, volatilization, reduction and the Maillard reaction. The

study strengthens the research on the processing chemistry of RG, and therefore should be helpful for

elucidating the scientific basis of RG preparation and application.

Development and validation of an ICP-MS method for the determination of elemental impurities

in TP-6076 active pharmaceutical ingredient (API) according to USP 〈232〉/〈233〉

Osama Chahrour, John Malone, Mark Collins, Vrushali Salmon, Nick Dunwoody

ABSTRACT

The new guidelines of the United States pharmacopeia (USP), European pharmacopeia (EP) and

international conference on harmonization (ICH) regulating elemental impurities limits in

pharmaceuticals signify the end of unspecific analysis of metals as outlined in USP 〈231〉. The new

guidelines specify both daily doses and concentration/limits of elemental impurities in pharmaceutical

final products, active pharmaceutical ingredients (API) and excipients. In chapter USP 〈233〉

method implementation, validation and quality control during the analytical process are described. We

herein report the use of a stabilising matrix that overcomes low spike recovery problem encountered

with Os and allows the determination of all USP required elemental impurities (As, Cd, Hg, Pb, V, Cr,

Ni, Mo, Cu, Pt, Pd, Ru, Rh, Os and Ir) in a single analysis. The matrix was used in the validation of a

method to determine elemental impurities in TP-6076 active pharmaceutical ingredient (API) by ICP-

MS according to the procedures defined in USP〈233〉 and to GMP requirements. This validation

will support the regulatory submission of TP-6076 which is a novel tetracycline analogue effective

against the most urgent multidrug-resistant gram-negative bacteria. Evaluation of TP-6076 in IND-

enabling toxicology studies has led to the initiation of a phase 1 clinical trial.

271

Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1


Oluwatosin O. Dada, Romesh Rao, Natalie Jones, Nomalie Jaya, Oscar Salas-Solano

ABSTRACT

Fragmentation of monoclonal antibodies is a critical quality attribute routinely monitored to assess the

purity and integrity of the product from development to commercialization. Cleavage in the upper

hinge region of IgG1 monoclonal antibodies is a common fragmentation pattern widely studied by size

exclusion chromatography (SEC). Capillary electrophoresis with sodium dodecylsulfate (CE-SDS) is a

well-established technique commonly used for monitoring antibody fragments as well, but its

comparability to SEC in monitoring hinge fragments has not been established until now. We report a

characterization strategy that establishes the correlation between hinge region fragments analyzed by

SEC and CE-SDS. Monoclonal antibodies with elevated hinge fragments were generated under low pH

stress conditions and analyzed by SEC and CE-SDS. The masses of the fragments generated were

determined by LC-MS. Electrophoretic migration of the hinge fragmentation products in CE-SDS

were determined based on their mass values. Comparative assessment of fragments by SEC, and CE-

SDS showed similar correlation with incubation time. This study demonstrates that CE-SDS can be

employed as a surrogate technique to SEC for monitoring hinge region fragments. Most importantly,

combination of these techniques can be used to obtain comprehensive understanding of fragment

related characteristics of therapeutic protein products.

Revisiting blood-brain barrier: A chromatographic approach

Xavier Subirats, Laura Muñoz-Pascual, Michael H. Abraham, Martí Rosés

ABSTRACT

Drugs designed to reach a pharmacological CNS target must be effectively transported across the

blood-brain barrier (BBB), a thin monolayer of endothelial cells tightly attached together between the

blood and the brain parenchyma. Because of the lipidic nature of the BBB, several physicochemical

partition models have been studied as surrogates for the passive permeation of potential drug

candidates across the BBB (octanol-water, alkane-water, PAMPA...). In the last years, biopartition

chromatography is gaining importance as a noncellular system for the estimation of biological

properties in early stages of drug development. Microemulsions (ME) are suitable mobile phases,

because of their ease of formulation, stability and adjustability to a large number of compositions

mimicking biological structures. In the present work, several microemulsion liquid chromatographic

(MELC) systems have been characterized by means of the Abraham‘s solvation parameter model, in

order to assess their suitability as BBB distribution or permeability surrogates. In terms of similarity

between BBB and MELC systems (dispersion forces arising from solute non-bonded electrons,

dipolarity/polarizability, hydrogen-bond acidity and basicity, and molecular volume), the passive

permeability surface area product (log PS) for neutral (including zwitterions), fully and partially

ionized drugs was found to be well correlated with the ME made of 3.3% SDS (w/v; surfactant) 0.8%

heptane (w/v; oil phase) and 6.6% 1-butanol (w/v; co-surfactant) in 50 mM aqueous phosphate buffer,

pH 7.4.

272

Liquid chromatographic enantioseparation of carbocyclic β-amino acids possessing limonene

skeleton on macrocyclic glycopeptide-based chiral stationary phases

Tímea Orosz, Nóra Grecsó, Gyula Lajkó, Zsolt Szakonyi, Antal Péter

ABSTRACT

Polar-ionic and reversed-phase high-performance liquid chromatographic separations of limonene-

based cyclic β-amino acid enantiomers were carried out by using macrocyclic glycopeptide-based

chiral selectors applying Chirobiotic T, TAG and R columns. The effects of additives, concentration of

the co- and counter-ions and the temperature in polar-ionic mobile phase systems were studied. The

influence of pH, MeOH content and alcohol additives were investigated in the reversed-phase mode.

The difference in the change in standard enthalpy Δ(ΔH°), entropy Δ(ΔS°), and free energy Δ(ΔG°)

was calculated from the linear van't Hoff plots derived from the ln α vs 1/T curves in the temperature

range 5–40 °C. Unusual temperature behavior was observed on Chirobiotic TAG for most of the

analytes: decreased retention times were accompanied with increased separation factors with

increasing temperature, and separation was entropically-driven. For two of the studied analytes

enthalpically-driven enantioseparations were observed. The elution sequence was determined in all

cases, but no general rule could be established.

On-line coupling of molecularly imprinted solid phase extraction with liquid chromatography

for the fast determination of coumarins from complex samples

Andrea Machyňáková, Ivona Lhotská, Katarína Hroboňová, Dalibor Ńatínský

ABSTRACT

In this work, an on-line SPE-HPLC method with spectrophotometric detection was developed for the

determination of coumarins in complex samples. For the on-line cleanup of samples, a molecularly

imprinted polymer was packed into the column cartridge and coupled directly with HPLC (MISPE-

HPLC) using a column switching system. The separation of coumarins was performed on a C18 core-

shell column (100 × 4.6 mm, 5 μm) with a mobile phase consisting of 0.3% acetic acid/acetonitrile

with gradient elution at a flow-rate of 1 mL min−1. The total time of the whole analytical run,

including the extraction step, was 13.25 min. The on-line MISPE-HPLC method was optimized and

validated. The results showed good linearity (0.10–100 μg mL−1) with correlation coefficients higher

than 0.99. The LOD values were from 0.03 to 0.15 μg mL−1. The proposed method was successfully

applied for analysis of real samples (Cassia cinnamon, chamomile tea, and Tokaj specialty wines) and

obtained recoveries varied from 78.7% to 112.2% with an RSD less than 9%.

273

Metabolic profiling analysis of Siraitia grosvenorii revealed different characteristics of green

fruit and saccharified yellow fruit

Chengnan Fang, Qingqing Wang, Xinyu Liu, Guowang Xu

ABSTRACT

Siraitia grosvenorii is an economic and medicinal plant, its fruit is considered to be good to health for

its diverse bioactive ingredients. However, the clarification of chemical composition and their changes

after saccharification procedure are not well performed. In present study, a metabolomics method

based on ultra-high-performance liquid chromatography tandem quadrupole time-of-flight mass

spectrometry was developed for metabolic profiling acquisition of Siraitia grosvenorii extract.

Furthermore, information dependent analysis (IDA) combined with self-constructed LC–MS/MS

identification system for metabolites were employed to identify primary and secondary metabolites in

Siraitia grosvenorii. A total of 126 metabolites were identified or tentatively identified. The obvious

differences of metabolic profiling between green fruit and saccharified yellow fruit were observed, and

metabolites showed their own distribution characteristics in peel, flesh and seed. The majority of the

nutrients and effective components were more distributed in flesh and peel, and saccharification was

conducive to accumulation of sweet glycosides. This study not only expanded metabolite composition

information of Siraitia grosvenorii, but also specified distribution characteristics of identified

metabolites.

Comparison of α-glucosidase inhibitory effect and bioactive constituents of Anemarrhenae

Rhizoma and Fibrous Roots

Si-Hui Nian, Hui-Jun Li, E-Hu Liu, Ping Li

ABSTRACT

Comprehensive utilization of medicinal plant resources is of great significance for sustainable

development of traditional Chinese medicines. In the present study, the α-glucosidase inhibitory

activities of the rhizome and fibrous root of Anemarrhena Asphodeloides Bunge, were compared

detailedly, and a high performance liquid chromatography coupled with electrospray ionization tandem

triple quadrupole mass spectrometry (HPLC-QQQ/MS) method was developed for simultaneous

quantification of seven bioactive constituents including neomangiferin, mangiferin, isomangiferin,

timosaponin BII, timosaponin B, timosaponin AIII, and timosaponin N in 40 batches of samples. The

results demonstrated that fibrous root extracts had more potent α-glucosidase inhibitory activity than

rhizome extracts. Mangiferin and isomangiferin were abundant in fibrous root, while the analyzed

saponins were rich in rhizome. Based on the chemometrics methods including principal component

analysis (PCA), orthogonal partial least square discriminant analysis (OPLS-DA), and partial least

square (PLS), mangiferin and isomangiferin might be mainly responsible for α-glucosidase inhibitory

activity of the genus. These findings indicate that the established HPLC-QQQ/MS method was proven

to be useful and efficient for quality control of Anemarrhena materials, and fibrous root had the

potential to be utilized as anti-diabetic medicinal resource.

274

Characterization of forced degradation products of torasemide through MS tools and

explanation of unusual losses observed during mass fragmentation of drug and degradation

products through density functional theory

Moolchand Kurmi, Neha Patel, Shalu Jhajra, Prasad V. Bharatam, Saranjit Singh

ABSTRACT

Mass spectrometry tools (HRMS/LC-HRMS, MSn, and/or on-line H/D exchange) were employed to

establish mass fragmentation pattern of torasemide and to characterize its degradation products.

During collision-induced dissociation, multiple rearrangement processes and unusual losses of sulfur

(S), sulfanyl (HS), sulfur dioxide (SO2), sulphinic acid radical (HSO2), sulfur monoxide (SO), carbon

monoxide (CO), formyl radical (CHO) and C5H3NOS were observed. The same were successfully

explained by study of energy profiles, established by application of density functional theory (DFT).

Metabolic profiling of the traditional Chinese medicine formulation Yu Ping Feng San for the


in U937 cells

Stefanie Nikles, Marlene Monschein, Huiqin Zou, Yong Liu, Rudolf Bauer

ABSTRACT

Yu Ping Feng San (YPFS) is a classical TCM formulation which has been traditionally used for

treatment of immune system related diseases such as chronic bronchitis, allergic rhinitis and asthma.

The formula is a mixture of Radix Saposhnikoviae (Fangfeng), Radix Astragali (Huangqi), and

Rhizoma Atractylodis macrocephalae (Baizhu). TLC- and LC-DAD-ESI-MS/MS methods have been

developed for the analysis of the metabolic profiles of the single herbs and of the formula. Decoctions

and ASE extracts were analyzed in order to trace components of the individual herbs in YPFS. Nine

constituents of Radix Saposhnikoviae, ten constituents of Radix Astragali and five constituents of

Rhizoma Atractylodis macrocephalae have been assigned in the chemical profiles of the formula,

which now allow the standardisation of YPFS. The pharmacological testing showed that all extracts

significantly inhibited expression of TNF-α, IFN-γ, and IL-1β in U937 cells, while the inhibition of IL-

4 was consistently low. Compared to conventional analyses which are focused on a limited set of

compounds, metabolomics approaches, together with novel data processing tools, enable a more

holistic comparison of the herbal extracts. In order to identify the constituents which are relevant for

the immunomodulatory effects of the formula, metabolomics studies (PCA, OPLS-DA) have been

performed using UPLC/QTOF MS data.

275

A comprehensive stability-indicating HPLC method for determination of chloroquine in active

pharmaceutical ingredient and tablets: Identification of oxidation impurities

Ana Silva Coelho, Clara Elisa Pontes Chagas, Rodrigo Maia de Pádua, Gerson Antônio Pianetti,

Christian Fernandes

ABSTRACT

Malaria is the most common parasitic disease in humans. It is estimated that 3 billion people live under

the risk of contracting this disease in the world. Chloroquine (CQ) is the drug of choice to treat cases

of non-complicated malaria. Forced degradation studies are important to know the drug‘s potentials

degradation products and to develop a stability indicating method. Thus, chloroquine active

pharmaceutical ingredient (API), chloroquine tablets and placebo were submitted to a detailed forced

degradation study, using several stressing agents. The results were used on the development of a

stability indicating method, using high performance liquid chromatography. The method was validated

showing selectivity, precision, accuracy, robustness and linearity in the range of 30–360 μg/mL of

chloroquine. Chloroquine API and tablets were susceptible to alkaline hydrolysis with NaOH 1 mol/L,

and to oxidation with H2O2 3.0%. Two degradation products were formed in oxidative test. Kinetics

of chloroquine degradation in alkaline hydrolysis was performed for both API and tablets. The

calculated decay constant (k1) was 0.223 days−1 for API and 0.182 days−1 for tablets, while the half-

life (t1/2) was 3.1 days for API and 3.8 days for tablets. Chemical structures have been proposed for

the two degradation products formed in the presence of H2O2, using an UHPLC-UV-MS/MS

approach.

Identification of impurities in macrolides by liquid chromatography–mass spectrometric


retention relationship (QSRR)

Xia Zhang, Jin Li, Chen Wang, Danqing Song, Changqin Hu

ABSTRACT

Macrolides are multicomponent drugs whose impurity control is always a challenge demanding

analysis method with good sensitivity and selectivity. Three separate, sensitive, accurate liquid

chromatography tandem mass spectrometry methods (LC–MS) were developed for the measurement

of three 16-membered ring macrolides (josamycin, josamycin propionate and midecamycin acetate)

and related substances in commercial samples. The characteristics of impurities in macrolides were

summarized as useful guidance for the impurity analysis of this class of drugs. For each drug, a large

number of unknown components have been detected with the high-sensitive MS detector and possible

structures of the majority of them were postulated based on the summarized fragmentation rules of 16-

membered ring macrolides. A QSRR model was constructed by multilinear regression to predict the

retention times of identified impurities which were not detected by the LC–MS methods, without

obtaining their reference standards. Satisfactory performance was obtained during leave-one-out cross-

validation with a predictive ability (Q2) of 0.95. The generalisation ability of the model was further

confirmed by an average error of 2.3% in external prediction. The best QSRR model, based on eight

molecular descriptors, exhibited a promising predictive performance and robustness.

276

The impact of ZnO and TiO2 on the stability of clotrimazole under UVA irradiation:

Identification of photocatalytic degradation products and in vitro cytotoxicity assessment

Agata Kryczyk, Paweł Żmudzki, Paulina Koczurkiewicz, Joanna Piotrowska, Urszula Hubicka

ABSTRACT

In order to ensure the safe and effective use of pharmaceutical products especially for topical

administration photostability testing is necessary. The current paper presents an in-depth analysis of

the stability of one of the most common antifungal agents, namely clotrimazole. Clotrimazole has

proven to be stable under UVA irradiation in applied experimental conditions, but the presence of

catalysts such as ZnO and TiO2 has contributed significantly to the degradation of this compound. The

findings indicate that its photocatalytic degradation reactions followed the pseudo first-order kinetics

with rate constant depending on the pH and the used solvent. Using LC–MS/MS, 14 presumable

degradation products of clotrimazole were identified and the plausible transformation pathways were

proposed. The in vitro cytotoxicity risk evaluation based on photostability of clotrimazole was also

performed using the Human skin fibroblast cell line (BJ) ATCC™ CRL-2522. There was no

statistically significant difference between cells viability in all analyzed combinations of clotrimazole,

TiO2/ZnO, and UVA irradiation (p < 0.05).

Chemometrically assisted development and validation of LC–MS/MS method for the analysis of

potential genotoxic impurities in meropenem active pharmaceutical ingredient

Katerina Grigori, Yannis L. Loukas, AnĎelija Malenović, Vicky Samara, Yannis Dotsikas

ABSTRACT

A sensitive Liquid Chromatography tandem mass spectrometry (LC–MS/MS) method was developed

and validated for the quantitative analysis of three potential genotoxic impurities (318BP, M9, S5) in

meropenem Active Pharmaceutical Ingredient (API). Due to the requirement for LOD values in ppb

range, a high concentration of meropenem API (30 mg/mL) had to be injected. Therefore, efficient

determination of meropenem from its impurities became a critical aim of this study, in order to divert

meropenem to waste, via a switching valve. ‎ After the selection of the important factors affecting

analytes‘ elution, a Box-Behnken design was utilized to set the plan of experiments conducted with

UV detector. As responses, the separation factor s between the last eluting impurity and meropenem,

as well as meropenem retention factor k were used. Grid point search methodology was implemented

aiming to obtain the optimal conditions that simultaneously comply to the conflicted criteria. Optimal

mobile phase consisted of ACN, methanol and 0.09% HCOOH at a ratio 71/3.5/15.5 v/v. All

impurities and internal standard omeprazole were eluted before 7.5 min and at 8.0 min the eluents were

directed to waste. The protocol was transferred to LC–MS/MS and validated according to ICH

guidelines.

277

Identification and interconversion of isomeric 4,5-functionalized 1,2,3-thiadiazoles and 1,2,3-

triazoles in conditions of electrospray ionization

D.M. Mazur, M.E. Zimens, V.A. Bakulev, A.T. Lebedev

ABSTRACT

1,2,3-Triazoles and 1,2,3-thiadiazoles have been receiving permanent interest due to their exciting

chemical reactivity and interesting biological properties including antibacterial, anticancer and

antiviral activities. There are four compounds bearing 1H-1,2,3-triazole core in clinical studies which

may appear in the market of drugs in nearest future. Definitely reliable methods of their identification

and quantification should be developed by that time. Mass spectrometry showed itself as the most

reliable method of analysis when dealing with trace levels of organic compounds in the mixtures and

in the most complex matrices, including biological ones. In the present study tandem mass

spectrometry was used to study fragmentation pathways of protonated and deprotonated molecules of

isomeric 4,5-functionalized 1,2,3-thiadiazoles and 1,2,3-triazoles in conditions of electrospray

ionization (ESI). A group of marker ions allowing differentiation between the targeted isomeric

compounds was established. Besides, interconversion of these isomers into one another was studied in

the gas phase in conditions mimicking these processes in solution.

Dissolution assessment of allopurinol immediate release tablets by near infrared spectroscopy

Jelena Smetińko, Sneņana Miljanić

ABSTRACT

The purpose of this study was to develop a NIR spectroscopic method for assessment of drug

dissolution from allopurinol immediate release tablets. Thirty three different batches of allopurinol

immediate release tablets containing constant amount of the active ingredient, but varying in excipients

content and physical properties were introduced in a PLS calibration model. Correlating allopurinol

dissolution reference values measured by the routinely used UV/Vis method, with the data extracted

from the NIR spectra, values of correlation coefficient, bias, slope, residual prediction determination

and root mean square error of prediction (0.9632, 0.328%, 1.001, 3.58, 3.75%) were evaluated. The

obtained values implied that the NIR diffuse reflectance spectroscopy could serve as a faster and

simpler alternative to the conventional dissolution procedure, even for the tablets with a very fast

dissolution rate (>85% in 15 minutes). Apart from the possibility of prediction of the allopurinol

dissolution rate, the other multivariate technique, PCA, provided additional data on the non-chemical

characteristics of the product, which could not be obtained from the reference dissolution values.

Analysis on an independent set of samples confirmed that a difference between the UV/Vis reference

method and the proposed NIR method was not significant. According to the presented results, the

proposed NIR method may be suitable for practical application in routine analysis and for continuously

monitoring the product's chemical and physical properties responsible for expected quality.

278

Enhanced and green extraction polyphenols and furanocoumarins from Fig (Ficus carica L.)

leave using deep eutectic solvents

Tong Wang, Jiao Jiao, Qing-Yan Gai, Peng Wang, Yu-Jie Fu

ABSTRACT

Nowadays, green extraction of bioactive compounds from medicinal plants has gained increasing

attention. As green solvent, deep eutectic solvent (DES) have been highly rated to replace toxic

organic solvents in extraction process. In present study, to simultaneous extraction five main bioactive

compounds from fig leaves, DES was tailor-made. The tailor-made DES composed of a 3:3:3 molar

ratio of glycerol, xylitol and D-(−)-Fructose showed enhanced extraction yields for five target

compounds simultaneously compared with traditional methanol and non-tailor DESs. Then, the tailor-

made DES based extraction methods have compared and microwave-assisted extraction was selected

and optimized due to its high extraction yields with lower time consumption. The influencing

parameters including extraction temperature, liquid-solid ratio, and extraction time were optimized

using response surface methodology (RSM). Under optimal conditions the extraction yield of

caffeoylmalic acid, psoralic acid-glucoside, rutin, psoralen and bergapten was 6.482 mg/g, 16.34 mg/g,

5.207 mg/g, 15.22 mg/g and 2.475 mg/g, respectively. Macroporous resin D101 has been used to

recovery target compounds with recovery yields of 79.2%, 83.4%, 85.5%, 81.2% and 75.3% for

caffeoylmalic acid, psoralic acid-glucoside, rutin, psoralen and bergapten, respectively. The present

study suggests that DESs are truly designer and efficient solvents and the method we developed was

efficient and sustainable for extraction main compounds from Fig leaves.mg/g

Site- and species-specific hydrolysis rates of cocaine

Levente Szöcs, Gergely Völgyi, Ákos Urai, Sándor Hosztafi, Béla Noszál

ABSTRACT

The hydroxide-catalyzed non-enzymatic hydrolysis of cocaine is quantified in terms of ten site- and

species-specific rate constants in connection with also ten site- and species-specific acid-base

equilibrium constants, comprising all the twelve coexisting species in solution. This characterization

involves the major and minor decomposition pathways via benzoylecgonine and ecgonine methyl

ester, respectively, leading to ecgonine, the final product. Hydrolysis has been found to be 10–330

times faster at site 2 than at site 3, depending on the ionization status of the amino moiety and the rest

of the molecule. Nitrogen protonation accelerates the hydrolyses approximately ten times both at site 2

and site 3.

279

Comparative stability-indicating chromatographic methods for determination of 4-

hexylresorcinol in pharmaceutical formulation and shrimps

Rafeek F. Shokry, Lories I. Bebawy, Mohamed R. Elghobashy, Samah S. Abbas

ABSTRACT

Three chromatographic stability-indicating methods were developed for determination of 4-

hexylresorcinol in pure form and in a pharmaceutical formulation. Method A was based on a gradient

elution liquid chromatographic HPLC determination of 4-hexylresorcinol, its related impurities and in

presence of its degradation products. UPLC–MS/MS (Method B) was described for determination of

the cited drug in presence of its degradation products. Method C was a thin- layer chromatography

(TLC)-densitometry method for the separation and determination of the active ingredient, one of its

related impurities and in presence of its degradation products. The mechanism of alkali, oxidative and

photodegradation of 4-hexylresorcinol was studied according to ICH guidelines. The degradation

products were characterized by the LC–MS/MS method. Methods A and B were applicable for

determination of 4-hexylresorcinol residues in shrimp meat. The studied drug was easily degraded in

alkali medium giving toxic compounds. The results obtained by the proposed methods were

statistically analyzed and compared with those obtained by applying a reported method.

Enantiomeric separation of seven β-agonists by NACE—Study of chiral selectivity with

diacetone-d-mannitol–boric acid complex

Lili Lv, Lijuan Wang, Jun Li, Yajun Jiao, Hongyuan Yan

ABSTRACT

A rapid and effective nonaqueous capillary electrophoresis (NACE)–ultraviolet (UV) method was

developed for the enantiomeric separation of seven β-agonists. Diacetone-d-mannitol–boric acid

complex was used as a new chiral selector. It was in situ synthesized by the reaction of diacetone-d-

mannitol and boric acid in methanol medium containing triethylamine. The effects of diacetone-d-

mannitol, boric acid, and triethylamine concentrations on the enantioseparation were carefully

investigated. Under the optimized conditions, baseline enantioseparation could be obtained for six of

the tested β-agonists within 12 min. These results were better than that obtained with d-mannitol–boric

acid complex in previous work. 11B nuclear magnetic resonance (11B NMR) was applied to determine

the fraction of boron species and confirm the formation of diacetone-d-mannitol–boric acid complex.

Validation of the established NACE method was also carried out according to ICH guidelines.

Calibration curves showed good linearity with correlation coefficients (r) ≥ 0.9992 over a certain

concentration range for each enantiomer of the tested five β-agonists. The relative standard deviations

(RSDs) of intra-day precisions and inter-day precisions of migration times were ≤1.4% (n = 6), and

≤6.3% (n = 10), respectively. That of peak areas were ≤3.7% (n = 6), and ≤5.6% (n = 10), respectively.

The limits of detection (LODs) and the limits of quantitation (LOQs) based on the signal-to-noise

ratios of 3 and 10 were found below 1.25 μg mL−1 and 5.00 μg mL−1, respectively. The proposed

method was successfully applied to the determination of clenbuterol enantiomers in a multi-component

pharmaceutical dosage form called ―Ambroxol Hydrochloride and Clenbuterol Hydrochloride Oral

Solution‖.

280

An optimized HPLC-UV method for quantitatively determining sesquiterpenes in

Nardostachyos Radix et Rhizoma

Vu Ngoc Han Le, Trong Quan Khong, Min Kyun Na, Kyung Tae Kim, Jong Seong Kang

ABSTRACT

Nardostachyos Radix et Rhizoma (NR), the root and rhizome from either Nardostachys jatamansi

Batal or Nardostachys jatamansi DC, is known to have biological functions including neuro-protective

and anti-pancreatitis activity. The main bioactive compounds within NR are all classified as

sesquiterpenes, and include desoxo-narchinol A, nardosinonediol, and nardosinone. Although NR is a

valuable herb that is widely used in many Asian countries, robust quality control protocols for NR are

still in question, especially those that can analyze the three main active compounds. Current

quantitative methods within the Chinese Pharmacopoeic use nardosinone as a marker compounds. One

compound cannot represent a complicated matrix, and is thus insufficient to control the quality of this

herbal medicine. Moreover, there are no high-performance liquid chromatography (HPLC) methods

that can simultaneously analyze desoxo-narchinol A (DA), nardosinonediol (NE), and nardosinone

(ND) within NR. This study aimed to establish an efficient quality control protocol by developing an

analytical method that simultaneously detects the three sesquiterpenes with HPLC using response

surface methodology (RSM) to optimize sample preparation. Optimized HPLC conditions included a

mobile phase of 0.1% formic acid in water (A), and a 0.1% formic acid in acetonitrile (B) under an

elution program of 20% B–80% B for 30 min at 254 nm. The method was well validated,

demonstrating satisfactory linearity, accuracy, precision, recovery, repeatability, and stability.

Optimized conditions for creating the analytical sample were predicted by RSM using a Box-Behnken

design. These conditions included reflux at 70 °C for 3 h using 24.98% ethanol as the extraction

solvent (solvent: solid ratio = 78.81 mL/g).

Photostability testing using online reactor HPLC hyphenation and mass spectrometric

compound identification illustrated by ketoprofen as model compound

Jaber Assaf, Diego Zulkiewicz Gomes, Bernhard Wuest, Maria Kristina Parr

ABSTRACT

Investigations on the photochemical stability of pharmaceutical substances are mandatory in drug

development and licensing as photo-induced degradation of an active pharmaceutical ingredient (API)

may not only lead to decreased API concentrations but also to toxic or reactive products. Thus, the US

Food and Drug Administration (FDA) and the International Council for Harmonisation of Technical

Requirements for Pharmaceuticals for Human Use (ICH) issued Guidance for Industry Q1B

―Photostability Testing of New Drug Substances and Products‖ for testing of pure but also packed

drugs. However, photoproducts are also known to be generated in vivo under sunlight exposure of the

skin and lead to considerable amounts of adverse drug effects. Herein we present an alternative system

that may be used for photostability testing mimicking both situations. It combines a tailored

photoreactor with an exchangeable pen light source and a modified HPLC system with online-SPE.

Identification of photoproducts may be performed using mass spectrometry. The potential of accurate

mass spectrometry as a tool for identification of photoproducts was demonstrated as well. A

comparison of the online photoreactor system and the traditional photochamber irradiation was

performed using ketoprofen for proof of concept. In both designs acetylbenzophenone and

ethylbenzophenone were detected as main photoproducts.

281

Optoelectronic iron detectors for pharmaceutical flow analysis

Natalia Rybkowska, Robert Koncki, Kamil Strzelak

ABSTRACT

Compact flow-through optoelectronic detectors fabricated by pairing of light emitting diodes have

been applied for development of economic flow analysis systems dedicated for iron ions

determination. Three analytical methods with different chromogens selectively recognizing iron ions

have been compared. Ferrozine and ferene S based methods offer higher sensitivity and slightly lower

detection limits than method with 1,10‐phenantroline, but narrower ranges of linear response. Each

system allows detection of iron in micromolar range of concentration with comparable sample

throughput (20 injections per hour). The developed flow analysis systems have been successfully

applied for determination of iron in diet supplements. The utility of developed analytical systems for

iron release studies from drug formulations has also been demonstrated.

Isoconversional approach for non-isothermal decomposition of un-irradiated and photon-

irradiated 5-fluorouracil

Hala Sh. Mohamed, AbdelRahman A. Dahy, Refaat M. Mahfouz

ABSTRACT

Kinetic analysis for the non-isothermal decomposition of un-irradiated and photon-beam-irradiated 5-

fluorouracil (5-FU) as anti-cancer drug, was carried out in static air. Thermal decomposition of 5-FU

proceeds in two steps. One minor step in the temperature range of (270–283 °C) followed by the major

step in the temperature range of (285–360 °C). The non-isothermal data for un-irradiated and photon-

irradiated 5-FU were analyzed using linear (Tang) and non-linear (Vyazovkin) isoconversional

methods. The results of the application of these free models on the present kinetic data showed quite a

dependence of the activation energy on the extent of conversion. For un-irradiated 5-FU, the non-

isothermal data analysis indicates that the decomposition is generally described by A3 and A4 modeles

for the minor and major decomposition steps, respectively. For a photon-irradiated sample of 5-FU

with total absorbed dose of 10 Gy, the decomposition is controlled by A2 model throughout the

coversion range. The activation energies calculated in case of photon-irradiated 5-FU were found to be

lower compared to the values obtained from the thermal decomposition of the un-irradiated sample

probably due to the formation of additional nucleation sites created by a photon-irradiation.

282

Characterization of impurities in sodium cromoglycate drug substance and eye drops using LC-

ESI-ion trap MS and LC-ESI-QTOF MS

Peixi Zhu, Jingxian Lu, Zhijian Wang, Weike Su, Erwin Adams

ABSTRACT

As requested by regulatory authorities, impurity profiling is an important issue of quality control. In

this work, a simple and sensitive liquid chromatographic (LC) method compatible with mass

spectrometry (MS) was developed to study related substances and degradation products in sodium

cromoglycate drug substance and eye drops. The method used a Sunfire column (4.6 mm × 150 mm,

3.5 μm). Mobile phase A consisted of 10 mM ammonium formate and mobile phase B were

acetonitrile. Linear gradient elution with a post-run time of 8 min was performed as follows: 0–30 min,

3% B to 50% B; 30–35 min, 50% B. The flow rate was set at 1.0 mL/min. Degradation experiments

were performed to check the stability indicating properties of the developed method. Based on MSn

spectral data and exact mass measurements, the chemical structures of 2 unknown impurities and 6

unknown degradation products were characterized, including impurity C listed in the European

Pharmacopoeia as unknown structure.

Method validation and nanoparticle characterization assays for an innovative amphothericin B

formulation to reach increased stability and safety in infectious diseases

Maraine Catarina Tadini, Ana Maria de Freitas Pinheiro, Daniel Blascke Carrão, Ana Luiza Scarano

Aguillera Forte, Franciane Marquele-Oliveira

ABSTRACT

Drug Delivery Systems (DDS) of known drugs are prominent candidates towards new and more-

effective treatments of various infectious diseases as they may increase drug bioavailability, control

drug delivery and target the site of action. In this sense, the encapsulation of Amphotericin B (AmB) in

Nanostructured Lipid Carriers (NLCs) designed with pH-sensible phospholipids to target infectious

tissues was proposed and suitable analytical methods were validated, as well as, proper nanoparticle

characterization were conducted. Characterization assays by Dinamic Light Scattering (DLS) and

Atomic Force Microscopy demonstrated spherical particles with nanometric size 268.0 ± 11.8 nm and

Zeta Potential −42.5 ± 1.5 mV suggestive of important stability. DSC/TGA and FT-IR assessments

suggested mechanical encapsulation of AmB. The AmB aggregation study indicated that the

encapsulation provided AmB at the lowest cytotoxic form, polyaggregate. Analytical methods were

developed and validated according to regulatory agencies in order to fast and assertively determine

AmB in nanoparticle suspension and, in Drug Encapsulation Efficiency (EE%), release and stability

studies. The quantification method for AmB in NLC suspension presented linearity in 5.05–60.60 μg

mL−1 range (y = 0.07659x + 0.05344) and for AmB in receptor solution presented linearity in 0.15–

10.00 μg mL−1 range (y = 54609x + 263.1), both with r ≥ 0.999. EE% was approximately 100% and

according to the release results, at pH 7.4, a sustained controlled profile was observed for up 46 h. In

the meantime, a micellar AmB solution demonstrated an instability pattern after 7 h of contact with the

medium. Degradation and release studies under acid conditions (infectious condition) firstly depicted a

prominent degradation of AmB (raw-material), with 20.3 ± 3.5% at the first hour, reaching 43.3 ±

7.0% after 7 h of study.

283

Surfactants enhance recovery of poorly soluble drugs during microdialysis sampling:

Implications for in vitro dissolution-/permeation-studies

Sebastian Koplin, Mont Kumpugdee-Vollrath, Annette Bauer-Brandl, Martin Brandl

ABSTRACT

Aim of this project was to investigate the applicability of a recently developed in vitro microdialysis-

sampling approach in connection with a dissolution-/permeation (D/P) system, especially the impact of

surfactants within the perfusion fluid. The D/P-system is based on side-by-side chambers, separated by

a barrier that simulates the intestinal barrier. Here, in contrast to conventional D/P-systems, the

dissolution of the drug (donor chamber concentration) is followed by microdialysis sampling. This

approach appears promising, because it is expected not to disturb the dynamic interplay between drug-

dissolution (-release) and drug permeation. Furthermore, it should allow quantification of the unbound

(free) drug concentration. In the first step, it was assessed, if the addition of the anionic surfactant

sodium dodecyl sulphate (SDS) to the perfusate of the microdialysis system affects the recovery of the

(slightly) water-soluble model drug acyclovir and the poorly water soluble model drug celecoxib

(CXB). SDS had no influence on acyclovir-recovery, but substantially enhanced CXB-recovery, partly

due to improved extraction efficiency, partly due to inhibition of loss of CXB due to non-specific

binding to surfaces and the probe. The fraction of CXB recovered from aqueous CXB-solutions by

microdialysis sampling using SDS-containing perfusates correlated well with the celecoxib

concentration in the samples, but was found independent of the SDS-concentrations (above critical

micelle concentration). In the next step microdialysis sampling with SDS-containing perfusates was

assessed for celecoxib solutions in fasted state simulated intestinal fluid (FaSSIF) and compared to that

in buffer. In FaSSIF, the measured CXB-concentrations were far below the overall CXB concentration,

likely representing the free celecoxib, i.e. the fraction of drug, which is not associated with

taurocholate surfactant micelles. In buffer, the measured concentrations matched the overall CXB

concentrations.

Simultaneous identification and quantification of polymethoxyflavones, coumarin and phenolic

acids in Ageratum conyzoides by UPLC-ESI-QToF-MS and UPLC-PDA

Larissa G. Faqueti, Louis P. Sandjo, Maique W. Biavatti

ABSTRACT

Ageratum conyzoides L. is a plant widely used in traditional medicine of tropical and subtropical

regions for its anti-inflammatory and antinociceptive properties. Nevertheless, the chemical

composition of its medicinal preparation has not been yet accurately established. In this study,

chromatographic methods were developed for the simultaneous identification and quantification of the

main compounds in A. conyzoides aqueous extract, using UPLC-PDA-ESI-QToF-MS. The qualitative

analyses defined by MS/MS analysis allowed the identification of 27 compounds in the aqueous

extract, including the toxic pyrrolizidine alkaloids, phenolic acids, coumarin and

polymethoxyflavones. Among the metabolites, twelve were detected for the first time in the species.

The quantitative method was validated according to the official guidelines, demonstrating to be

specific, linear, precise, accurate and sensitive for the quantification of chlorogenic acid, coumaric

acid, coumarin (1,2-benzopyranone), 5,6,7,3′,4′,5′-hexamethoxyflavone, nobiletin, 5′-methoxynobiletin

and eupalestin.

284

Degradation and metabolite formation of estrogen conjugates in an agricultural soil

Li Ma, Scott R. Yates

ABSTRACT

Estrogen conjugates are precursors of free estrogens such as 17ß-estradiol (E2) and estrone (E1),

which cause potent endocrine disrupting effects on aquatic organisms. In this study, microcosm

laboratory experiments were conducted at 25 °C in an agricultural soil to investigate the aerobic

degradation and metabolite formation kinetics of 17ß-estradiol-3-glucuronide (E2-3G) and 17ß-

estradiol-3-sulfate (E2-3S). The aerobic degradation of E2-3G and E2-3S followed first-order kinetics

and the degradation rates were inversely related to their initial concentrations. The degradation of E2-

3G and E2-3S was extraordinarily rapid with half of mass lost within hours. Considerable quantities of

E2-3G (7.68 ng/g) and E2-3S (4.84 ng/g) were detected at the end of the 20-d experiment, particularly

for high initial concentrations. The major degradation pathway of E2-3G and E2-3S was oxidation,

yielding the primary metabolites 17ß-estrone-3-glucuronide and 17ß-estrone-3-sulfate, respectively.

Common metabolites were E2, the second primary metabolite, and E1, the secondary metabolite.

Additionally, ring B unsaturated estrogens and their sulfate conjugates were tentatively proposed as

minor metabolites. The persistence of E2-3G and E2-3S (up to 20 d) suggests that the high rate of

application of conjugated estrogen-containing substances could be responsible for the frequent

detection of free estrogens in surface and subsurface water.

Chemical analysis and potential endocrine activities of aluminium coatings intended to be in

contact with cosmetic water

Elias Bou-Maroun, Laurence Dahbi, Marie-Pierre Gomez-Berrada, Philippine Pierre, Marie-Christine

Chagnon

ABSTRACT

The objective of the work was to check the presence of Non-Intended Added Substances (NIAS) with

hormonal activities in aluminium coatings extracts coded: AA, BBF, MC and RR, furnished by four

different suppliers. Water samples were prepared at room temperature or at 40 °C for three months to

verify the storage effect on the coatings. Solid phase extraction was used to concentrate and to extract

coating substances. Hormonal activities were checked in vitro using reporter gene bioassays. Except

BBF, all extracts induced a weak but significant estrogenic agonist activity in the human cell line.

Using an estrogenic antagonist (ICI-182, 780), the answer was demonstrated specific in the bioassay.

RR was the only extract to induce a concentration dependent anti-androgenic response in the MDA-

KB2 cell line. Analysis performed using GC–MS and HPLC–MS detected 12 substances in most of the

extracts. 8 NIAS were present. Among them, 4 were identified with certainty: HMBT, BGA, DCU and

BPA. Estrogenic potency was BPA > DCU > BGA > HMBT. HMBT was also anti-androgenic at high

concentration. Combining chemical analysis and bioassays data, we demonstrated that in the RR and

the RR40 extracts, the observed estrogenic response was mainly due to BPA, the anti-androgenic

activity of RR could be due to a synergism between HMBT and BPA. For MC and AA, estrogenic

responses appear to be due to the presence of DCU. Except BBF, storage conditions tended to increase

estrogenic activities in all extracts. However, in term of risk assessment, activities observed were

negligible.

285

Identification and determination of related substances of ceftaroline fosamil in medicinal

product by high performance liquid chromatography with diode array detection and tandem

mass spectrometry

Izabela Karpiuk, Katarzyna Michalska, Katarzyna Bus, Monika Kiljan, Stefan Tyski

ABSTRACT

Ceftaroline fosamil, the prodrug of ceftaroline, is an advanced-generation cephalosporin antibacterial

agent approved for treatment in the European Union in 2012. The drug is dedicated to curing

complicated skin and soft tissue infections and community-acquired pneumonia. The developed

analytical method of high performance liquid chromatography (HPLC) followed by diode array

detector (DAD) set at 243 nm was used to identify and determine degradation products of ceftaroline

fosamil. The elaborated method demonstrated good selectivity and linearity [r > 0.998 for the range

0.8–1.2 mg/mL (80–120%) and r > 0.997 for the range 0.005–0.015 mg/mL (0.5–1.5%)]. Limit of

detection (LOD) and limit of quantification (LOQ) values were equal to 0.15 μg/mL and 0.5 μg/mL,

respectively. The forced decomposition of ceftaroline fosamil carried out under acidic, basic,

oxidative, photolytic and thermal conditions revealed the high sensitivity of ceftaroline on

photodegradation and thermolysis processes. Representative ceftaroline samples were selected and

analysed by LC coupled with electrospray ionisation time-of-flight mass spectrometry (LC-ESI-Q-

TOF-MS) to identify the related substances that appeared under stress conditions. The LC-DAD

method was transferred without any modification to LC–MS/MS system, allowing it to correlate

results back to the LC-DAD method. Eight unknown signals were detected in the photolytic and

thermal stress solutions for ceftaroline fosamil. The evaluated method was applied to the analysis of a

medicinal product containing ceftaroline fosamil − Zinforo™, 600 mg, powder for concentrate for

solution for infusion. Moreover, the optimised conditions allowed for the successful separation of eight

cephalosporin antibiotics possessing a molecular structure similar to ceftaroline (cefepime, cefapirin,

ceftazidime, cefpirome, cefalonium, cefotaxime, cefquinome, cefalotin).

Quantification of concentrated Chinese medicine granules by quantitative polymerase chain

reaction

Yat-Tung Lo, Pang-Chui Shaw

ABSTRACT

Determination of the amount of constituent in a multi-herb product is important for quality control. In

concentrated Chinese medicine granules (CCMG), no dregs are left after dissolution of the CCMG.

This study is the first to examine the feasibility of using quantitative polymerase chain reaction

(qPCR) to find the amount of CCMG in solution form. DNA was extracted from Hirudo and Zaocys

CCMG mixed at different ratios and amplified in qPCR using species-specific primers. The threshold

cycle (CT) obtained was compared with the respective standard curves. Results showed that

reproducible quantification results could be obtained (1) for 5–50 mg CCMG using a modified DNA

extraction protocol, (2) amongst DNA extracted from the same batch of CCMG and (3) amongst

different batches of CCMG from the same company. This study demonstrated the constitute amount of

CCMG in a mixture could be determined using qPCR. This work has extended the application of DNA

techniques for the quantification of herbal products and this approach may be developed for quality

assurance in the CCMG industry.

286

Chiral separation of terbutaline and non-steroidal anti-inflammatory drugs by using a new

lysine–bridged hemispherodextrin in capillary electrophoresis

V. Cucinotta, M. Messina, A. Contino, G. Maccarrone, A. Giuffrida

ABSTRACT

A method for the separation of a mixture of terbutaline and non-steroidal anti-inflammatory drugs was

developed using capillary electrophoresis with a new hemispherodextrin, ad hoc designed, the lysine −

bridged hemispherodextrin (THLYSH). The use of lysine residues to bridge the trehalose capping unit

moiety to the cyclodextrin cavity gives rise to a receptor with two long chains with amine nitrogen

atoms, whose charge can be easily tuned as a function of the solution pH. The new hemispherodextrin

was accurately characterised by ESI–MS and NMR spectroscopy, also highlighting its protonation

behaviour. Circular dichroism and ESR spectroscopy measurements were also carried out to test its

inclusion ability towards anthraquinone-3-sulfonate and its metal coordination ability towards

copper(II) ion, respectively. Analogously to the other hemispherodextrins, the main skill of this new

derivative lies in its chiral selector properties, as shown by the separation of the enantiomeric pairs of

terbutaline and ibuprofen, flurbiprofen, suprofen and tiaprofenic acid by capillary electrophoresis. The

focused use of the solution equilibria involved in the separations made it possible to understand the

phenomena occurring in solution, and to finely tune the charge status of the receptor. In this way the

chiral separation of the racemic mixture was successfully obtained, even if the receptor was

individually used, differently by the other hemispherodextrins previously studied whose chiral

separation capabilities are present only if used as binary mixtures.

Separation and characterization of allergic polymerized impurities in cephalosporins by 2D-

HPSEC × LC-IT-TOF MS

Yu Xu, DanDan Wang, Lan Tang, Jian Wang

ABSTRACT

Eleven unknown allergic impurities in cefodizime, cefmenoxime and cefonicid were separated and

characterized by a trap-free two-dimensional high performance size exclusion chromatography

(HPSEC) and reversed phase liquid chromatography (RP-HPLC) coupled to high resolution ion

trap/time-of-flight mass spectrometry (2D-HPSEC × LC-IT-TOF MS) with positive and negative

modes of electrospray ionization method. Separation and characterization the allergic polymerized

impurities in β-lactam antibiotics were on the basis of column-switching technique which effectively

combined the advantages of HPSEC and the ability of RP-HPLC to identify the special impurities. In

the first dimension HPSEC, the column was Xtimate SEC-120 analytical column (7.8 mm × 30 cm, 5

μm), and the gradient elution used pH 7.0 buffer-acetonitrile as mobile phase And the second

dimension analytical column was ZORBAX SB-C18 (4.6 × 150 mm, 3.5 μm) with ammonium formate

solution (10 mM) and ammonium formate (8 mM) in [acetonitrile-water (4:1, v/v)] solution as mobile

phase. Structures of eleven unknown impurities were deduced based on the high resolution MSn data

with both positive and negative modes, in which nine impurities were polymerized impurities. The

forming mechanism of β-lactam antibiotic polymerization in cephalosporins was also studied. The

question on incompatibility between non-volatile salt mobile phase and mass spectrometry was solved

completely by multidimensional heart-cutting approaches and online demineralization technique,

which was worthy of widespread use and application for the advantages of stability and repeatability.

287

Phytochemical analysis and anti-inflammatory evaluation of compounds from an aqueous

extract of Croton cajucara Benth

Adamara M. Nascimento, Daniele Maria-Ferreira, Fernando T. Dal Lin, Alexandre Kimura, Lauro M.

de Souza

ABSTRACT

Croton cajucara Benth is a medicinal plant popularly used in the Brazilian Amazonia, where it is

known as sacaca, being consumed as tea, decoction or infusion of the leaves and stem bark. From a

decoction of the leaves, a comprehensive phytochemical analysis was developed by liquid

chromatography-mass spectrometry. Many compounds were identified for the first time in C. cajucara,

such as O-glycosides of kaempferol and quercetin, flavonoid-C-glycosides, tannins and cinnamic acid

derivatives. These compounds were fractionated by polarity and assayed for their anti-inflammatory

activity, using a model of mice edema, induced by an intraplantar injection of carrageenan. All

fractions exhibited anti-inflammatory properties.

Supercritical fluid chromatography approach for a sustainable manufacture of new

stereoisomeric anticancer agent

Alina Ghinet, Yasmine Zehani, Emmanuelle Lipka

ABSTRACT

Two routes aimed at the manufacture of unprecedented stereoisomeric combretastatin A-4 analogue

were described: flash chromatography vs supercritical fluid chromatography. The latter has many

advantages over liquid chromatography and was therefore chosen for the small scale separation of

methyl 1-[(3-hydroxy-4-methoxyphenyl) (3,4,5-trimethoxyphenyl)methyl]-5-oxo-l-prolinate 5, with

potential antitumoral activity. After a screening of six different polysaccharide based chiral stationary

phases and four co-solvents, the percentage of co-solvent, the flow-rate and the outlet pressure were

optimized through a design of experiments (DoE). The preparation of 50 mg of each stereoisomer was

achieved successfully on a Chiralpak AD-H with isopropanol as a co-solvent. Productivity (kkd),

solvent usage and environmental factor (E Factor) were calculated. Flash chromatography and

supercritical fluid chromatography approaches were compared in terms of yield and purity of each

stereoisomer manufactured.

288

Use of ionic liquids as headspace gas chromatography diluents for the analysis of residual

solvents in pharmaceuticals

Omprakash Nacham, Tien D. Ho, Jared L. Anderson, Gregory K. Webster

ABSTRACT

In this study, two ionic liquids (ILs), 1-butyl-3-methylimidazolium bis[(trifluoromethyl)

sulfonyl]imide ([BMIM][NTf2]) and trihexyltetradecylphosphonium bis[(trifluoromethyl)sulfonyl]

imide ([P66614][NTf2]) were examined as contemporary diluents for residual solvent analysis using

static headspace gas chromatography (SHS-GC) coupled with flame ionization detection (FID). ILs are

a class of non-molecular solvents featuring negligible vapor pressure and high thermal stabilities.

Owing to these favorable properties, ILs have potential to enable superior sensitivity and reduced

interference, compared to conventional organic diluents, at high headspace incubation temperatures.

By employing the [BMIM][NTf2] IL as a diluent, a 25-fold improvement in limit of detection (LOD)

was observed with respect to traditional HS-GC diluents, such as N-methylpyrrolidone (NMP). The

established IL-based method demonstrated LODs ranging from 5.8 parts-per-million (ppm) to 20 ppm

of residual solvents in drug substances. The optimization of headspace extraction conditions was

performed prior to method validation. An incubation temperature of 140 °C and a 15 min incubation

time provided the best sensitivity for the analysis. Under optimized experimental conditions, the mass

of residual solvents partitioned in the headspace was higher when using [BMIM][NTf2] than NMP as

a diluent. The analytical performance was demonstrated by determining the repeatability, accuracy,

and linearity of the method.

Adduct ion-targeted qualitative and quantitative analysis of polyoxypregnanes by ultra-high

pressure liquid chromatography coupled with triple quadrupole mass spectrometry

Xu Wu, Lin Zhu, Jiang Ma, Yang Ye, Ge Lin

ABSTRACT

Polyoxypregnane and its glycosides (POPs) are frequently present in plants of Asclepiadaceae family,

and have a variety of biological activities. There is a great need to comprehensively profile these

phytochemicals and to quantify them for monitoring their contents in the herbs and the biological

samples. However, POPs undergo extensive adduct ion formation in ESI-MS, which has posed a

challenge for qualitative and quantitative analysis of POPs. In the present study, we took the advantage

of such extensive adduct ion formation to investigate the suitability of adduct ion-targeted analysis of

POPs. For the qualitative analysis, we firstly demonstrated that the sodium and ammonium adduct ion-

targeted product ion scans (PIS) provided adequate MS/MS fragmentations for structural

characterization of POPs. Aided with precursor ion (PI) scans, which showed high selectivity and

sensitivity and improved peak assignment confidence in conjunction with full scan (FS), the

informative adduct ion-targeted PIS enabled rapid POPs profiling. For the quantification, we used

formic acid rather than ammonium acetate as an additive in the mobile phase to avoid simultaneous

formation of sodium and ammonium adduct ions, and greatly improved reproducibility of MS response

of POPs. By monitoring the solely formed sodium adduct ions [M+Na]+, a method for simultaneous

quantification of 25 POPs in the dynamic multiple reaction monitoring mode was then developed and

validated. Finally, the aforementioned methods were applied to qualitative and quantitative analysis of

POPs in the extract of a traditional Chinses medicinal herb, Marsdenia tenacissima (Roxb.) Wight et

Arn., and in the plasma obtained from the rats treated with this herb.

289

Quantitative determination of dobutamine in newborn pig plasma samples by HPLC–MS/MS

O.E. Albóniga, M.L. Alonso, M.E. Blanco, O. González, R.M. Alonso

ABSTRACT

A novel gradient reverse phase high performance liquid chromatography tandem mass spectrometry

(HPLC/MS-MS) was performed as a method for the determination of dobutamine hydrochloride

(DOB) in newborn pig plasma samples. It was developed and validated after optimization of sample

treatment and various chromatographic and mass spectrometric conditions. Trimethoxydobutamine

(TMD) was used as internal standard. Heptafluorobutyric acid (HFBA) and ethyl acetate were used for

the treatment of plasma samples. The separation of dobutamine and internal standard was done using a

Kinetex F5 (50 × 2.1 mm, 2.6 μm, 100 Å) analytical column. The mobile phase was a mixture of

acetonitrile and HCOOH 0.01%. The column oven temperature was optimized at 40° C and the flow

rate was 0.25 mL/min. DOB and TMD were detected by multiple reaction monitoring (MRM) mode in

ESI+, using a cone voltage (CV) of 25 V and a collision energy (CE) of 25 eV. The weighted

calibration curve (1/x2) was found to be linear over the concentration range of 1–100 ng/mL (r2 >

0.999). The limit of quantification (LLOQ) of the method was 1 ng/mL. The values of selectivity,

carryover, LLOQ, linearity, accuracy, precision, matrix effect, stability and recovery obtained meet the

acceptable range according to European Medicines Agency (EMA) and Food and Drug Administration

(FDA) guidelines. The method was efficiently applied to quantify DOB in plasma samples from a

pharmacokinetic/pharmacodynamic study in a disease model of newborn piglet.

An integrative investigation of the toxicity of Aconiti kusnezoffii radix and the attenuation effect

of its processed drug using a UHPLC-Q-TOF based rat serum and urine metabolomics strategy

Zhenyu Sui, Qing Li, Lin Zhu, Zhenru Wang, Kaishun Bi

ABSTRACT

Aconiti kusnezoffii radix (AKR), the root of Aconitum kusnezoffii Reichb, is commonly used in the

treatment of the rheumatoid arthritis. However, the clinical application is limited due to its potential

toxicity. Therefore, to investigate the mechanism of its potential neurotoxicity and nephrotoxicity, a

comprehensive metabolomics study combined with serum biochemistry and histopathology

measurements was carried out. A UHPLC-Q-TOF mass spectrometry based metabolomics approach

was applied to characterize the AKR toxicity, while the toxicity attenuation effects of Aconiti

kusnezoffii radix cocta (AKRC) on Wistar rats were also investigated. Two chromatographic

techniques involving reversed-phase chromatography and hydrophilic interaction chromatography

were combined for the serum and urine detection, which balanced the integrity and selectivity of the

two matrices. Principal component analysis was used to determine the groups, and principal

component analysis discriminant analysis was carried out to confirm the important variables. Then, the

developed integrative toxicity evaluation method was applied to assess the toxicity of AKR and the

attenuation effect of AKRC. The highly sensitive and specific toxic biomarkers, which can provide

practical bases were identified for the diagnosis of the neurotoxicity and nephrotoxicity induced by

AKR. In all, a total of 19 putative biomarkers were characterized, and related metabolic pathways were

identified.

290

Calibration and validation of a MCC/IMS prototype for exhaled propofol online measurement

Felix Maurer, Larissa Walter, Martin Geiger, Jörg Ingo Baumbach, Sascha Kreuer

ABSTRACT

Propofol is a commonly used intravenous general anesthetic. Multi-capillary column (MCC) coupled

Ion-mobility spectrometry (IMS) can be used to quantify exhaled propofol, and thus estimate plasma

drug concentration. Here, we present results of the calibration and analytical validation of a MCC/IMS

pre-market prototype for propofol quantification in exhaled air. Calibration with a reference gas

generator yielded an R2 ≥ 0.99 with a linear array for the calibration curve from 0 to 20 ppbv. The

limit of quantification was 0.3 ppbv and the limit of detection was 0.1 ppbv. The device is able to

distinguish concentration differences > 0.5 ppbv for the concentration range between 2 and 4 ppbv and

> 0.9 ppbv for the range between 28 and 30 ppbv. The imprecision at 20 ppbv is 11.3% whereas it is

3.5% at a concentration of 40 ppbv. The carry-over duration is 3 min. The MCC/IMS we tested

provided online quantification of gaseous propofol over the clinically relevant range at measurement

frequencies of one measurement each minute.

Biochemical and functional analysis of corticotropin releasing factor purified from an aqueous

extract of human placenta used as wound healer

Namrata Singh, Debasish Bhattacharyya

ABSTRACT

Human placental extract constitutes of innumerable therapeutically important components mostly used

in wound healing arising from the skin and burn injuries. However, there is still some bioactive present

in the placental extracts yet to be characterized to better under the complex process of wound healing

mediated by the placental extract. In this study, the presence of corticotropin releasing factor (CRF) in

an aqueous extract of human placenta was detected and quantified by dot blot and CRF-ELISA

immunoassay kit respectively. Subsequently, it was purified by immuno-affinity chromatography and

quantified as 0.45 ± 0.05 μg of CRF per ml of placental extract where its molecular weight found to be

4.78 kDa by MALDI-TOF. To study functional analysis of CRF, an in vitro WI-38 lung fibroblast cell

scratch wound model was used which indicated proliferation, motility of cells after treatment with

purified CRF. Moreover, reduction in apoptosis rate of cells during closure of wound was observed

from microscopy studies and FACS analysis. Also, Antalarmin, an antagonist of CRF type 1 receptor

inhibited the wound closure potency of the purified component. Faster healing of wound with an

elevation of IL-6 and TGF-β during early stages of repair by placental CRF was observed on excision

rat model. The process of healing was accompanied by the decrease in the level of TNF-α and IFN-γ.

291

Toxic compounds from tobacco in placenta samples analyzed by UPLC-QTOF-MS

Somayeh Mohammadi, Celia Domeno, Isabel Nerin, Margarita Aznar, Cristina Nerin

ABSTRACT

Polycyclic aromatic hydrocarbons (PAHs), tobacco-specific nitrosamines (TSNAs) and aromatic

amines are carcinogens present in cigarette smoke. These compounds are distributed in the human

body and they could be transferred to the foetus during the pregnancy. Placenta is the main barrier to

these toxic compounds and its study is the objective of this work. A method based on solid-phase

extraction (SPE) with ultra-performance liquid chromatography−tandem quadrupole-time-of-flight

mass spectrometry (UPLC-QTOF-MS) has been examined and optimized for the analysis of 9 target

analytes (4 tobacco-specific nitrosamines and some of their metabolites, 3 aromatic amines, nicotine

and cotinine) in 26 placenta samples from smoking and non-smoking women. Limits of detection

(LODs) were in the range of 3–27 ng/g of placenta. Nicotine, cotinine, N-nitrosoanatabine (NAT) and

4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK) metabolite, 4-(methylnitrosamino)-1-(3-

pyridyl)-1-butanol (NNAL) were detected in the placenta samples of smoking woman. Nicotine was

detected in 3 out of 8 placentas from smoking women, always below the limit of quantification (88

ng/g). This could be expected, as the half-life of nicotine in the body is limited to about 0.5–3 h.

Cotinine, the main metabolite from nicotine, was detected in all placentas from smoking women at

concentrations between 17.2 and 61.8 ng/g, reaching the highest values for those women that smoked

the highest number of cigarettes. NAT and NNAL were detected in all placentas from smoking

women, always below the limit of quantification (40 ng/g and 33 ng/g respectively).

HPLC-HG-AFS determination of arsenic species in acute promyelocytic leukemia (APL) plasma

and blood cells

Meihua Guo, Wenjing Wang, Xin Hai, Jin Zhou

ABSTRACT

Arsenic trioxide (ATO) has been successfully used in the treatment of acute promyelocytic leukemia

(APL). To clarify the arsenic species in APL patients, high performance liquid chromatography-

hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) and HG-AFS methods were

developed and validated to quantify the plasma concentrations of inorganic arsenic (As(III) and As(V))

and methylated metabolites (MMA and DMA), and the total amounts of arsenic in blood cells and

plasma. Blood cells and plasma were digested with mixtures of HNO3H2O2 and analyzed by HG-

AFS. For arsenic speciation, plasma samples were prepared with perchloric acid to precipitate protein.

The supernatant was separated on an anion-exchange column within 6 min with isocratic elution using

13 mM CH3COONa, 3 mM NaH2PO4, 4 mM KNO3 and 0.2 mM EDTA-2Na. The methods provided

linearity range of 0.2–20 ng/mL for total arsenic and 2.0–50 ng/mL for four arsenic species. The

developed methods for total arsenic and arsenic species determination were precise and accurate. The

spiked recoveries ranged from 81.2%-108.6% and the coefficients of variation for intra- and inter-

batch precision were less than 9.3% and 12.5%, respectively. The developed methods were applied

successfully for the assay of total arsenic and arsenic species in 5 APL patients. The HPLC-HG-AFS

may be a good alternative for arsenic species determination in APL patients with its simplicity and

low-cost in comparison with HPLC-ICP-MS.

292

Quantification of IDP-73152, a novel antibiotic, in plasma from mice, rats and humans using an


pharmacokinetic studies

Myongjae Lee, Dohee Kim, Jeongcheol Shin, Hee-Yeol Lee, Suk-Jae Chung

ABSTRACT

IDP-73152, a novel inhibitor of a bacterial peptide deformylase, was recently approved as a new,

investigational drug in Korea for the clinical management of infections caused by Gram positive

bacteria. The objective of this study was to develop/validate a simple and robust analytical method for

the determination of IDP-73152 in plasma samples from rodents and humans, and to assess the

feasibility of the assay for use in pharmacokinetic studies using animal models. Plasma samples were

processed using a standard method for protein precipitation and an aliquot of the extract then injected

onto an UHPLC–MS/MS system. The drug and IDP-117293, an internal standard, were analyzed in

the positive ion-mode by electrospray ionization and quantified by monitoring the transition at m/z

555.2 → 245.2 for IDP-73152 and 563.3 → 253.1 for the internal standard, respectively. The lower

and upper limit of the assay was determined to be 5 and 10000 ng/ml, respectively, with an acceptable

linearity (R > 0.999) in the response-concentration relationship. Validation parameters, including

accuracy, precision, dilution, recovery, matrix effect and stability were found to be within the

acceptable ranges recommended by the assay validation guidelines of the United States FDA. The

method was successfully applied to the quantification of IDP-73152 in plasma from mice/rats that had

received a single oral administration of 80 mg/kg IDP-73152, in the form of the mesylate salt.

New approach for the diagnosis of histamine intolerance based on the determination of

histamine and methylhistamine in urine

Oriol Comas-Basté, M.Luz Latorre-Moratalla, Roberta Bernacchia, M.Teresa Veciana-Nogués,

M.Carmen Vidal-Carou

ABSTRACT

Histamine intolerance is a disorder in the homeostasis of histamine due to a reduced intestinal

degradation of this amine, mainly caused by diamine oxidase (DAO) enzyme deficiency, which

provokes its accumulation in plasma and the appearance of adverse health affects. A new approach for

the diagnosis of this intolerance could be the determination of histamine and its metabolites in urine.

The aim of this work was to develop and validate a rapid method to determine histamine and

methylhistamine in human urine by Ultra High Performance Liquid Chromatography and Fluorimetric

detection (UHPLC-FL). The proposed method is a consistent procedure to determine histamine and

methylhistamine in less than 11 min with adequate linearity and sensitivity. Relative standard

deviation was always lower than 5.5%, ensuring method precision; and mean recovery was greater

than 99% for both analytes. The structure of histamine and methylhistamine conjugated with OPA

were confirmed by UHPLC-ITD-FTMS which enabled to unequivocally identify both analytes in

standards and also in urine samples. The analysis of histamine and methylhistamine in urine samples

could be a potential new approach for the routine diagnosis of histamine intolerance, more patient-

friendly and with clear advantages in terms of equipment and personnel demand for sample collection

in comparison with current plasmatic DAO activity determination.

293

Development and validation of sensitive LC–MS/MS method for the quantification of SUVN-502

and its metabolite and its application for first in human pharmacokinetic study

Ramakrishna Nirogi, Devender Reddy Ajjala, Raghupathi Aleti, Lakshmiprasanna Rayapati, Naga

Surya Prakash Padala

ABSTRACT

A sensitive and rapid LC–MS/MS method was developed and validated for the quantification of

SUVN-502 and M1 of SUVN-502, a 5-HT6 receptor antagonist for the treatment of dementia

associated with Alzheimer‘s disease. Following solid-phase extraction, SUVN-502 and M1 of SUVN-

502 and IS were eluted with 10 mM ammonium acetate (pH 4.0) and acetonitrile using a rapid gradient

program on reverse phase column. Multiple reaction monitoring mode was used to monitor the

respective transitions of m/z 478.2 → 377.7 for SUVN-502 and m/z 464.1 → 377.7 for M1 of SUVN-

502. The assay exhibited a linear dynamic range of 10–10000 pg/mL for SUVN-502 and 20–20000

pg/mL for M1 of SUVN-502 in human plasma. Acceptable precision and accuracy were obtained for

concentrations over the standard curve range. The within batch accuracy and precision were within

acceptable limits. All the other validation parameters were within the acceptable limits. The validated

method was applied to analyze human plasma samples obtained from a human pharmacokinetic study

consisting single and multiple ascending doses.

Glycosylation patterns of selected proteins in individual serum and cerebrospinal fluid samples

Isabella Karlsson, Lorena Ndreu, Alessandro Quaranta, Gunnar Thorsén

ABSTRACT

A method we previously developed has been applied to the determination of the glycosylation pattern

of specific proteins in biological samples. Six proteins (alpha-1-antitrypsin, transferrin, haptoglobin,

C1 inhibitor, alpha-1 acid glycoprotein, and immunoglobulin G) were studied in serum samples from

five individuals and cerebrospinal fluid (CSF) samples from three individuals, to investigate the

expected normal distribution of glycosylation patterns and to assess whether this methodology can be

used to discriminate between samples from different individuals. For serum samples, the differences

were shown to be small, while much larger differences were found for the CSF samples, with a greater

number of glycoforms present. This can be linked to the occurrence of differential glycosylation in

proteins expressed in the brain compared with proteins expressed elsewhere in the body. The

developed method could distinguish differences in the glycosylation pattern of specific proteins in the

individual samples, which was not reflected in the glycan content of total CSF. This is the first time

that the glycoforms of several of these proteins have been investigated in CSF.

294

MIL-101(Cr)@GO for dispersive micro-solid-phase extraction of pharmaceutical residue in

chicken breast used in microwave-assisted coupling with HPLC–MS/MS detection

Yudan Wang, Xinpeng Dai, Xi He, Lin Chen, Xiaohong Hou

ABSTRACT

In this work, MIL-101(Cr)@GO (Graphite Oxide) was synthesized using a hydrothermal synthesis

method and was applied as a dispersive micro-solid-phase extraction (D-μ-SPE) sorbent for the

efficient concentration of four residual drugs (metronidazole, MNZ; tinidazole, TNZ;

chloramphenicol, CAP; sulfamethoxazole, SMX). Meanwhile, the extraction process was optimized by

combining it with microwave-assisted extraction. Factors affecting the D-μ-SPE efficiency, such as

selection of sorbent materials, pH of the sample solution, salting-out effect, amount of used material,

extraction time, desorption solvent and desorption time, were studied. Under the optimal extraction

conditions, the linearity ranged from 10 to 1000 ng kg−1 and 1–100 ng kg−1 (r2 ≥ 0.9928) for the

target analytes. The limits of detection were between 0.08 and 1.02 ng kg−1, and the limits of

quantitation were between 0.26 and 3.40 ng kg−1. Additionally, the developed method also exhibited

good precision (RSD ≤ 2.5%), repeatability (RSD ≤ 4.3%), high recoveries (88.9%–102.3%) and low

matrix effects (78.2%–95.1%). The proposed method proved to be an efficient and reliable approach

for the determination of the analytes. Finally, we successfully detected the four drugs in chicken

breast.

Development of a robust reporter gene assay to measure the bioactivity of anti-PD-1/anti-PD-L1

therapeutic antibodies

Lan Wang, Chuanfei Yu, Yalan Yang, Kai Gao, Junzhi Wang

ABSTRACT

Being regarded as the ‗cancer panacea‘, the anti-PD-1/anti-PD-L1 monoclonal antibodies (mAbs) have

become the R&D focus of biopharmaceutical industries. Several marketed such mAbs have been

proved particularly effective in treating various cancers. However, the cell-based bioassay to measure

the biological activities of the anti-PD-1/anti-PD-L1 mAbs as the lot release or stability test has been a

great challenge to quality control laboratories due to the immunomodulating nature of the mAbs. Here,

we describe the development and validation of a reporter gene assay consisting of two-cell systems to

measure the bioactivity of the anti-PD-1/anti-PD-L1 mAbs. We have generated two cell lines, the

CHO-PD-L1-CD3L cell line that stably expresses PD-L1 and the membrane-anchored anti-CD3 single

chain antibody fragment (scFv) named CD3L and the Jurkat-PD-1-NFAT cell line that stably

expresses PD-1 and the luciferase gene under the control of the NFAT response elements from the IL-

2 promoter. The results show good dose-dependent responsiveness to the mAbs and excellent

performance characteristics including specificity, accuracy and precision. The biological relevance of

the assay, the passage stability of the two cell lines, and the capability of measuring various anti-PD-

1/anti-PD-L1 mAbs render this assay applicable not only in lot release and stability test but also in

characterization and development of new anti-PD1/anti-PD-L1 mAbs.

295

LC–MS/MS-ESI method for simultaneous quantification of darolutamide and its active

metabolite, ORM-15341 in mice plasma and its application to a pharmacokinetic study

Sreekanth Dittakavi, Pavan Kumar V.S.P. Nagasuri, Suresh P. Sulochana, Syed Mohd Saim, Ramesh

Mullangi

ABSTRACT

A sensitive and rapid LC–MS/MS method was developed and validated for the simultaneous

quantitation of darolutamide and its active metabolite i.e. ORM-15341 in 50 μL mice plasma using

bicalutamide as an internal standard (I.S.) as per regulatory guidelines. Sample processing was

accomplished through liquid-liquid extraction. Chromatographic separation was achieved using an

Atlantis C18 column with an isocratic mobile phase comprising 0.2% formic acid:acetonitrile (35:65,

v/v) at a flow rate of 0.8 mL/min within 2.5 min. Detection and quantitation were done by multiple

reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 397 →

202, 395 → 202 and 429 → 255 for darolutamide, ORM-15341 and I.S, respectively in the negative

ionization mode. The calibration curve was linear from 0.61–1097 ng/mL for both darolutamide and

ORM-15341. The intra- and inter-day precisions were in the range of 1.34–13.8 and 4.85–12.9 and

3.91–13.7 and 6.54–14.2%, for darolutamide and ORM-15341, respectively. Darolutamide and ORM-

15341 were found to be stable under different stability conditions. The validated method was applied

to a pharmacokinetic study in mice.

Metabolite identification of AZD8055 in Sprague-Dawley rats after a single oral administration

using ultra-performance liquid chromatography and mass spectrometry

Md Mamunur Rashid, Hyun-A Oh, Hyunbeom Lee, Byung Hwa Jung

ABSTRACT

AZD8055 is an ATP-competitive specific dual mTOR inhibitor and exhibited potent antitumor activity

on several types of solid tumors. However, the metabolism of AZD8055 in the body still remains

unknown. In this study, metabolite identification of AZD8055 was performed using ultra high-

performance liquid chromatography-ion trap mass spectrometry (UHPLC-IT-MS) through both in

vitro and in vivo approaches using rat liver microsomes (RLMs) and rat plasma, urine and feces,

respectively. A total of eight putative metabolites (five phase I and three phase II) were identified, and

a tentative metabolic pathway was suggested for the first time. Considering the accurate mass and

mass fragmentations of the detected metabolites, their plausible structures were suggested.

Demethylation, hydroxylation, oxidation and morpholine ring opening were the major

biotransformation processes for the phase-I metabolism, while phase-II metabolites were merely

generated by the glucuronide conjugation reaction. The cumulative excretion of AZD8055 in urine and

feces was 0.13% and 1.11% of the dose, respectively. When the semi-quantitative analysis of the

metabolites was performed using UHPLC–MS/MS (ultra-performance liquid chromatography tandem

mass spectrometry) to evaluate the overall trend of metabolites formation and excretion, AZD8055

was excreted more in the form of the metabolites than itself and their formation was very fast.

Therefore it was presumed that biotransformation was playing a crucial role in its elimination.

Ultimately, this study provides novel insights regarding the in vitro and in vivo biotransformations of

AZD8055. Further investigations of metabolites of this potent anti-cancer compound could be

beneficial for the antitumor drug design and development process.

296

Confirmation of metabolites of the neuroleptic drug prothipendyl using human liver

microsomes, specific CYP enzymes and authentic forensic samples—Benefits for routine drug

testing

M. Krämer, S. Broecker, B. Madea, C. Hess

ABSTRACT

Metabolism of the tricyclic azaphenothiazine neuroleptic drug prothipendyl was investigated with in

vitro studies using human liver microsomes but also specific isoforms of cytochrome P450 (CYP)

enzymes. Identification and analysis of metabolites was done by liquid chromatography (LC) coupled

with quadrupole time of flight mass spectrometry (LC-QTOF-MS) as well as triple quadrupole mass

spectrometry (LC-QQQ-MS). Results of the herein presented study revealed the proof of various

demethylated and oxidized metabolites (-CH2, -C2H4, four derivatives of prothipendyl +O and three

derivatives of prothipendyl -CH2 + O). Metabolic reactions of prothipendyl were mainly catalyzed by

CYP enzymes CYP1A2, CYP2D6, CYP2C19 and CYP3A4. N-demethyl-prothipendyl was

predominantly formed by isoforms CYP2C19 and CYP1A2, while particularly the CYP isoenzyme

3A4 was responsible for the formation of prothipendyl sulfoxide. To confirm the formation of

previously identified metabolites in vivo, cardiac blood samples that were tested positive for

prothipendyl during routine drug testing and serum and urine samples, collected after a voluntary

intake of prothipendyl, were analyzed by LC-QQQ-MS.

Quantification of 16 β-lactams in chicken muscle by QuEChERS extraction and UPLC-Q-

Orbitrap-MS with parallel reaction monitoring

Qing Chen, Xiao-Dong Pan, Bai-Fen Huang, Jian-Long Han

ABSTRACT

A method is described for the analysis of 16 β-lactams in chicken muscle by UPLC-quadrupole(Q)-

Orbitrap-MS with parallel reaction monitoring (PRM). QuEChERS approach includes clean-up step by

sorbent of primary-secondary amine (PSA) and C18 was adopted for sample preparation. Q-Orbitrap

with PRM showed high sensitivity with limits of detection (LODs) ranged from 0.01 μg kg−1 to 0.35

μg kg−1. The method was further validated by intra- and inter-day test with spiking levels less than

MRLs (maximum residue limits, the European Union). Recovery (83–112%) and precision values

(RSDs <15%) for all studied analytes were obtained. The result indicates that UPLC-Q-Orbitrap

coupled with QuEChERS preparation can serve as a routine quantification method for β-lactam

residues in chicken muscles.

297

Characterization of the phase I and phase II metabolic profile of tolvaptan by in vitro studies

and liquid chromatography–mass spectrometry profiling: Relevance to doping control analysis

Monica Mazzarino, Valeria Buccilli, Xavier de la Torre, Ilaria Fiacco, Francesco Botrè

ABSTRACT

Phase I and phase II biochemical reactions involved in the biotransformation pathways of tolvaptan

were characterized by LC–MS-based techniques and in vitro models to identify the most appropriate

marker(s) of intake. The effects of physiological and non-physiological factors on the metabolic profile

of tolvaptan were also evaluated. In vitro approaches were based on the use of pooled human liver

microsomes and recombinant isoforms of cytochrome P450 and uridine diphospho glucuronosyl-

transferase. Sample preparation included liquid/liquid extraction at neutral pH with tert-butyl methyl-

ether. In the case of the study of phase II metabolism an additional enzymatic hydrolysis step was

performed. The chromatographic separation was carried out using reversed-phase chromatography,

whereas detection was performed by either triple-quadrupole or time-of-flight analyzers in positive

electrospray ionization and different acquisition modes. Our data show that tolvaptan is metabolized to

at least 20 phase I metabolites, the biotransformation reactions being catalyzed mainly by CYP3A4

and CYP3A5 isoforms. The phase-I reactions include hydroxylation (in different positions),

carboxylation, oxidation, hydrogenation, dealkylation, isomerization and a combination of the above.

Most of the phase I metabolites undergo glucuronidation, carried out mostly by UGT2B7 and

UGT2B17 isoforms. Dealkylated, mono-hydroxylated and carboxylated metabolites both in the free

and in the glucuronidated form appear to be the most suitable urinary diagnostic markers for the

detection of tolvaptan intake in doping control.

An LC–MS/MS method for simultaneous determination of nine steroidal saponins from Paris

polyphylla var. in rat plasma and its application to pharmacokinetic study

Guangyi Yang, Wei Lu, Meng Pan, Chenning Zhang, Gao Song

ABSTRACT

Paris polyphylla var is an herbal plant herb widely used in Traditional Chinese Medicine. The purpose

of this study is to develop an Ultra Performance Liquid Chromatography-tandem mass spectrometer

(UPLC–MS) method to quantify the major components (i.e., nine saponins) from P. polyphylla in

plasma samples. A UItra BiPh column (100 × 2.1 mm, 5 μm) was used with acetonitrile/0.1% formic

acid in water as mobile phases. The analytes were quantified using a Waters XEVO TQ mass

spectrometer via multiple reaction monitoring (MRM) with positive scan mode. A protein precipitation

method was used to extract the analytes from rat plasma. The inter/intra-day precision, accuracy,

recovery, matrix effect, and stability were evaluated per the FDA guidance. The method showed

linearity in the concentration ranges of 2.4–1250 ng/mL. The intra-day and inter-day precisions (RSD)

of these analytes at three different levels were less than 15.0%. The extraction recoveries of these

analytes were from 83.8% to 109.4% and the matrix effects ranged from 87.4% to 105.4%. The

stabilities of these compounds in plasma were evaluated by analyzing three different concentrations

following storage at 25 °C for 6 h, and −80 °C for 30 days. All the samples displayed less than 15.0%

variations.

298

N-glucuronidation catalyzed by UGT1A4 and UGT2B10 in human liver microsomes: Assay

optimization and substrate identification

Danyi Lu, Qian Xie, Baojian Wu

ABSTRACT

N-glucuronidation is an important pathway for metabolism and disposition of tertiary amines in

humans. This reaction is mainly catalyzed by the enzymes UGT1A4 and UGT2B10. However, the

metabolic patterns of UGT1A4- and UGT2B10-mediated N-glucuronidation are not fully clear. In this

study, we first optimized in vitro reaction conditions for N-glucuronidation by using specific substrates

(i.e., trifluoperazine for UGT1A4, cotinine and amitriptyline for UGT2B10). Furthermore, we found

that hepatic N-glucuronidation showed significant species differences. In addition, UGT1A4 and

UGT2B10 were primarily responsible for N-glucuronidation of many tertiary amines, including

asenapine, loxapine, clozapine, chlorpromazine, dothiepin, doxepin, mirtazapine, mianserin,

chlorcyclizine, cyclizine, promethazine, cyclobenzaprine, imatinib, retrorsine, strychnine and brucine.

In conclusion, this study provides an in vitro assay system for evaluating N-glucuronidation of amines.

Also, UGT1A4- and UGT2B10-mediated N-glucuronidation might play significant roles in

metabolism and detoxification of tertiary amines in humans.

A simple high performance liquid chromatography–mass spectrometry method for Therapeutic

Drug Monitoring of isavuconazole and four other antifungal drugs in human plasma samples

Giovanna Fatiguso, Fabio Favata, Ilaria Zedda, Amedeo De Nicolò, Antonio D‘Avolio

ABSTRACT

Triazoles chanced the prevention and treatment of invasive fungal infections, but their

pharmacokinetic properties are still unclear. In particular, isavuconazole (ISC) is a new broad-

spectrum antifungal triazole approved in 2015 as first-line treatment for intravenous and oral use

against invasive aspergillosis and for mucormycosis. Nowadays, the optimal management of the

treatments with triazoles requires the use of Therapeutic Drug Monitoring (TDM), in order to prevent

sub-therapeutic or toxic concentrations. In turn, the routine use of TDM requires reliable quantification

methods The aim of this work was the development and full validation of a HPLC-mass spectrometry

assay for the simultaneous quantification of fluconazole, itraconazole, isavuconazole, posaconazole

and voriconazole in human samples. Both standards and quality controls were prepared in human

plasma. After the addition of internal standard (6,7-dimethyl-2,3-di(2-pyridyl)quinaxoline for

voriconazole, posaconazole and itraconazole; stable isotope labeled compounds for fluconazole and

isavuconazole), protein precipitation with acetonitrile and dilution with water were performed.

Chromatographic separation was performed on Atlantis® T3 5 μm 4.6 × 150 mm column, with a

gradient of water and acetonitrile, both added with 0.05% formic acid. Accuracy intra-day and inter-

day imprecision fitted FDA and EMA guidelines, while matrix effects and recoveries resulted stable

between samples for each analyte. Stability results were in accordance with previously published data.

Finally, we tested this method by monitoring plasma concentrations in real patients and using external

quality controls with good results. This method resulted very simple, fast, cheap and very useful for

TDM application, to improve clinical management of antifungal therapy in critically ill patients.

299

Metabolic profiling of dehydrodiisoeugenol using xenobiotic metabolomics

Qian-Qian Lv, Xiao-Nan Yang, Dong-Mei Yan, Wei-Qing Liang, Fei Li

ABSTRACT

Dehydrodiisoeugenol (DDIE), a representative and major benzofuran-type neolignan in Myristica

fragrans Houtt., shows anti-inflammatory and anti-bacterial actions. In order to better understand its

pharmacological properties, xenobiotic metabolomics was used to determine the metabolic map of

DDIE and its influence on endogenous metabolites. Total thirteen metabolites of DDIE were identified

through in vivo and in vitro metabolism, and seven of them were reported for the first time in the

present study. The identity of DDIE metabolites was achieved by comparison of the MS/MS

fragmentation pattern with DDIE using ultra-performance chromatography electrospray ionization

quadrupole time-of-flight mass spectrometry (UPLC-ESI- QTOFMS). Demethylation and ring-

opening reaction were the major metabolic pathways for in vivo metabolism of DDIE. Recombinant

cytochrome P450 s (CYPs) screening revealed that CYP1A1 is a primary enzyme contributing to the

formation of metabolites D1-D4. More importantly, the levels of two endogenous metabolites 2,8-

dihydroxyquinoline and its glucuronide were significantly elevated in mouse urine after DDIE

exposure, which explains in part its modulatory effects on gut microbiota.

Development and validation of a reliable method for thiopurine methyltransferase (TPMT)


genotypic correlations

Supaporn Wiwattanakul, Santirhat Prommas, Nuttawut Jenjirattithigarn, Siwalee Santon, Chonlaphat

Sukasem

ABSTRACT

A liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for the

determination of thiopurine methyltransferase (TPMT) activity in human whole blood lysate, based on

conversion of 6-mercaptopurine (6-MP) by TPMT to 6-methylmercaptopurine (6-MMP) using S-

adenosyl-l-methionine (SAM) as the methyl donor. This method was improved from the previous

laborious method for washing of red cell lysate preparation to develop whole blood EDTA lysate. In

addition, the TPMT incubation was optimized and the chromatography was performed in a short

runtime of 7 min on a C18-column by detection via triple quadrupole mass spectrometry. The MS/MS

was optimally tuned to monitor mass to charge a ratio (m/z) for 6-MMP 167.2 → 151.9 and the isotope

6-MMP-d3 with m/z of 170.5 → 152.2 were applied as an internal standard. The calibration curve

covered the range of 2.5–360 ng/ml and the correlation coefficient was greater than 0.999. The

accuracy of this method was determined in four concentrations of control of quality that ranged

between 99.33 and 106.33%. The intra-assay coefficient of variation (CV) was less than 4.41% and the

inter-assay was less than 5.43%. This method developed for measuring TPMT by LC–MS/MS is a

reliable, safe, and simple with a small volume requirement (100 μl of whole blood EDTA). The assay

was used to study TPMT activity in 132 Thai children with a range from 29.0 to 89.1 nmol 6-MMP/g

Hb/h with means and median values of TPMT activity 55.9 ± 12.47 nmol 6-MMP/g Hb/h and 54.2

nmol 6-MMP/g Hb/h. The genotype-phenotype association of TPMT was evaluated for common

ethnic Thai single nucleotide polymorphisms (SNP) in 30 samples and demonstrated good

concordance.

300

Development and validation of a GC–MS method for the determination of hydroxyzine and its

active metabolite, cetirizine, in whole blood

Maria Katselou, Sotiris Athanaselis, Panagiota Nikolaou, Artemisia Dona, Ioannis Papoutsis

ABSTRACT

A simple, rapid, sensitive and accurate gas chromatography–mass spectrometric method was

developed and validated for the simultaneous determination of hydroxyzine and cetirizine in whole

blood. Solid-phase extraction procedure using Bond Elut LRC Certify II columns was used for the

isolation of hydroxyzine and cetirizine from 1 mL whole blood followed by derivatization with a

mixture of acetic anhydride:n-propanol (1:1, v/v). Limits of detection and quantification were 1.50 and

5.00 ng/mL, respectively. The assay was linear within the concentration range of 5.00–1000.0 ng/mL

and the correlation coefficient was R2 ≥ 0.993 for both analytes. Absolute recovery was determined at

three quality control concentration levels and was found to be at least 87.2% for both substances. Intra-

day and inter-day accuracy values for both hydroxyzine and cetirizine were ranged from −1.2 to 3.8%

and −2.7 to 2.0%, respectively, at the three concentration levels studied, whereas their respective intra-

day and inter-day precision values were less than 9.9 and 6.5%, respectively, in terms of relative

standard deviation (%RSD). The developed method was successfully applied for the quantification of

hydroxyzine and cetirizine concentrations in whole blood, during the investigation of clinical cases

where these two antihistamines were detected.

An on-spot internal standard addition approach for accurately determining colistin A and

colistin B in dried blood spots using ultra high-performance liquid chromatography–tandem

mass spectrometry

I-Lin Tsai, Ching-Hua Kuo, Hsin-Yun Sun, Yu-Chung Chuang, Yun-Jung Tsai

ABSTRACT

Outbreaks of multidrug-resistant Gram-negative bacterial infections have been reported worldwide.

Colistin, an antibiotic with known nephrotoxicity and neurotoxicity, is now being used to treat

multidrug-resistant Gram-negative strains. In this study, we applied an on-spot internal standard

addition approach coupled with an ultra high-performance liquid chromatography-tandem mass

spectrometry (LC–MS/MS) method to quantify colistin A and B from dried blood spots (DBSs). Only

15 μL of whole blood was required for each sample. An internal standard with the same yield of

extraction recoveries as colistin was added to the spot before sample extraction for accurate

quantification. Formic acid in water (0.15%) with an equal volume of acetonitrile (50:50 v/v) was used

as the extraction solution. With the optimized extraction process and LC–MS/MS conditions, colistin

A and B could be quantified from a DBS with respective limits of quantification of 0.13 and 0.27 μg

mL−1, and the retention times were < 2 min. The relative standard deviations of within-run and

between-run precisions for peak area ratios were all < 17.3%. Accuracies were 91.5-111.2% for lower

limit of quantification, low, medium, and high QC samples. The stability of the easily hydrolyzed

prodrug, colistin methanesulfonate, was investigated in DBSs. Less than 4% of the prodrug was found

to be hydrolyzed in DBSs at room temperature after 48 h. The developed method applied an on-spot

internal standard addition approach which benefited the precision and accuracy. Results showed that

DBS sampling coupled with the sensitive LC–MS/MS method has the potential to be an alternative

approach for colistin quantification, where the bias of prodrug hydrolysis in liquid samples is

decreased.

301

Comparative study of single/combination use of Huang-Lian-Jie-Du decoction and berberine on

their protection on sepsis induced acute liver injury by NMR metabolic profiling

Yan Lv, Junsong Wang, Dingqiao Xu, Shanting Liao, Lingyi Kong

ABSTRACT

Sepsis is a serious clinical disease with a high mortality rate all around the world. Liver organ

dysfunction is an important sign for the severity and outcome of sepsis in patients. In this study, 1H

NMR-based metabolomics approach and biochemical assays were applied to investigate the metabolic

profiling for cecal ligation and puncture (CLP) induced acute liver injury, the therapeutical effect of

single/combination use of Huang-Lian-Jie-Du decoction (HLJDD) and berberine, and the interaction

of them. Metabolomics analysis revealed significant perturbations in livers of septic rats, which could

be ameliorated by HLJDD, berberine and their combination treatment. Berberine could better rectified

glycolysis and nucleic acid metabolism in the liver. HLJDD had exceptional better anti-inflammatory,

antibacterial and antioxidative effects than berberine. The interaction of berberine and HLJDD could

further strengthen the anti-inflammation and anti-oxidation, but with poor effect on amino acids

metabolism. These findings highlighted the feasibility of the integrated NMR based metabolomics

approach to understand the pathogenesis of diseases, the action mechanisms of therapy and the herb-

drug interaction.

Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices:


separations

Ádám Tölgyesi, Enikő Barta, Andrea Simon, Thomas J. McDonald, Virender K. Sharma

ABSTRACT

Veterinary drugs containing synthetic anabolic steroid and nitroimidazole active agents are not allowed

for their applications in livestock of the European Union (EU). This paper presents analyses of twelve

selected steroids and six nitroimidazole antibiotics at low levels (1.56 μg/L–4.95 μg/L and 0.17 μg/kg–

2.14 μg/kg, respectively) in body fluids and egg incurred samples. Analyses involved clean-up

procedures, high performance liquid chromatography (HPLC) separation, and tandem mass

spectrometric screening and confirmatory methods. Target steroids and nitroimidazoles in samples

were cleaned by two independent supported liquid extraction and solid phase extraction procedures.

Separation of the selected compounds was conducted on Kinetex XB C-18 HPLC column using

gradient elution. The screening methods utilised supported liquid extraction that enabled fast and cost

effective clean-up. The confirmatory methods were improved by extending the number of matrices and

compounds, and by introducing an isotope dilution mass spectrometry for nitroimidazoles. The new

methods were validated according to the recommendation of the European Union Reference

Laboratories and the performance characteristics evaluated met fully the criteria. The methods were

applied to incurred samples in the proficiency tests. The obtained results of Z-scores demonstrated the

applicability of developed protocols of the methods to real samples. The confirmatory methods were

applied to the national monitoring program and natural contamination of prednisolone could be

detected in urine at low concentration in few samples.

302

Validation of an HPLC-UV method for analysis of Kaempferol-loaded nanoemulsion and its

application to in vitro and in vivo tests

Mariana Colombo, Gabriela de Lima Melchiades, Fabrício Figueiró, Ana Maria Oliveira Battastini,

Letícia Scherer Koester

ABSTRACT

A simple and reliable HPLC-UV method for Kaempferol (KPF) determination in a Kaempferol-loaded

nanoemulsion (KPF-NE), samples from mucosa permeation/retention studies, and murine brain was

developed and validated according to international guidelines. The analyses were performed on a

reversed-phase C18 column at 35 °C and under UV detection at 368 nm. The mobile phase was

composed of methanol:formic acid 0.1% (75:25, v/v) and was eluted at an isocratic flow rate of 1.0

mL/min. The method was selective and sensitive for KPF analysis in matrix extracts, and linear in the

range of 0.25–7.5 μg/mL. The method was also considered precise, accurate, and robust. The recovery

rates of KPF from the porcine nasal mucosa and murine brain were higher than 85%. Low matrix

effect was observed to determine KPF, including biological matrices. The applicability of the method

was confirmed in all different approaches, i.e., quantification of KPF in nanoemulsion, in vitro

permeation/retention of KPF across porcine nasal mucosa, and in vivo quantification of KPF in brain

samples after nasal administration in rats. Thus, the method is effective and reliable to determine KPF

in different real samples. The proposed method, therefore, provides a useful quantification approach to

routine processes, to the development of drug delivery systems, and to KPF quantification in different

biological matrices. Furthermore, the method is applicable in bioavailability studies and the developed

formulation (KPF-NE) is suitable for preclinical trials in different brain disorders.

Development of direct assays for Toxoplasma gondii and its use in genomic DNA sample

Lívia M. Alves, Vinícius R. Rodovalho, Ana C.H. Castro, Márcia A.R. Freitas, Ana G. Brito-Madurro

ABSTRACT

This work describes an approach for the selection and detection of specific DNA probes related to

Toxoplasma gondii, a protozoan parasite responsible for toxoplasmosis. The detection system was

developed on graphite carbon electrode modified with poly(3-hydroxybenzoic acid) sensitized with

ToxG1 probe. The hybridization of the specific genomic DNA related to T. gondii showed good

response by direct detection of guanine residue oxidation using differential pulse voltammetry (DPV).

The biosensor was able to distinguish both the complementary and non-complementary targets and

detect up to 100 ng μL−1 of the T. gondii genomic DNA. The hybridization (ToxG1: T. gondii

genomic DNA) was confirmed by optical measurement. Optical assays using gold

nanoparticles:ToxG1 probe showed a significant change in the absorbance peak in the presence of the

T. gondii genomic DNA according to the electrochemical results. This novel biosensor shows potential

as electrochemical transducer and was successfully applied in the biological sample.

303

Investigation and structural elucidation of a new impurity in bulk drug of cilostazol by

LC/MS/MS, FT-IR and NMR

Zhaoxia Hu, Sanguo Gao, Jue Gao

ABSTRACT

A new impurity was detected in bulk cilostazol (CIL) crude during routine analysis. The impurity

(∼4%, the specification for unknown impurity in crude is not more than 0.20%) has a relative retention

time of 1.46. Based on MS, NMR and IR spectral data, the impurity was identified as 6,6′-bis(4-(1-

cyclohexyl-1H-tetrazol-5-yl) butoxy)-3,3′,4,4′-tetrahydro-[7,7′-biquinoline]-2,2′(1H,1′H)-dione(CIL-

dimer). The precursor of CIL-dimer is an oxidative product of starting material 6-Hydroxy-3,4-

dyhydro-1H-quinolin–2-one(6-HQ), CIL-dimer was formed in the following reaction with 5-(3-

Chloro-propyl)-1-cyclohexyl-1H-tetrazol(CHCBT).

Selective screening of glutaric acid acidurias by capillary electrophoresis-mass spectrometry

Jaime Fernández-Bravo, Fernando de Andrés, Mohammed Zougagh, Ángel Ríos

ABSTRACT

A sensitive and selective method for the separation and quantification of the three organic acids 3-

hydroxy-3-methylglutaric acid, 3-methylglutaric acid, and glutaric acid in human urine samples by CE

with mass spectrometry detection has been developed. This methodology is faster, simpler and less

time-consuming, than other methodologies previously described, and requires of reduced amounts of

reagents as well. Samples are first filtered and then diluted in water. For the electrophoretic separation,

a 20 mM ammonium acetate and 10% methanol solution at pH 9.1 was selected as the running

electrolyte. With 5-s hydrodynamic injection, detection limits ranging from 15.5 to 39.3 μM and linear

responses ranging from the LOQ calculated for each analyte to more than 400 μM were obtained for

the analysis of the different organic acids in less than 13 min. Remarkable selectivity is achieved by

mass spectrometry detection using 0.25% of formic acid in 50% v/v 2-propanol-water solution as

sheath liquid, and enough sensitivity without interferences from the matrices was obtained as well.

This methodology has revealed as an efficient approach to help the 3-hydroxy-3-methylglutaric

aciduria diagnoses in order to discard or confirm the occurrence of the disease as of the presence or

absence of the expected increased levels of these analytes in samples of potential patients.

304

Online turbulent flow extraction coupled with liquid chromatography–tandem mass

spectrometry for high throughput screening of anabolic steroids in horse urine

Hyun Du Shin, Joon Hyuk Suh, Junghyun Kim, Hyun-Deok Cho, Sang Beom Han

ABSTRACT

A high throughput method for simultaneous screening of anabolic steroids and their metabolites (4-

esterendione, trenbolone, boldenone, oxandrolone, nandrolone, methandrostenolone, testosterone, 1-

androstendione, ethisterone, normethandrolone, methyltestosterone, 16β-Hydroxystanozolol,

epitestosterone, bolasterone, norethandrolone, danazol, stanozolol and androstadienone) in equine

urine by online turbulent flow extraction coupled with liquid chromatography-tandem mass

spectrometry was developed. The use of turbulent flow chromatography could simplify pretreatment of

horse urine, which has complex matrices as well as high viscosity. The urine was extracted by mixed-

mode cation exchange solid phase extraction, and hydrolyzed using β-glucuronidase/arylsulfatase.

Then, the sample was automatically loaded on the TurboFlow Cyclone extraction column for removal

of further matrix, followed by separation on a fused core C18 column before MS/MS detection.

Optimization and validation of the method were discussed in detail. All analytes were rapidly detected

within 10 min with high sensitivity (picogram to nanogram per milliliter level), and no interference

was observed. The linearity range was from 0.1–10 ng/mL for nine steroids and 1.0–50 ng/mL for the

others, with correlation of coefficient values over 0.995. Precision and accuracy ranged from 0.1 to

14.5% and 1.7 to 12.4%, respectively. The developed method was successfully applied to the analysis

of anabolic steroids in horse urine after administration of a model drug.

Development of a mixed-mode chromatography with tandem mass spectrometry method for the

quantitative analysis of 23 underivatized amino acids in human serum

Min Sun Choi, Shaheed Ur Rehman, In Sook Kim, Hi-Joon Park, Hye Hyun Yoo

ABSTRACT

In this study, a robust, selective and simplified method was developed and validated for the

simultaneous quantitative analysis of 23 underivatized amino acids in human serum using mixed-mode

chromatography with tandem mass spectrometry (LC–MS/MS). Serum samples were deproteinized

with acetonitrile and subjected to LC–MS/MS analysis. The chromatographic separation of amino

acids was achieved using a mixed-mode column (150 × 3 mm, 3 μm) with a gradient elution system;

the mobile phase consisted of 50 mM ammonium formate and 0.1% formic acid in acetonitrile. The

total run time was 22 min. Eluted compounds were detected in the electrospray ionization-positive

mode with multiple reaction monitoring. The validation study evaluated linearity, repeatability, intra

and inter-day accuracy and precision, and matrix effect. The validation results were satisfactory in all

the tested parameters. This method was successfully applied to the analysis of amino acids in the

clinical sample of human serum.

305

Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance

sensor

Atsushi Shoji, Yumiko Suenaga, Atsushi Hosaka, Yuuki Ishida, Masao Sugawara

ABSTRACT

We describe a simple method for evaluating the inhibition of collagen IV degradation by cathepsin B

with a surface plasmon resonance (SPR) biosensor. The change in the SPR signal decreased with an

increase in the concentration of cathepsin B inhibitors. The order of the inhibitory constant (Ki)

obtained by the SPR method was CA074Me ≈ Z-Phe-Phe-FMK < leupeptin. This order was different

from that obtained by benzyloxycarbonyl-Phe-Phe-Fluoromethylketone (Z-Phe-Phe-FMK) as a peptide

substrate. The comparison of Ki suggested that CA074 and Z-Phe-Phe-FMK inhibited exopeptidase

activity, and leupeptin inhibited the endopeptidase activity of cathepsin B more strongly.

Liquid chromatography-tandem mass spectrometry assay to quantify plitidepsin in human

plasma, whole blood and urine

L. van Andel, H. Rosing, S. Fudio, P. Avilés, J.H. Beijnen

ABSTRACT

Plitidepsin is an anti-cancer drug currently evaluated in phase I/II/III clinical trials. This article

describes the development and validation of a bioanalytical assay to quantify plitidepsin in human

plasma, urine and whole blood using HPLC–MS/MS. The analyte was extracted from the matrix by

liquid–liquid extraction using tert-butyl methyl ether. Final extracts were injected onto a C18 column,

gradient elution was applied for chromatographic separation and detection was performed on a triple

quadrupole mass spectrometer operating in the positive ion mode. The assay was linear over the range

0.1–100 ng/mL, with acceptable accuracy and precision values. This is the first reported bioanalytical

assay quantifying plitidepsin using a stable isotopically labelled standard, achieving a lower limit of

quantification of 0.1 ng/mL in all three matrices, allowing the quantification of trace levels of

plitidepsin and accomplishing this in an analysis time of two minutes only. The presented method was

successfully applied in a mass balance study with plitidepsin in patients with advanced cancer.

306

Rapid analysis of benzalkonium chloride using paper spray mass spectrometry

Jingjing Liu, Wenjie Deng, Muqian Yu, Ruizhi Wen, Bo Chen

ABSTRACT

A paper spray mass spectrometry (PS-MS) method for rapid and reliable analysis of benzalkonium

chloride (BAC) in compound eye drops and body surface disinfectant was developed. The sample was

dropped onto triangular filter paper, and high voltage (3.5 kV) was applied to form an electrospray.

This method can provide the composition of benzalkonium chloride in samples without pretreatment,

solvent or chromatographic separation, and the analysis time is only 10 s. The primary homologues

C12-BAC, C14-BAC and C16-BAC of benzalkonium chloride were quantitatively analyzed using PS-

MS. Samples were subjected to simple dilution and quantified using the internal standard method. Ion

trap mass spectrometry was scanned using SIM mode. The linear ranges of C12-BAC, C14-BAC and

C16-BAC were 1–100 μg mL−1; the linear regression coefficients were 0.998–0.999; the detection

limits (LODs) were 0.1 μg mL−1; the limit of quantifications (LOQs) was <1 μg mL−1, and the

method validation indicated that the method precision and accuracy were good. Compared with HPLC-

UV methods, there was no significant difference in the quantitative determination of the actual

samples, but the analysis time for PS-MS is shorter (2 min). In addition, reagent consumption in PS-

MS is small, and no chromatographic separation is needed, suggesting that PS-MS is especially

suitable for high-throughput analysis.

Rapid screening of non-steroidal anti-inflammatory drugs illegally added in anti-rheumatic

herbal supplements and herbal remedies by portable ion mobility spectrometry

Mengjiao Li, Haiyan Ma, Jinglin Gao, Lina Zhang, Ye Jiang

ABSTRACT

In this work, for the first time, a high-performance ion mobility spectrometry with electrospray

ionization (ESI-HPIMS) method has been employed as a rapid screening tool for the detection of

acetaminophen, ibuprofen, naproxen, diclofenac sodium and indomethacin illegally added in anti-

rheumatic herbal supplements and herbal remedies. Samples were dissolved and filtered through a 0.45

μm microporous membrane, then the filtrate was directly injected into the high-performance ion

mobility spectrometry for analysis. Using this approach, the screening of illegal additions can be

accomplished in as rapid as two to three minutes with no pretreatment required. The proposed method

provided a LOD of 0.06–0.33 μg mL−1, as well as a good seperation of the five NSAIDs. The

precision of the method was 0.1–0.4% (repeatability, n = 6) and 0.9–3.3% (reproducibility, n = 3). The

proposed method appeared to be simple, rapid and highly specific, thus could be effective for the in-

situ screening of NSAIDs in anti-rheumatic herbal supplements and herbal remedies.

307

Simultaneous quantitative analysis of polyethylene glycol (PEG), PEGylated paclitaxel and


spectrometry

Heping Sun, Qi Zhang, Zhi Zhang, Jin Tong, Jingkai Gu

ABSTRACT

PEGylation is practically one of most important modifications of drugs including small molecules,

peptides and proteins, which has been proven to dramatically improve physicochemical properties and

pharmacokinetic behavior of the PEGylated drugs. However, it is a challenge currently to

quantitatively analyze PEG and PEGylated drugs by various analytical methods, even mass

spectrometry because of multiple parent ion distribution of PEG caused by its polydispersity of

molecular weight. Here we developed a robust method with MS/MSALL technique using electrospray

ionization (ESI) source coupled high resolution Quadrupole Time-of-Flight (Q-TOF) mass

spectrometry for the quantification of PEG2K-Paclitaxel (PEG-PTX) and its two metabolites, PEG and

Paclitaxel (PTX). The analysis was performed on a 300SB-C18 column with acetonitrile and 0.1%

formic acid as the mobile phase. Samples were simply prepared by protein precipitation in a small

quantity of plasma (50 μL). Calibration curve was linear within the range of 50.0–4000 ng/mL for

PEG and PEG-PTX and 1.0–1000 ng/mL for PTX. The intra- and inter-day precisions were 3.2–6.9%

and 3.1–6.9% for PEG, 4.1–7.8% and 4.0–9.9% for PEG-PTX, and 3.3–4.8% and 3.1–6.9% for PTX,

respectively. The recoveries were greater than 90% with low matrix effects. Afterwards, the newly

developed method was successfully applied to support a preclinical pharmacokinetic study in six rats

after single intravenous injection of PEG-PTX (51.7 mg/kg).

Lyophilic matrix method for dissolution and release studies of nanoscale particles

Jenni Pessi, Sami Svanbäck, Ilkka Lassila, Edward Hæggström, Jouko Yliruusi

ABSTRACT

We introduce a system with a lyophilic matrix to aid dissolution studies of powders and particulate

systems. This lyophilic matrix method (LM method) is based on the ability to discriminate between

non-dissolved particles and the dissolved species. In the LM method the test substance is embedded in

a thin lyophilic core-shell matrix. This permits rapid contact with the dissolution medium while

minimizing dispersion of non-dissolved particles without presenting a substantial diffusion barrier. The

method produces realistic dissolution and release results for particulate systems, especially those

featuring nanoscale particles. By minimizing method-induced effects on the dissolution profile of

nanopowders, the LM method overcomes shortcomings associated with current dissolution tests.

308

Incorporation of 14C-cholesterol in human adrenal corticocarcinoma H295R cell line and

online-radiodetection of produced 14C-steroid hormone metabolites

Jonas Abdel-Khalik, Erland Björklund, Frederik Knud Nielsen, Martin Hansen

ABSTRACT

This study demonstrates the addition of 14C-cholesterol to the human cell line H295R will in-situ form

radiolabeled steroid hormones allowing for new mechanistic and metabolic insights. The aim of the

present study was to in-situ radiolabel steroid hormones from cell line-incorporated 14C-cholesterol

using the OECD guideline 456, H295R steroidogenesis in-vitro assay. Radiodetection of the steroid

metabolites of the steroidogenic pathway allows for an improved understanding of the various

enzymatic mechanisms involved without necessarily being dependent on quantification. Generated

radiolabeled steroids were analyzed using HPLC hyphenated with a Flow Scintillation Analyzer

(FSA). H295R cells were incubated with radiolabeled cholesterol and cell media were collected and

prepared by solid phase extraction and analyzed with HPLC-FSA. For successful radiolabeling of the

steroids in the steroidogenesis of H295R cells, radioactive cholesterol may potentially only need to be

added just before the cells are incubated for 72 h in well plates. Based on the obtained HPLC-FSA

chromatograms, and confirmation of the observations by studies in the literature, a qualitative time

profile for the production of steroid hormones was estimated. Multiple radiolabeled steroid hormones

were identified by means of analytical standards and UV (ultraviolet) co-chromatography, though the

elucidation of multiple metabolites remains unresolved. Although online radiodetection proved to

suffer from suboptimal sensitivity, the concept of radiolabeling the steroidogenesis in H295R cells

with 14C-cholesterol and detecting the radiolabeled steroid hormones online was proved and may

assist in further toxicological studies.

Verification of the effectiveness of the Fourier transform infrared spectroscopy computational

model for colorectal cancer

J. Depciuch, E. Kaznowska, A. Koziorowska, J. Cebulski

ABSTRACT

Colorectal cancer is one of the most common cancers. Its formation is influenced by genetic and

environmental factors. Despite the continuous development of diagnostic tools and cancer therapies,

there are no methods that allow a real-time estimation of treatment efficiency. This method can be a

vibrational spectroscopy. The resulting infrared spectrum (FTIR) of the tissue gives us information

about the chemical composition and the content of the individual components. We have noticed that

tumor tissues, healthy and after chemotherapy tissues, have different vibrational spectra. It was also

shown that spectra acquired from normal (benign) tissues were similar to those derived from tissues

post-chemotherapy. The similarity was greater, when the effectiveness of chemotherapy, confirmed by

medical documentation, was better. Therefore, we decided to use the physical model proposed in our

earlier paper to verify its correctness and to show whether a particular type of chemotherapy was

effective or not. Comparison of the results obtained from the physical model with patients data have

been found as close to the physical condition.

309

Quantification of paracetamol and 5-oxoproline in serum by capillary electrophoresis:

Implication for clinical toxicology

Tomáń Hloņek, Tomáń Kříņek, Petr Tůma, Miroslava Bursová, Radomír Čabala

ABSTRACT

High anion gap metabolic acidosis frequently complicates acute paracetamol overdose and is generally

attributed to lactic acidosis or compromised hepatic function. However, metabolic acidosis can also be

caused by organic acid 5-oxoproline (pyroglutamic acid). Paracetamol‘s toxic intermediate, N-acetyl-

p-benzoquinoneimine irreversibly binds to glutathione and its depletion leads to subsequent disruption

of the gamma glutamyl cycle and an excessive 5-oxoproline generation. This is undoubtedly an

underdiagnosed condition because measurement of serum 5-oxoproline level is not readily available. A

simple, cost effective, and fast capillary electrophoresis method with diode array detection (DAD) for

simultaneous measurement of both paracetamol (acetaminophen) and 5-oxoproline in serum was

developed and validated. This method is highly suitable for clinical toxicology laboratory diagnostic,

allowing rapid quantification of acidosis inducing organic acid 5-oxoproline present in cases of

paracetamol overdose. The calibration dependence of the method was proved to be linear in the range

of 1.3–250 μg mL−1, with adequate accuracy (96.4–107.8%) and precision (12.3%). LOQ equaled 1.3

μg mL−1 for paracetamol and 4.9 μg mL−1 for 5-oxoproline.

Quantification of cyclocreatine in mouse and rat plasma using hydrophilic-interaction ultra-

performance liquid chromatography-tandem mass spectrometry

Amy Q. Wang, Emma Hughes, Wenwei Huang, Edward H. Kerns, Xin Xu

ABSTRACT

An accurate, rapid and selective method was developed to quantify cyclocreatine in mouse and rat

plasma using hydrophilic interaction (HILIC) ultra-performance liquid chromatography-tandem mass

spectrometry (UPLC–MS/MS). The plasma samples were prepared by protein precipitation with

acetonitrile:methanol (70:30). Chromatographic separation was performed on a HILIC BEH amide

column (2.1 mm × 50 mm, 1.7 μm) with a 3 min gradient elution at a flow rate of 0.5 mL/min. For

mass spectrometric detection, selected reaction monitoring (SRM) was used; the SRM transitions were

m/z 144 → 98 and m/z 144 → 56 for cyclocreatine and m/z 148 → 102 for the internal standard (D4-

cyclocreatine) in the positive ionization mode. No endogenous components interfered with the analysis

of cyclocreatine and the internal standard in mouse and rat plasma. Plasma calibration curves were

constructed in the range of 0.01–25 μM. The correlation coefficient of the calibration curves was

greater than 0.99. The mean intraday assay accuracy for all quality control (QC) replicates was

between 93 and 105%. The mean intraday assay precision (CV%) was 1.9-11% for all QC levels. The

HILIC–UPLC–MS/MS method was successfully applied in pharmacokinetic (PK) studies of

cyclocreatine in mice and rats for the first time. After a single 30 mg/kg oral administration in mice

and rats, the AUC0-∞ (area under the curve) was 84.1 μg h/mL and 91.7 ± 18.0 μg h/mL, respectively.

310

Volumetric adsorptive microsampling-liquid chromatography tandem mass spectrometry assay


validation and comparison with dried blood spot

Sebastiano Barco, Elio Castagnola, Andrea Moscatelli, James Rudge, Giuliana Cangemi

ABSTRACT

Summary: In this paper we show the development and validation of a volumetric absorptive

microsampling (VAMS™)-LC–MS/MS method for the simultaneous quantification of four antibiotics:

piperacillin-tazobactam, meropenem, linezolid and ceftazidime in 10 μL human blood. The novel

VAMS-LC–MS/MS method has been compared with a dried blood spot (DBS)-based method in terms

of impact of hematocrit (HCT) on accuracy, reproducibility, recovery and matrix effect. Antibiotics

were extracted from VAMS and DBS by protein precipitation with methanol after a re-hydration step

at 37 °C for 10 min. LC–MS/MS was carried out on a Thermo Scientific™ TSQ Quantum™ Access

MAX triple quadrupole coupled to an Accela ™UHPLC system. The VAMS-LC–MS/MS method is

selective, precise and reproducible. In contrast to DBS, it allows an accurate quantification without any

HCT influence. It has been applied to samples derived from pediatric patients under therapy. VAMS is

a valid alternative sampling strategy for the quantification of antibiotics and is valuable in support of

clinical PK/PD studies and consequently therapeutic drug monitoring (TDM) in pediatrics.

PLGA Ethionamide Nanoparticles for Pulmonary Delivery: Development and in vivo evaluation

of dry powder inhaler

Sujit Kumar Debnath, Srinivasan Saisivam, Abdelwahab Omri

ABSTRACT

PLGA (50:50) nanoparticles were prepared to sustain the release of Ethionamide in order to decrease

the dose and dosing frequency. It further modified in the form of dry powder inhaler to make suitable

for pulmonary administration and increase drug residency in lungs. Ethionamide loaded PLGA

nanoparticles were prepared by solvent evaporation method. Freeze dried nanoparticles and anhydrous

inhalable grade lactose were mixed manually using geometrical dilution process to modify the

nanoparticles in the form of dry powder inhaler. Animal study was conducted to correlate between in-

vivo and in-vitro. PLGA nanoparticles showed initial burst release followed by zero order release up to

95.17 ± 3.59% in 24 h. Aerodynamic particle size of optimized dry powder inhaler was found as 1.79

μm. There was no significant aggregation of dry powder inhaler during 6 months of stability study.

Area under the concentration-time curve from 0 h to infinity (AUC0−∞) signifies the prolong

residency of ETH in body compartment, revealed from animal study. PLGA 50:50 coated

nanoparticles released Ethionamide for the period of 24 h in simulated lungs fluid. Correlation

between in-vitro dissolution and in-vivo study was established after performing animal study. Prepared

dry powder inhaler maintained Ethionamide concentration above minimum inhibitory concentration

for more than 12 h after single dose administration.

311

Simultaneous determination and pharmacokinetics of danshensu, protocatechuic aldehyde, 4-


MS/MS after oral administration of Compound Danshen Dripping Pills

Wei Li, Hongjie Zhou, Yang Chu, Xiangyang Wang, He Sun

ABSTRACT

Compound Danshen Dripping Pills (CDDP), a herbal patent medicine, is widely used in China for the

prevention and treatment of cardiovascular diseases. A simple, sensitive and reliable method for

simultaneous determination of danshensu (DSS), protocatechuic aldehyde (PCA), and their related

metabolites, 4-hydroxy-3-methyloxyphenyl lactic acid (HMLA) and protocatechuic acid (PAA) in

human plasma was developed and validated based on liquid chromatography tandem mass

spectrometry (LC–MS/MS). The analytes and internal standard (IS), vanillic acid (VAA), were

extracted from plasma with ethyl acetate and separated on a C18 column by using the mobile phase

consisted of methanol-0.1% formic acid via gradient elution. The electrospray ionization (ESI) source

was applied and operated under the multiple reaction monitoring (MRM) mode. The linear calibration

curves were obtained at the concentration ranges of 0.46–1000 ng/mL for DSS and PAA, and 1.38–

1000 ng/mL for PCA and HMLA, respectively. The inter- and intra-day precisions (RSD%) were less

than 13.5%, and the accuracy (±RE%) was within 13.4%. The described method was successfully

applied for the clinical pharmacokinetics of CDDP in Chinese healthy volunteers.

LC–MS bioanalysis of Trastuzumab and released emtansine using nano-surface and molecular-


emtansine-treated breast cancer patients

Noriko Iwamoto, Akihiko Shimomura, Kenji Tamura, Akinobu Hamada, Takashi Shimada

ABSTRACT

Antibody-drug conjugates (ADCs) consist of monoclonal antibody and cytotoxic drugs covalently

attached via stable crosslinkers, and are prospective antibody drugs for cancer therapy. To cover the

overall pharmacokinetic understanding of ADCs, both the antibody and the released drugs are

necessary for practical clinical observation. The nano-surface and molecular-orientation limited

(nSMOL) proteolysis is a universal approach for antibody bioanalysis that enable Fab-selective

proteolysis, which maintains antibody sequence specificity while decreasing excess analyte peptides.

In this study, we describe quantitative assays for ADC in human plasma using nSMOL for the

antibody and polarity-selective liquid–liquid partition with a methanol/ethyl acetate mixed solvent for

the cytotoxic drugs. This approach led to the successful development of LC–MS validated bioanalysis

of the antibody and released drugs within 20% for lower limit of quantitation and 15% for another

concentration setting of Trastuzumab emtansine (T-DM1), Trastuzumab antibody and emtansine

conjugated with crosslinker (DM1-MCC). The validated concentration ranges in human plasma were

0.06–250 μg/mL for T-DM1 and 0.39–200 ng/mL for DM1-MCC. These results indicate that LC–MS

method with a two-sided approach, using nSMOL and liquid–liquid partition, show potential for the

precise pharmacokinetic study for ADC development and treatment.

312

Separation of furostanol saponins by supercritical fluid chromatography

Jie Yang, Lingling Zhu, Yang Zhao, Yongwei Xu, Baiping Ma

ABSTRACT

Supercritical fluid chromatography (SFC) has good separation efficiency and is suitable for separating

weakly polar compounds. Furostanol saponins, as an important kind of steroidal saponins, generally

have two sugar chains, which are polar and hydrophilic. The hydroxyl group at the C-22 position of

furostanol saponins is active and easily reacts with lower alcohols under appropriate conditions. The

separation of hydrophilic furostanol saponins was tested by SFC in this study. The effects of

chromatographic conditions on the separation of the mixed furostanol saponins and their hydroxyl

derivatives at the C-22 position were studied. The conditions for SFC, which included different

column polarity, modifier, additive, and column temperature, were tested. After optimization, the

mixed 10 similar structures of furostanol saponins were separated in 22 min on the Diol column at a

temperature of 40 °C. The mobile phase was CO2 (mobile phase A) and methanol (containing 0.2%

NH3∙H2O and 3% H2O) (mobile phase B). The backpressure was maintained isobarically at 11.03

MPa. SFC was found to be effective in separating the furostanol saponins that shared the same

aglycone but varied in sugar chains. SFC was sensitive to the number and type of sugars. The

resolution of furostanol saponin isomers was not ideal. The extract of Dioscorea zingiberensis C. H.

Wright was profiled by SFC–quadrupole time-of-flight mass spectrometry. The main saponins of the

extract were well separated. Therefore, SFC could be used for separating hydrophilic furostanol

saponins and analyzing traditional Chinese medicines that mainly contained steroidal saponins.

Macroporous monoliths for biodegradation study of polymer particles considered as drug

delivery systems

M.V. Volokitina, V.A. Korzhikov-Vlakh, T.B. Tennikova, E.G. Korzhikova-Vlakh

ABSTRACT

Nanostructures based on biodegradable polymers are often considered as drug delivery systems. The

properties of these nanomaterails towards in vitro biodegradation are very important and usually are

studied using the model physiological conditions. In this work the novel approach based on application

of monolithic immobilized enzyme reactors (IMERs) as the systems for biodegradation study of the

nanoobjects of different nature and morphology was suggested. Rigid nanospheres based on

poly(lactic acid) and self-assembled nanoobjects formed from block-copolymer of glutamic acid and

phenylalanine were applied as model nanomaterials. For that, two enzymes, namely, esterase and

papain were chosen for preparation of the monolithic IMERs. The properties of immobilized enzymes

were compared to those obtained for soluble biocatalysts in the reaction of poly(lactic acid) and

poly(glutamic acid) degradation. The monitoring of substrate destruction process was carried out using

different HPLC modes (anion-exchange, cation-exchange or precipitation-redissolution based process)

also based on application of the same modern stationary phase, namely, macroporous monoliths (CIM

disks and lab-made column). Finally, the applicability of monolithic immobilized enzyme reactors for

degradation of polyester and polypetide-based particles was demonstrated and compared to the process

observed in human blood plasma.

313

A novel enantioseparation approach based on liposome electrokinetic capillary chromatography

Xiaoqi Li, Yingxiang Du, Zijie Feng, Xiaodong Sun, Zhifeng Huang

ABSTRACT

As a novel separation mode of capillary electrophoresis (CE), liposome electrokinetic capillary

chromatography (LEKC) has aroused considerable attention in recent years; however, the

enantioseparation based on this new system has not been previously investigated. In this study, we

proposed a brand-new LEKC chiral separation approach using liposomes comprised of

phosphatidylcholine (PC) and cholesterol as pseudo-stationary phase and sulfobutyl ether-β-

cyclodextrin (SBE-β-CD) as chiral selector. Compared with the single CD system and CD-SDS-

MEKC system, this LEKC method presented an obviously preferable enantioseparation of four model

drugs (naproxen, warfarin, ketoprofen and amlodipine). In this new established system, all the

enantiomers represented baseline separations with the resolution and selectivity respectively achieving

1.584/1.067 (for naproxen), 2.226/1.045 (for warfarin), 1.537/1.038 (for ketoprofen) and 2.592/1.097

(for amlodipine), while other two comparative systems demonstrated no separation or a poor

separation. Several important parameters affecting the enantioseparation, such as buffer pH,

concentration of liposomes, phosphate buffer solution (PBS) and chiral selector (SBE-β-CD), and

applied voltage were systematically investigated. Satisfactory repeatability was achieved through intra-

day, inter-day and batch-to-batch investigations with relative standard deviations less than 3.40%.

Furthermore, the established method was successfully applied to test the chiral impurity of naproxen

sample.

Comparative study on the anticancer activities and binding properties of a hetero metal

binuclear complex [Co(dipic)2Ni(OH2)5]·2H2O (dipic = dipicolinate) with two carrier proteins

Somaye Shahraki, Fereshteh Shiri, Mostafa Heidari Majd, Zohreh Razmara

ABSTRACT

Recognizing of binding mechanisms between drugs and carrier proteins is basic for us to understand

the pharmacokinetics and pharmacodynamics of them. In this research, the anticancer activities of a

binuclear complex [Co(dipic)2Ni(OH2)5]·2H2O (dipic = dipicolinate) against MDA-MB-231 cell

lines were studied. Results of MTT assay and flow cytometry analysis revealed that above complex

can induce the cytotoxicity and the apoptosis in breast cancer cell lines. So, this complex was selected

to investigate its binding to human serum albumin (HSA) and bovine β-lactoglobulin (βLG) by

spectroscopic methods (UV–visible, fluorescence and FT-IR) along with molecular docking technique.

The fluorescence data showed Co-Ni complex quench the fluorescence of both proteins by a static

quenching mechanism and HSA has stronger binding affinity toward Co-Ni complex than βLG. The

binding constant (Kb), number of binding sites (n) and thermodynamic parameters were calculated and

showed that the Co-Ni complex binds to protein (HSA and βLG) through hydrogen bonding and van

der Waals forces with one binding site. The results of UV–visible measurements indicated that the

binding of above complex to HSA and βLG may induce conformational and micro-environmental

changes of studied proteins. Protein–ligand docking analysis confirmed that the Co-Ni complex binds

to residues located in the subdomain IIA of HSA and site II of βLG.

314

Characterization and quantitative analysis of phenolic derivatives in Longxuetongluo Capsule by

HPLC-DAD-IT-TOF-MS

Jing Sun, Yuelin Song, Hui Sun, Wenjing Liu, Jun Li

ABSTRACT

Longxuetongluo Capsule (LTC), which is derived from the total phenolic extract of Chinese dragon‘s

blood, has been proved to be safe as well as effective towards ischemic stroke. However, the effective

material basis remains unclear. The present study thereby focused on the clarification of the qualitative

and quantitative properties for the phenolic derivatives in LTC. Regarding homolog-focused chemical

profiling, the mass fragmentation patterns of the primary subtypes of phenolic compounds such as

homoisoflavanones, flavanes, chalcones, and flavonoid oligomers were summarized by assaying

authentic references with hybrid ion trap time-of-flight mass spectrometry, and the chemical structures

of 124 phenolic compounds, in total, were unambiguously or tentatively annotated in LTC by

matching the accurate mass spectral profiles with the proposed mass cracking rules and those reference

substances. Afterwards, simultaneous determination of 12 primary phenolic compounds was carried

out in different batches of LTC using HPLC-DAD, after that the method was proved to be accurate,

precise, and reproducible according to diverse method validation assays. The obtained findings are

expected to be meaningful for clarifying the effective substances and quality assessment of LTC.

Volume 146 November 2017

Analytical characterization of human milk oligosaccharides – potential applications in

pharmaceutical analysis

Márkó Grabarics, Orsolya Csernák, Réka Balogh, Szabolcs Béni

ABSTRACT

Human breast milk is the gold standard for infant feeding and the best possible nourishment a new-

born could have. Breastfeeding is the natural way to provide optimal nutritional, immunological and

emotional nurturing for the healthy growth and development of infants. Human milk is a complex and

dynamic biofluid comprised of many hundreds to thousands of distinct bioactive structures, among

which one of the most abundant substances are the non-conjugated complex carbohydrates referred to

as human milk oligosaccharides (HMOs). Due to their structural diversity and abundance, HMOs

possess many beneficial biological functions. In order to understand human milk composition and

HMO functions, state-of-the-art glycomic methods are inevitable. The industrial, large scale

chemoenzymatic production of the most abundant HMOs became a reality in the last years and it

evokes the need for straightforward and genuine analytical procedures to monitor the synthetic process

and the quality of the products. It is obvious, that HMOs represent the next breakthrough in infant

nutrition, as the addition of HMOs (such as 2′-fucosyllactose or lacto-N-neotetraose) to infant- and

follow-on formulas, processed cereal-based food and baby foods for infants and young children etc.

will revolutionize this field. This review highlights the potential applications of HMOs in the (bio)

pharmaceutical industry, also summarizes the analytical methods available for the characterization of

HMOs. An overview of the structure and function of HMOs along with their determination methods in

complex matrices are provided. Various separation methods including liquid- and gas chromatography

and capillary electrophoresis for the characterization and novel approaches for the quantitation of

HMOs are discussed.

315

Analysis of macrolide antibiotics in water by magnetic solid-phase extraction and liquid

chromatography–tandem mass spectrometry

Rosa Ana Pérez, Beatriz Albero, Macarena Férriz, José Luis Tadeo

ABSTRACT

Macrolides are one of the most commonly used families of antibiotics employed in human and

veterinary treatment. These compounds are considered emerging contaminants with potential

ecological and human health risks that could be present in surface water. This paper describes the

development and application of a simple and efficient extraction procedure for the determination of

tilmicosin; erythromycin, tylosin and erythromycin-H2O from water samples. Sample extraction was

carried out using magnetic solid-phase extraction using oleate functionalized magnetic nanoparticles

followed by LC–MS/MS analysis. The effects of several parameters on the extraction efficiency of

MLs from water were evaluated. The recovery results obtained were >84% for most of the compounds,

except for erytromycin. The LOD and LOQ values ranged from 11.5 to 26 ng L−1 and from 34 to 77

ng L−1, respectively. The selected method was applied to monitor these contaminants in water samples

from different sources. Tilmicosin and tylosin were not detected in any of the samples, but

erythromycin and erythromycin-H2O were found in 50% of the surface water samples at levels from

<LOQ to 264 ng L−1 and 149 ng L−1, respectively.

Second harmonic generation microscopy as a tool for the early detection of crystallization in

spray dried dispersions

Clara Correa-Soto, Niraj S. Trasi, Paul D. Schmitt, Yongchao Su, Lynne S. Taylor

ABSTRACT

Various techniques have been used to detect crystallization in amorphous solid dispersions (ASD).

However, most of these techniques do not enable the detection of very low levels of crystallinity

(<1%). The aim of the current study was to compare the sensitivity of second harmonic generation

(SHG) microscopy with powder X-ray diffraction (XRPD) in detecting the presence of crystals in low

drug loading amorphous solid dispersions. Amorphous solid dispersions of the poorly water soluble

compounds, flutamide (FTM, 15 wt.% drug loading) and ezetimibe (EZT, 30 wt.% drug loading) with

hydroxypropyl methylcellulose acetate succinate (HPMCAS) were prepared by spray drying. To

induce crystallization, samples were subsequently stored at 75% or 82% relative humidity (RH) and 40

°C. Crystallization was monitored by XRPD and by SHG microscopy. Solid state nuclear magnetic

resonance spectroscopy (ssNMR) was used to further investigate crystallinity in selected samples. For

flutamide, crystals were detected by SHG microscopy after 8 days of storage at 40 °C/82% RH,

whereas no evidence of crystallinity could be observed by XRPD until 26 days. Correspondingly, for

FTM samples stored at 40 °C/75% RH, crystals were detected after 11 days by SHG microscopy and

after 53 days by XRPD. The evolution of crystals, that is an increase in the number and size of

crystalline regions, with time could be readily monitored from the SHG images, and revealed the

formation of needle-shaped crystals. Further investigation with scanning electron microscopy indicated

an unexpected mechanism of crystallization, whereby flutamide crystals grew as needle-shaped

projections from the surface of the spray dried particles. Similarly, EZT crystals could be detected at

earlier time points (15 days) with SHG microscopy relative to with XRPD (60 days). Thus, SHG

microscopy was found to be a highly sensitive method for detecting and monitoring the evolution of

crystals formed from spray dried particles, providing much earlier detection of crystallinity than XRPD

under comparable run times.

316

Sensitive analysis of bioactive secondary metabolites in lichen species using liquid

chromatography–mass spectrometry

Syed Ghulam Musharraf, Fareeha Siddiqi, Arslan Ali, Vinitha Moolchand Thadhani

ABSTRACT

Lichens are a large group of valuable lower plants with unique features and diverse applications

worldwide such as in medicine, cosmetics, food, and textile industries. They are also well known for

their potential in observing climate and environmental monitoring. Their successful exploitations

require reliable analytical methods to check and maintain quality and efficacy of the products based on

them. This study focuses on the development of a sensitive and reliable quantification method for the

analysis of important depsides, depsidones, dibenzofuran and monocyclic phenols inseven known and

an unidentified lichen species. Multiple Reaction Monitoring (MRM) approach using UHPLC-QqQ-

MS instrument was employed for the development of the quantitative method. Both LC and MS

parameters were optimized to ensure maximum separation. High sensitivity, and selectivity. LODs and

LOQs were found to be in the range of 2.1–71.5 ng/mL and 6.3–212.9 ng/mL, respectively. The

accuracy (% bias) and precision (% RSD) were found to be <5% in most cases. Metabolites 1–9 were

found in the range of 0.5–41429 μg/g in the analysed lichen extracts. The analysis revealed that

metabolites 1, 2 and 3 are the predominant ones. This method can be used for the identification and

absolute quantification of secondary metabolites in lichen extracts, and herbal or consumer products

based upon them.

Semiautomated determination of neonicotinoids and characteristic metabolite in urine samples

using TurboFlow™ coupled to ultra high performance liquid chromatography coupled to

Orbitrap analyzer

Marina López-García, Roberto Romero-González, Marina Lacasaña, Antonia Garrido Frenich

ABSTRACT

A semiautomated method based on ultra-high performance liquid chromatography (UHPLC) coupled

to Orbitrap high resolution mass spectrometry has been developed for the determination of

neonicotinoids (imidacloprid, acetamiprid, clothianidin, dinotefuran, nitenpyram, thiacloprid and

thiamethoxam) and the metabolite acetamiprid-n-desmethyl in urine samples. Two automated methods

were tested (solid-phase extraction ―SPE‖ and turbulent flow chromatography ―TurboFlow™‖),

obtaining the best results when TurboFlow™ was applied. The total analysis time for the developed

method was 14 min. The optimized method was validated, obtaining suitable results for all validation

parameters. Recoveries ranged from 78% to 116% meanwhile repeatability and reproducibility were

evaluated obtaining values lower than 10% and 20% respectively (except for dinotefuran and

nitenpyram at 0.2 μg L−1). The limit of quantification (LOQ) for all compounds was established at 0.2

μg L−1. The proposed analytical methodology was applied to analyze the target compounds in thirty

six urine samples from pregnant women living in agricultural areas of Almería (Spain). Imidacloprid,

acetamiprid and acetamiprid-n-desmethyl were detected in some of the samples at concentrations

ranging from 0.23 to 1.57 μg L−1. Furthermore, dinotefuran was identified in two samples at trace

levels.

317

A new polymorph of ciprofloxacin saccharinate: Structural characterization and pharmaceutical

profile

Pawanpreet Singh, Renu Chadha

ABSTRACT

In this study, a new polymorph of ciprofloxacin saccharinate has been thoroughly evaluated with

respect to structural as well as biopharmaceutical properties. Preliminary characterization of the new

polymorph was performed by differential scanning calorimetry and thermogravimetric analysis, later

confirmed by Fourier transform infra-red spectroscopy. The crystal structure was determined from the

powder X-ray diffraction pattern using the direct-space algorithm. It lies in the triclinic P-1 space

group. It is having lattice parameters different from previously reported forms. The solid-state 13C

NMR data calculated from the crystal structure by exploiting density functional theory were found to

be in excellent agreement with corresponding experimental 13C NMR data, thus providing a robust

validation of the authenticity of the structure. The prepared polymorph shows enhanced aqueous

solubility and dissolution rate in contrast to previously reported polymorph. The new form

demonstrated improved oral bioavailability and inhibition of bacterial species.

Qualitative and quantitative measurement of cannabinoids in cannabis using modified

HPLC/DAD method

Bhupendra Patel, Daniel Wene, Zhihua (Tina) Fan

ABSTRACT

This study presents an accurate and high throughput method for the quantitative determination of

various cannabinoids in cannabis plant material using high pressure liquid chromatography (HPLC)

with a diode array detector (DAD). Sample extraction and chromatographic analysis conditions for the

measurement of cannabinoids in the complex cannabis plant material matrix were optimized. The

Agilent Poroshell 120 SB-C18 column provided high resolution for all target analytes with a short run

time (10 minutes) given the core shell technology. The aqueous buffer mobile phase was optimized

with ammonium acetate at pH 4.75. The change in the mobile phase and the new column ensured a

separation between cannabidiol (CBD and cannabigerol (CBG) along with cannabigerol and

tetrahydrocannabinolic acid (THCA), which were not well separated by previous publications,

improved buffering capacity, and provided analytical performance stability. Moreover, baseline

drifting was significantly minimized by the use of a low concentration buffer solution (25 mM

ammonium acetate). In addition, evaporation and reconstitution of the sample residue with a methanol-

organic pure (OP) water solution (65:35) significantly reduced the matrix interference. The modified

extraction produced good recoveries (>91%) for each of the eight cannabinoids. The optimized method

was validated for specificity, linearity, sensitivity, precision, accuracy, and stability. The combined

relative standard deviation (%RSD) for intra-day and inter-day precision for all eight analytes varied

from 2.5% to 5.2% and 0.28% to 5.5%, respectively. The %RSD for the repeatability study varied

from 1.1% to 5.5%. The recoveries from spiked cannabis matrix samples were greater than 90% for all

analytes, except delta-8-tetrahydrocannabinol (Δ8-THC), which was 80%. The recoveries varied from

81% to 107% with a precision of 0.7-8.1%RSD. Delta-9-tetrahydrocannabinol (Δ9-THC) in all of the

cannabis samples (n = 635) was less than 10%, which is in compliance with the NJ Medicinal

Marijuana regulation.

318

Quantification of gabapentin polymorphs in gabapentin/excipient mixtures using solid state 13C

NMR spectroscopy and X-ray powder diffraction

Radaduen Tinmanee, Sarah C. Larsen, Kenneth R. Morris, Lee E. Kirsch

ABSTRACT

Gabapentin was used as a model pharmaceutical compound with susceptibility to polymorphic

transformation as a function of environmental and mechanical stress. The utility of 13C CP/MAS

NMR and XRPD as stability-indicating methods to quantify polymorphic transformation kinetics was

investigated. Polymorphic Form II and III were distinguishable based on their chemical shift and

distinct diffraction peak differences. Reproducible and accurate quantification of polymorphic

composition in the presence of selected excipients was demonstrated using both signals from 13C

CP/MAS NMR spectra and XRPD patterns. The effect of excipients on polymorphic transformations

(Form II → III) was determined by measuring the transformation after co-milling. Both 13C CP/MAS

NMR and XRPD were capable of measuring polymorphic composition in co-milled excipient mixtures

without excipient peak interference. The amounts of Form III present in co-milled mixtures containing

colloidal silicon dioxide, starch, hydroxy propyl cellulose and dibasic calcium phosphate were 8.7, 21,

33, and 39 mol%, respectively. A quenching procedure for obtaining 13C CP/MAS NMR spectra and

environmentally-controlled XRPD were devised to determine polymorphic transformation kinetics of

co-milled excipient mixtures during storage.

Accurate recognition and feature qualify for flavonoid extracts from Liang-wai Gan Cao by

liquid chromatography-high resolution-mass spectrometry and computational MS/MS

fragmentation

Min He, Hai Wu, Juan Nie, Pan Yan, Rui Pei

ABSTRACT

In this study, Liquid Chromatography (LC) separation combined with quadrupole-Time-Of-Flight

Mass Spectrometry (qTOF-MS) detection was used to analyze the characteristic ions of the flavonoids

from Liang-wai Gan Cao (Radix Glycyrrhizae uralensis). First, accurate mass measurement and

isotope curve optimization could provide reliable molecular prediction after noise deduction, baseline

calibration and ―ghost peak recognition‖. Thus, some spectral features in the LC–MS data could be

clearly explained. Secondly, the chemical structure of flavonoids was deduced by MS/MS fragment

ions, and the in-silico spectra by MS-FINDER program provided strong support for overcoming the

bottleneck of phytochemical identification. For a predicted formula and experimental MS/MS

spectrum, the MS-FINDER program could sort the candidate compounds in the public database based

on a comprehensive weighted score, and we took the first 20 reliable compounds to seek the target

compound in an in-house database. Certainly, those fragmentation pathways could also be deduced

and described as Retro-Diels-Alder (RDA) fragmentation reaction, losses of C4H8, C5H8, CH3, CO,

CO2 and others. Accordingly, 63 flavonoids were identified, and their in-silico bioactivity were clearly

disclosed by some bioinformatics tools. In this experiment, the flavonoids obtained by the four

extraction processes were tested by LC-qTOF-MS. We looked for possible Q-markers from these data

matrices and then quantified them; their similarities/differences were also described. The results also

indicated that the Macroporous Adsorption Resins (MARs) purification is a low cost, environmentally

friendly and effective approach.

319

Sample preparation composite and replicate strategy case studies for assay of solid oral drug

products

Beverly Nickerson, Brent Harrington, Fasheng Li, Michele Xuemei Guo

ABSTRACT

Drug product assay is one of several tests required for new drug products to ensure the quality of the

product at release and throughout the life cycle of the product. Drug product assay testing is typically

performed by preparing a composite sample of multiple dosage units to obtain an assay value

representative of the batch. In some cases replicate composite samples may be prepared and the

reportable assay value is the average value of all the replicates. In previously published work by

Harrington et al. (2014) [5], a sample preparation composite and replicate strategy for assay was

developed to provide a systematic approach which accounts for variability due to the analytical method

and dosage form with a standard error of the potency assay criteria based on compendia and regulatory

requirements. In this work, this sample preparation composite and replicate strategy for assay is

applied to several case studies to demonstrate the utility of this approach and its application at various

stages of pharmaceutical drug product development.

Separation and characterization of chemical constituents in Ginkgo biloba extract by off-line

hydrophilic interaction × reversed-phase two-dimensional liquid chromatography coupled with

quadrupole-time of flight mass spectrometry

Shuai Ji, Dan-dan He, Tian-yun Wang, Jie Han, Dao-quan Tang

ABSTRACT

Ginkgo biloba extract (GBE), derived from the leaves of Ginkgo biloba L., is one of the most widely

used traditional Chinese medicines worldwide. Due to high structural diversity and low abundance of

chemical constituents in GBE, conventional reversed-phase liquid chromatography has limited power

to meet the needs of its quality control. In this study, an off-line hydrophilic interaction × reversed-

phase two-dimensional liquid chromatography (HILIC × RP 2D-LC) system coupled with diode array

detection (DAD) and quadrupole time-of-flight mass spectrometry (qTOF-MS) was established to

comprehensively analyze the chemical constituents of GBE. After optimizing the chromatographic

columns and mobile phase of 2D-LC, a Waters XBridge Amide column using

acetonitrile/water/formic acid as the mobile phase was selected as the first dimension to fractionate

GBE, and the obtained fractions were further separated on an Agilent Zorbax XDB-C18 column with

methanol/water/formic acid as the mobile phase. As a result, a total of 125 compounds were detected

in GBE. The orthogonality of the 2D-LC system was 69.5%, and the practical peak capacity was 3864

and 2994, respectively, calculated by two different methods. The structures of 104 compounds were

tentatively characterized by qTOF-MS analysis, and 21 of them were further confirmed by comparing

with reference standards. This established HILIC × RP 2D-LC-qTOF/MS system can greatly improve

the separation and characterization of natural products in GBE or other complicated herbal extracts.

320

Double-Track Electrochemical Green Approach for Simultaneous Dissolution Profiling of

Naproxen Sodium and Diphenhydramine Hydrochloride

Mostafa A. Shehata, Esraa M. Fawaz, Mohamed K.Abd El-Rahman, Ezzat M. Abdel-Moety

ABSTRACT

Acquisition of the dissolution profiles of more than single active ingredient in a multi-analyte

pharmaceutical formulation is a mandatory manufacturing practice that is dominated by utilization of

the off-line separation-based chromatographic methods. This contribution adopts a new ―Double-

Track‖ approach with the ultimate goal of advancing the in-line potentiometric sensors to their most

effective applicability for simultaneous acquisition of the dissolution profiles of two active ingredients

in a binary pharmaceutical formulation. The unique abilities of these sensors for real-time

measurements is the key driver for adoption of ―green analytical chemistry‖ (GAC) principles aiming

to expand the application of eco-friendly analytical methods With the aim of performing a side-by-side

comparison, this work investigates the degree of adherence of ISEs to the 12 principles of GAC in

multicomponent dissolution profiling with respect to the HPLC. For the proof of concept, a binary

mixture of naproxen sodium (NAPR) and diphenhydramine hydrochloride (DIPH) marketed as Aleve

pm® tablets was selected as a model for which dissolution profiles were attained by two techniques.

The first ―Double-Track‖ in-line strategy depends on dipping two highly integrated membrane sensors

for continuous monitoring of the dissolution of each active pharmaceutical ingredient (API) by tracing

the e.m.f change over the time scale. For the determination of NAPR, sensor I was developed using

tridodecyl methyl ammonium chloride as an anion exchanger, while sensor II was developed for the

determination of DIPH using potassium tetrakis (4-chlorophenyl) borate as a cation exchanger. The

second off-line strategy utilizes a separation-based HPLC method via off-line tracking the increase of

peak area by UV detection at 220 nm over time using a mobile phase of acetonitrile: water (90:10) pH

3. The advantages of the newly introduced ―Double-Track‖ approach regarding GAC principles are

highlighted, and the merits of these benign real-time analyzers (ISEs) that can deliver equivalent

analytical results as HPLC while significantly reducing solvent consumption/waste generation are

described.

A workflow for column interchangeability in liquid chromatography using modeling software

and quality-by-design principles

Róbert Kormány, Katalin Tamás, Davy Guillarme, Szabolcs Fekete

ABSTRACT

The goal of the present study was to develop a generic workflow to evaluate the chromatographic

resolution in a large design space and easily find some replacement column for the method. To attain

this objective from a limited number of initial experiments, modern LC modeling software (Drylab)

was employed to study the behaviour of the compounds and visually compare the parts of design

spaces obtained with different columns, where a given criterion of critical resolution is fullfilled. A

zone of robust space can then easily be found by overlapping design spaces. By using 50 × 2.1 mm

columns packed with sub–2 μm fully porous particles (UHPLC), the resolution in the entire design

space can be modeled on the basis of only 2–3 h experimental work per column. To demonstrate the

applicability of the developed procedure, amlodipine and its related pharmacopeia impurities were

selected as a case study. It was demonstrated that two columns from different providers (Waters

Acquity HSS C18, Thermo Hypersil Gold C18) can be interchanged, providing a sufficient resolution

at the same working point and a high degree of robustness around this condition.

321

Absolute quantification of poly(dl-lactide-co-glycolide) in microspheres using quantitative 1H

NMR spectroscopy

Qi Zhang, Ningzi Guo, Yue Sun, Xiaodong Li, Huaxin Yang

ABSTRACT

The complex nature and the manufacturing process of poly(dl-lactide-co-glycolide) (PLGA), a key

component of PLGA-based microspheres, have made the quantification of this copolymer difficult.

The main purpose of the current study was to investigate the potential of three different methods for

the quantitative analysis of the PLGA content of clinical products. In this regard, leuprorelin acetate

microspheres from different vendors were chosen as templates to validate quantitative 1H nuclear

magnetic resonance (qHNMR) spectroscopy, size exclusion chromatography (SEC), and high-

performance liquid chromatography (HPLC) methods qHNMR proved to be an excellent and rapid

PLGA quantification method compared to the other two. The recovery value was 99.12% and the

linearity correlation coefficient was 0.9999. The results obtained from the qHNMR method were found

to match the data provided by the vendor, suggesting that qHNMR can be utilized as a reliable quality

control and inspection tool for PLGA-based clinical products.

A novel LC-MS/MS method for the quantitative measurement of the acetate content in

pharmaceutical peptides

Rani J. Qasem, Ibrahim K. Farh, Mohammed A. Al Essa

ABSTRACT

Most pharmaceutical peptides are supplied as acetate salts and the relative amount of acetate to peptide

in the final product is one quality criterion required by regulatory agencies to approve the product for

clinical use. The objective of the present study was to develop a validated LC-MS/MS method that

allows the quantitative determination of the acetate content in pharmaceutical peptide preparations and

simultaneous monitoring and collection of qualitative and quantitative information on the peptide

during manufacture and in the final product. The method uses reversed phase C18-chromatography to

elute the acetate ions under acidic conditions, pH 3, followed by post-column infusion of ammonium

hydroxide 0.6 M, methanolic solution (30:70) at a rate of 0.5 mL/hr. The acetate ions were monitored

in negative polarity mass spectrometry by pseudo multiple reaction monitoring (pseudo MRM) against

1, 2- 13C labelled acetate, the internal standard used in the method. The method was linear for acetate

concentrations between 0.4 and 25 μg/mL with a coefficient of determination (r2) equal to 0.9999. The

minimum level of detection and minimum level of quantification were at 0.06 μg/mL and 0.18 μg/mL

respectively. Accuracy of the method was judged by determining the acetate content in a commercial

product of the peptide pharmaceutical tetracosactide (TCS) and parallel comparison to the amounts

determined by a reversed phase HPLC method with detection at a wavelength of 210 nm. The amounts

determined by the two methods were in agreement with a RSD that was less than 2%. Additional

confirmation of method accuracy was determined by spiking the pharmaceutical peptide with varying

amounts of acetate. The recoveries ranged on average between 101 and 102% for the spiked amounts.

Accuracy was also determined by calculating the percentage relative error of the predicted to actual

acetate concentration in quality controls and was determined to be less than 5%. The LC-MS/MS

method was precise with an intra- and inter-day RSD of less than 5%. The standard solutions were

stable for at least one month when kept frozen at −80 °C with no loss in response and an inter-day

RSD of less than 5%.

322

A quality by design-based approach to a capillary electrokinetic assay for the determination of

dextromepromazine and levomepromazine sulfoxide as impurities of levomepromazine

Stephan Niedermeier, Gerhard K.E. Scriba

ABSTRACT

Using a quality by design approach, a capillary electrophoresis method for the simultaneous

determination of dextromepromazine and the oxidation product levomepromazine sulfoxide in

levomepromazine was developed. The analytical target profile was defined that the method should be

able to quantify 0.1% of both impurities with a precision of ≤10%. Hydroxypropyl-γ-cyclodextrin was

used as chiral selector. The critical process parameters cyclodextrin concentration, buffer pH and

concentration as well as temperature and applied voltage were studied using a fractional factorial

resolution V+ design for defining the knowledge space. A central composite face centered design was

used as response surface methodology for deriving the design space by Monte Carlo simulations. The

selected working point was a 100 mM citric acid buffer, pH 2.85, containing 3.6 mg/mL

hydroxypropyl-γ-cyclodextrin, a temperature of 15 °C and a voltage of 25 kV. Robustness was

estimated using a Plackett-Burman design. The method was subsequently validated in the relative

concentration range of 0.1%–1.0% of the impurities for a solution containing 0.25 mg/mL

levomepromazine. The method was applied to the determination of the purity of the reference

substance of the European Pharmacopoeia and of the drug in a commercial injection solution.

An HPLC–MS/MS method for quantitation of Gly-MCA in mouse plasma: Application to a

pharmacokinetic study

Jia Liu, Guan Lian, Ting Wang, Yuanheng Ma, Yuxin Yin

ABSTRACT

A simple, sensitive and rapid method using high performance liquid chromatography-tandem mass

spectrometry (HPLC–MS/MS) was developed and validated for quantification of glycine-β-muricholic

acid (Gly-MCA) in mouse plasma for the first time. Plasma samples were pretreated with protein

precipitation. The analyte and internal standard were separated on a Shimadzu Shim-pack XR-ODS

column (4.6 × 50 mm, 2.2 μm) using 0.1% formic acid-water-methanol as mobile phase, with a

runtime of 5 min. Detection was performed using negative ion electrospray tandem mass spectrometry

via multiple reaction monitoring (MRM) scan mode. The linear range was 5 ng–2 μg/ml (correlation

coefficient >0.995) for Gly-MCA with a lower limit of quantitation of 5 ng/ml. The intra-day and

inter-day precision were less than 9.2% for the analyte and accuracy was from 0.4% to 7.0%. This

analytical method was then successfully applied to a pharmacokinetic study of Gly-MCA following

oral administration and intraperitoneal injection in mice.

323

Development and validation of a sensitive LC–MS/MS method without derivatization/ion-

pairing agents for etimicin quantification in rat plasma, internal ear and kidney

Lan Yao, Fang Zhou, Mingmin Cai, Ying Peng, Jingwei Zhang

ABSTRACT

Etimicin (ETM), which belongs to the newest generation of aminoglycosides (AGs), has been proven

to not only maintain but also strengthen the advantages of former AGs with relatively less toxicity.

Now, it is widely applied for the treatment of bacterial infections in the clinic. Nevertheless,

nephrotoxicity and ototoxicity are unavoidable issues for AGs, and while ETM is no exception, the

seriousness of these issues is different. To explore the reason why ETM exhibits less toxicity and to

better direct the optimization and development of new AGs, it is of great necessity and importance to

monitor the pharmacokinetic behaviors of ETM in its potential toxicity target organs, the kidney and

internal ear, as well as in plasma. Therefore, a novel, sensitive and efficient LC–MS/MS method

without derivatization or ion-pairing agents had been developed and validated for quantification of

ETM in rat plasma, kidney and internal ear for the first time. This method showed good linearity over

the range of 50–2000 ng/mL for rat plasma/internal ear and 100–5000 ng/mL for rat kidney. The

precision was less than 4.4% and the accuracy was below 4.8%. Recovery and matrix effects were

71.3%–82.8% and 97.6%–108.5%, respectively. After intravenous administration of a single dose of

ETM, plasma drug concentrations fit well with a two-compartmental model, and the AUC0-∞, t1/2α,

t1/2β, MRT and CL were 127.96 ± 5.52 μg *h/mL, 0.53 ± 0.03 h, 3.32 ± 1.11 h, 1.01 ± 0.03 h and

234.80 ± 10.05 mL/h/kg, respectively. Particularly, ETM showed a considerably long half-life in

kidney and internal ear, up to 155.96 ± 19.95 h and 83.11 ± 26.60 h, respectively, which might

contribute greatly to its toxicity.

Bioanalysis of monomethyl fumarate in human plasma by a sensitive and rapid LC–MS/MS

method and its pharmacokinetic application

Imam Pasha S, Murali Balaram Varanasi, Ibrahim Mohammed

ABSTRACT

Dimethyl fumarate (DMF) is the methyl ester of fumaric acid, after oral administration completely

converts to its active metabolite monomethyl fumarate (MMF). A simple, rapid and sensitive LC–

MS/MS method was developed and validated for the quantification of MMF in human plasma.

Monomethyl fumarate d3 was used as an internal standard (IS). The analyte and the IS were extracted

from plasma using a selective solid phase extraction technique. The clean samples were

chromatographed on a C18 column using formic acid and acetonitrile (25:75, v/v) as mobile phase. An

API-4000 LC–MS/MS system equipped with turbo ion spray (TIS) source and operated in multiple

reactions monitoring (MRM) mode was used for the study. The method was validated for linearity in

the range of 5.03-2006.92 ng/mL. Also, a number of stability tests were conducted to evaluate the

stability of analyte, IS in plasma samples and in neat samples, the results comply with recent

bioanalytical guidelines. A shortest run time helped us to analyze more than 300 samples in a day. The

method was applied to a pharmacokinetic study in ten healthy male Indian subjects and the study data

was authenticated by conducting incurred sample reanalysis (ISR).

324

Simultaneous determination of tenofovir alafenamide and its active metabolites tenofovir and

tenofovir diphosphate in HBV-infected hepatocyte with a sensitive LC–MS/MS method

Bingchen Ouyang, Fang Zhou, Le Zhen, Ying Peng, Jingwei Zhang

ABSTRACT

Tenofovir (TFV), a first-line anti-viral agent, has been prepared as various forms of prodrugs for better

bioavailability, lower systemic exposure and higher target cells loading of TFV to enhance efficacy

and reduce toxicity. TFV undergoes intracellular phosphorylation to form TFV diphosphate (TFV-DP)

in target cell to inhibit viral DNA replication. Hence, TFV-DP is the key active metabolite that exhibits

anti-virus activity, its intracellular exposure and half-life determine the final activity. Therefore,

simultaneous monitoring prodrug, TFV and TFV-DP in target cells will comprehensively evaluate

TFV prodrugs, both considering the stability of ester prodrug, and the intracellular exposure of TFV-

DP. Thus we intended to develop a convenient general analytical method, taking tenofovir alafenamide

(TAF) as a representative of TFV prodrugs. A sensitive LC–MS/MS method was developed, and TAF,

TFV and TFV-DP were separated on a XSelect HSS T3 column (4.6 mm × 150 mm, 3.5 μm, Waters)

with gradient elution after protein precipitation. The method provided good linearity for all the

compounds (2–500 nM for TFV and TAF; 20–5000 nM for TFV-DP) with the correlation coefficients

(r) greater than 0.999. Intra- and inter-day accuracies (in terms of relative error, RE < 10.4%) and

precisions (in terms of coefficient of variation, CV < 14.1%) satisfied the standard of validation. The

matrix effect, recovery and stability were also within acceptable criteria. Finally, we investigated the

intracellular pharmacokinetics of TAF and its active metabolites in HepG2.2.15 cells with this method.

LC–MS/MS method for preclinical pharmacokinetic study of QX-OH, a novel long-acting local

anesthetic, in sciatic nerve blockade in rats

YuJun Zhang, DeYing Gong, QingShan Zheng, Jin Liu, WenSheng Zhang

ABSTRACT

QX-OH, a new synthetic local anesthetic, produced concentration-dependent, reversible, and long-

acting local anesthesia in animal models, with moderate local toxicity. As part of preclinical research

for drug development, we developed and validated a method for the determination of QX-OH in the

plasma, muscle, and sciatic nerve using liquid chromatography–mass spectrometry. After a simple

protein precipitation procedure, analysis was performed on an Extend C18 column (100 mm × 3 mm,

3.5 μm) by isocratic elution with 0.05% formic acid/acetonitrile (78:22, v/v) at a flow rate of 0.3

mL/min. A multiple-reaction monitoring mode at the transitions of m/z 279.1 → 102.1 for QX-OH and

m/z 275.2 → 126.1 for an internal standard (ropivacaine hydrochloride) was used for the

quantification, with a positive electrospray ionization interface. The approach was validated as per the

U.S. Food and Drug Administration guidelines and successfully used in a pharmacokinetic study of

QX-OH after a sciatic nerve block with 0.2 mL of 35 mM QX-OH. The results demonstrated that the

new local anesthetic, QX-OH, had a high concentration in tissue, low systemic exposure, and long

duration in the sciatic nerve.

325

Capillary electrophoresis study on the base-catalyzed formation of bioactive oxidized metabolites

of 20-hydroxyecdysone

Halima Meriem Issaadi, Attila Hunyadi, Krisztina Németh

ABSTRACT

A novel capillary electrophoretic method was developed for the analysis and monitoring of the base-

catalyzed autoxidation of 20-hydroxyecdysone, a worldwide used non-hormonal anabolic food

supplement. An effective separation of the starting material and its bioactive oxidized derivatives was

achieved by using sulfobutyl-β-cyclodextrin as selector at pH 11 and by fixing the separation voltage

at +30 kV. Only a dilution step was inserted before injecting the sample, taken from the crude reaction

mixture, to the capillary electrophoresis instrument. The same alkaline pH was used for the analysis as

for the reaction, unlike the previously reported HPLC study where sample neutralization was required

prior to the measurement. Due to the very short analysis time (6 min) in capillary electrophoresis, more

frequent sampling and more detailed time scale analysis could be carried out. Furthermore, in contrast

with the preceding HPLC results, the previously unobserved calonysterone could also be detected by

capillary electrophoresis as a minor primary product. Our novel method demonstrated higher

resolution than the one before. Baseline separation could be achieved and the resolution values were in

the range of 1.9–7.0. The limit of detection was below 71 μg/ml, the relative standard deviation values

of the migration time and peak area for intra- and inter-day precision were less than 10%. The more

precise, direct monitoring of the time dependency of the oxidation process is expected to have a

significant impact on yield optimization initiatives to allow related pharmacological studies in the near

future.

Quantification of the antimalarial drug pyronaridine in whole blood using LC–MS/MS —

Increased sensitivity resulting from reduced non-specific binding

Daniel Blessborn, Karnrawee Kaewkhao, Lijiang Song, Nicholas J. White, Joel Tarning

ABSTRACT

Malaria is one of the most important parasitic diseases of man. The development of drug resistance in

malaria parasites is an inevitable consequence of their widespread and often unregulated use. There is

an urgent need for new and effective drugs. Pyronaridine is a known antimalarial drug that has

received renewed interest as a partner drug in artemisinin-based combination therapy. To study its

pharmacokinetic properties, particularly in field settings, it is necessary to develop and validate a

robust, highly sensitive and accurate bioanalytical method for drug measurements in biological

samples. We have developed a sensitive quantification method that covers a wide range of clinically

relevant concentrations (1.5 ng/mL to 882 ng/mL) using a relatively low volume sample of 100 μL of

whole blood. Total run time is 5 min and precision is within ±15% at all concentration levels.

Pyronaridine was extracted on a weak cation exchange solid-phase column (SPE) and separated on a

HALO RP amide fused-core column using a gradient mobile phase of acetonitrile–ammonium formate

and acetonitrile-methanol. Detection was performed using electrospray ionization and tandem mass

spectrometry (positive ion mode with selected reaction monitoring). The developed method is suitable

for implementation in high-throughput routine drug analysis, and was used to quantify pyronaridine

accurately for up to 42 days after a single oral dose in a drug-drug interaction study in healthy

volunteers.

326

Simultaneous extraction of propofol and propofol glucuronide from hair followed by validated

LC–MS/MS analyses

Alexandra Maas, Christoph Maier, Stefanie Iwersen-Bergmann, Burkhard Madea, Cornelius Hess

ABSTRACT

Besides its clinical application, the anaesthetic agent propofol is being increasingly misused, mostly by

healthcare professionals, and its abuse potential gained worldwide attention after the tragic death of

Michael Jackson in 2009. Due to the short duration of its narcotic effects, propofol abuse is especially

easy to hide compared with the use of other recreational drugs. However, propofol possesses a very

narrow therapeutic window between the desired effect and potentially fatal toxicity, making abuse of

the drug extremely dangerous even in experienced physicians. Consequently, it is important that

forensic laboratories possess a sensitive and specific method for the detection of chronic propofol

abuse. We present a simple, fast and reliable method to simultaneously extract propofol and its main

metabolite propofol glucuronide from hair, followed by sensitive LC–MS/MS analyses, allowing to

determine a chronic propofol abuse. Difficulties regarding the detection of propofol using LC–MS/MS

were solved by using a derivatization reaction with 2-fluoro-1-methylpyridinium-p-toluene-sulfonate

and triethylamine. Reliability of extraction method and subsequent LC–MS/MS analyses was

confirmed under consideration of the validation parameters selectivity, linearity, accuracy and

precision, analytical limits, processed sample stability, matrix effects and recovery. Appropriate

quantification (LLOQ = 10 pg/mg hair) and detection limits (3.6 pg/mg hair for propofol and 7.8

pg/mg hair for propofol glucuronide) could be achieved, enabling to detect even small amounts of both

analytes. Applicability of the method was confirmed by analysis of three human hair samples from

deceased with suspicion of chronic propofol abuse.

11-nor-9-carboxy-Δ9-tetrahydrocannabinol glucuronide exhibits acyl-migration isomers

Stephanie Hanisch, Alexander Paulke, Stefan W. Toennes

ABSTRACT

11-nor-Δ9-Tetrahydrocannabinol-9-carboxylic acid glucuronide (THCCOOH-glucuronide) is an 1-β-

O-acyl glucuronide which degrades not only to 11-nor-9-carboxy-Δ9-THC (THCCOOH) but,

additionally, to an isomer with a currently unknown structure. The present study was carried out to

examine whether acyl glucuronide isomers are formed by acyl migration and if they are involved in

formation of this isomer. THCCOOH-glucuronide was incubated in phosphate buffer (pH 7.4, 37 °C, 7

days) and a variety of glucuronide cleavage procedures were performed. Samples of the incubation

mixture and of different biological specimens from cannabis users were analyzed using liquid

chromatography-mass spectrometry (LC–MS/MS). A total of six chromatographically separated

isomeric acyl glucuronides were detected during incubation of THCCOOH-glucuronide reference

substance. In biological specimens of cannabis users, two additional isomers were found. However, the

main glucuronide present in human specimens was different from that of a commercially available

reference substance. Both, the commercial and the authentic glucuronide were cleaved by β-

glucuronidases, the other formed isomers by alkaline hydrolysis only. Mass spectrometric

investigations (i.e. product ion, precursor ion and neutral loss scans) confirmed identity. The

THCCOOH isomer was detected in all authentic samples, but not in those after buffer incubation. By

analyzing THCCOOH-glucuronide in authentic samples, it has to be taken into account that the

authentic glucuronide is different from that of the commercial reference standard.

327

Simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human

serum using an immobilized trypsin

Kaito Shibata, Takafumi Naito, Jun Okamura, Seiji Hosokawa, Junichi Kawakami

ABSTRACT

Proteomic approaches using liquid chromatography coupled to tandem mass spectrometry (LC-

MS/MS) without an immunopurification technique have not been applied to the determination of

serum cetuximab. This study developed a simple and rapid LC-MS/MS method for the absolute

determination of cetuximab in human serum and applied it to clinical settings. Surrogate peptides

derived from cetuximab digests were selected using a Fourier transform mass spectrometer. Reduced-

alkylated serum cetuximab without immunopurification was digested for 20 minutes using

immobilized trypsin, and the digestion products were purified by solid-phase extraction. The LC-

MS/MS was run in positive ion multiple reaction monitoring mode. This method was applied to the

determination of serum samples in head and neck cancer patients treated with cetuximab. The

chromatographic run time was 10 minutes and no peaks interfering with surrogate peptides in serum

digestion products were observed. The calibration curve of absolute cetuximab in serum was linear

over the concentration range of 4–200 μg/mL. The lower limit of quantification of cetuximab in human

serum was 4 μg/mL. The intra-assay and inter-assay precision and accuracy were less than 13.2% and

88.0–100.7%, respectively. The serum concentration range of cetuximab was 19–140 μg/mL in

patients. The serum cetuximab concentrations in LC-MS/MS were correlated with those in ELISA (r =

0.899, P < 0.01) and the mean bias was 1.5% in cancer patients. In conclusion, the present simple and

rapid method with acceptable analytical performance can be helpful for evaluating the absolute

concentration of serum cetuximab in clinical settings.

Development of quantitative immunochromatographic assay for rapid and sensitive detection of

carbohydrate antigen 19-9 (CA 19-9) in human plasma

Kwaku Baryeh, Sunitha Takalkar, Michelle Lund, Guodong Liu

ABSTRACT

A quantitative immunochromatographic assay (QIA) was developed by using gold nanoparticle

(GNP)-based lateral flow strip biosensor (LFSB) and a portable strip reader for rapid and sensitive

quantitation of Carbohydrate Antigen 19-9 (CA 19-9) in human plasma. CA 19-9 is a biomarker that

has been associated with cancers (such as pancreatic and colorectal cancers) and various non-

cancerous diseases. The principle is based on sandwich-type immunoreactions between gold

nanoparticle (GNP)-labelled detection antibody, anti-CA 19-9 capture antibody and CA 19-9to capture

the GNPs on the test zone of LFSB. The accumulation of GNPs on the test zone gave a red line whose

intensity was read with a portable strip reader to quantify the concentration of CA 19-9. Assay

parameters including the membrane type, antibody concentration, amount of GNP-anti-CA 19-9

conjugates and the components of the running buffer were optimized to obtain the best sensitivity and

reproducibility of the assay. The detection limit of the assay was determined to be 5 U mL−1 (S/N = 3)

with a linear range of 5 U mL−1–100 U mL−1. CA 19-9 concentrations in healthy human and

pancreatic cancer patient plasma samples were successfully evaluated using the developed quantitative

immunochromatographic assay (QIA), and the results were in accordance with that obtained with

enzyme linked immunosorbent assay (ELISA). The developed assay shows great promise for clinical

application and biomedical diagnosis, particularly in limited resource settings.

328

Simultaneous determination of pentoxifylline, metabolites M1 (lisofylline), M4 and M5, and

caffeine in plasma and dried blood spots for pharmacokinetic studies in preterm infants and

neonates

Madhu Page-Sharp, Tobias Strunk, Sam Salman, Julie Hibbert, Kevin T. Batty

ABSTRACT

Advances in bioanalytical methods are facilitating micro-volume and dried blood spot (DBS) analysis

of drugs in biological matrices for pharmacokinetic studies in children and neonates. We sought to

develop a UPLC–MS/MS assay for simultaneous measurement of caffeine, pentoxifylline (PTX) and

three metabolites of PTX in both plasma and DBS. Caffeine, PTX, the metabolites M1 (lisofylline),

M4 and M5, and the internal standards (caffeine-d9 and PTX-d6) were separated using a Waters

Aquity T3 UPLC C18 column and gradient mobile phase (water-methanol-formic acid). Retention

times for caffeine, M5, M4, PTX and M1 were 1.6, 1.7, 1.9, 2.0 and 2.1 min, respectively, with a run

time of 5 min. The precision (≤10%) and accuracy (≤15%) across the concentration range 0.1–50 mg/L

for caffeine, PTX and the three metabolites in plasma and DBS were within accepted limits, as were

the limits of quantification (100 μg/L for caffeine and 10 μg/L for PTX, M1, M4 and M5). Caffeine,

PTX and the metabolites were stable in DBS for >34 days at room and refrigerated temperatures.

Plasma and DBS samples were obtained from 24 preterm infants recruited into a clinical

pharmacokinetic study of PTX.

Pharmacokinetics and tissue distribution of five major triterpenoids after oral administration of

Rhizoma Alismatis extract to rats using ultra high-performance liquid chromatography–tandem

mass spectrometry

Wen Xu, Xiaoyan Li, Na Lin, Xue Zhang, Shuisheng Wu

ABSTRACT

Rhizoma Alismatis (RA) was wildly used for treatment of dysuria, pyelonephritis, hyperlipidemia,

enteritis diarrhea, diabetes, inflammation, and cancer. Triterpenoids are the major active components

of RA, and its extract is mainly composed of alisol A (ALA), alisol B (ALB), alisol C 23-acetate

(ALC-23A), alisol A 24-acetate (ALA-24A), and alisol B 23-acetate (ALB-23A). In this study, a

simple, reliable, and sensitive ultra high-performance liquid chromatography with triple quadrupole

mass spectrometry (UHPLC–MS/MS) method was created and validated for the quantification of the

five major triterpenoids in rat plasma and various tissues biosamples (including intestine, stomach,

liver, kidney, fat, muscle, brain, heart, lung, spleen, and testes). The plasma and tissues biosamples

were pretreated by direct precipitation deproteinization method with acetonitrile. 17α-

Hydroxyprogesterone was used as internal standard (IS). The chromatography was performed on a

Phenomenex C8 column (30 × 2.00 mm, 1.8 μm) at room temperature with gradient elution.

Compounds were quantified by selected multi-reactions monitoring (SRM) scanning with positive

electric spray ionization mode. The linearity of detection for each triterpene was respectively from 1 to

1000 ng/mL for ALC-23A and ALA, from 4 to 4000 ng/mL for ALA-24A, from 10 to 10,000 ng/mL

for ALB, and from 2 to 2000 ng/mL for ALB-23B (r > 0.99) with low quantification limits of 1–10

ng/mL for all analytes. All of the other validation parameters were also in an acceptable range. The

validated UHPLC–MS/MS method subsequently applied for the pharmacokinetic and tissue

distribution studies of RA extract. After orally given 100 mg/kg of RA extract, ALA was the most

exposed component, followed by ALB and ALA-24A.

329

Solid-phase microextraction coupled to gas chromatography–mass spectrometry followed by

multivariate data analysis for the identification of volatile organic compounds as possible

biomarkers in lung cancer tissues

Federica Bianchi, Nicolò Riboni, Paolo Carbognani, Letizia Gnetti, Maria Careri

ABSTRACT

Solid-phase microextraction and gas chromatography–mass spectrometry followed by multivariate

data analysis were used to analyze lung tissues from both healthy and carcinogenic patients. A total of

78 volatile compounds belonging to different chemical classes were identified, seven of which were

able to discriminate between the two groups. Discriminant analysis allowed to correctly classify 98.3%

of the cases. By using the leave-one-method, 100% of the cross-validated samples belonging to the

―tumor‖ group was correctly classified, whereas 2 cross-validated healthy samples out of 48 were

erroneously allocated in the ―tumor‖ group. Achieved results suggest the need of further investigation

to assess the role of the seven identified compounds as lung cancer biomarkers in breath analysis, thus

allowing the development of low-cost diagnostic devices.

UPLC–MS/MS assay of riluzole in human plasma and cerebrospinal fluid (CSF): Application in

samples from spinal cord injured patients

Mahua Sarkar, Robert G. Grossman, Elizabeth G. Toups, Diana S.-L. Chow

ABSTRACT

In the present study, a sensitive and robust LC–MS/MS method has been developed and validated for

the quantification of riluzole in human plasma and cerebrospinal fluid (CSF) in clinical samples from

patients with spinal cord injury (SCI). Riluzole and its labeled internal standard (IS) were isolated from

plasma and CSF by liquid–liquid extraction using ethyl acetate. Riluzole (m/z 235 → 166) and IS (m/z

238 → 169) were detected by electrospray ionization (ESI) using multiple reaction monitoring (MRM)

in a positive mode. The assay was linear in the concentration range of 0.5 (LLOQ, signal/noise ratio >

10)–800 ng/ml in plasma, and 1.0 (LLOQ)–800 ng/ml in CSF samples. The intra- and inter-day

accuracy in plasma were 94.2–110.0% and 97.8–102.0%, respectively, and those in CSF were 87.6–

105.1% and 91.9–98.8%, respectively. The intra- and inter-day precision were 2.2–7.2% and 4.0–

9.1%, respectively, in plasma, and 1.4–14.1% and 2.6–11.5%, respectively in CSF. Matrix effect was

negligible from both matrices with signal percentages of 97.6–100.6% in plasma and 99.4–106.4% in

CSF. The recoveries were >75% in plasma, >84% in CSF with low protein (53.9 mg/dl), and >68% in

CSF with high protein (348.2 mg/dl). This method was successfully applied to quantify riluzole

concentrations in plasma and CSF from patients with SCI.

330

Simultaneous determination and pharmacokinetic study of twelve bioactive compounds in rat

plasma after intravenous administration of Xuebijing injection by UHPLC-Q-Orbitrap HRMS

Lihua Zuo, Qiuyue Zhong, Zhenhui Wang, Zhi Sun, Xiaojian Zhang

ABSTRACT

Xuebijing injection (XBJ) is a traditional Chinese herbal prescription widely used in the treatment of

sepsis. Extensive studies revealed that the major bioactive constituents of XBJ injection, including

phenolic acids, flavonoids, monoterpene glycosides, lactones and organic acids, play an important role

in the treatment. In this study, a rapid, sensitive and accurate ultra high performance liquid

chromatography – Q Exactive hybrid quadrupole – orbitrap high-resolution accurate mass

spectrometry (UHPLC-Q-Orbitrap HRMS) method was developed for simultaneous determination of

twelve bioactive compounds in rat plasma after intravenous administration of XBJ injection. A

gradient elution for separation was achieved on a waters ACQUITY UHPLC® BEH C18 column (2.1

mm × 50 mm, 1.7 μm) column with acetonitrile-water (containing 0.1% formic acid) as mobile phase

at a flow rate of 0.2 mL/min. All compounds and IS were monitored by parallel reaction monitoring

assay both in positive and negative ion mode. The developed method was validated for intra-day and

inter-day accuracy and precision whose values fell in the acceptable limits. Extraction recoveries at

three levels of QC concentrations were all more than 80% for all compounds and IS, and matrix effects

were found in the range of 80.0–120.0%. Stability results showed that all analytes were stable at the

investigated conditions. The validated method was successfully applied to a pharmacokinetic study of

Xuebijing injection following intravenous administration of 2.5, 5.0, 10.0 mL/kg to rats respectively.

And the result indicated that the pharmacokinetic behavior of XBJ injection is positively related to

dosage at the range of 2.5–10 mg/kg. This study will provide a meaningful basis for evaluating the

rationality of XBJ injection for clinical application.

A surrogate analyte-based liquid chromatography-tandem mass spectrometry method for the

determination of endogenous cyclic nucleotides in rat brain

Jie Chen, Ali Tabatabaei, Doug Zook, Yan Wang, Kathe Stauber

ABSTRACT

A robust high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) assay was

developed and qualified for the measurement of cyclic nucleotides (cNTs) in rat brain tissue. Stable

isotopically labeled 3′,5′-cyclic adenosine-13C5 monophosphate (13C5-cAMP) and 3′,5′-cyclic

guanosine-13C,15N2 monophosphate (13C15N2-cGMP) were used as surrogate analytes to measure

endogenous 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate

(cGMP). Pre-weighed frozen rat brain samples were rapidly homogenized in 0.4 M perchloric acid at a

ratio of 1:4 (w/v). Following internal standard addition and dilution, the resulting extracts were

analyzed using negative ion mode electrospray ionization LC–MS/MS. The calibration curves for both

analytes ranged from 5 to 2000 ng/g and showed excellent linearity (r2 > 0.996). Relative surrogate

analyte-to-analyte LC–MS/MS responses were determined to correct concentrations derived from the

surrogate curves. The intra-run precision (CV%) for 13C5-cAMP and 13C15N2-cGMP was below

6.6% and 7.4%, respectively, while the inter-run precision (CV%) was 8.5% and 5.8%, respectively.

The intra-run accuracy (Dev%) for 13C5-cAMP and 13C15N2-cGMP was <11.9% and 10.3%,

respectively, and the inter-run Dev% was <6.8% and 5.5%, respectively. Qualification experiments

demonstrated high analyte recoveries, minimal matrix effects and low autosampler carryover.

331

Determination of ketamine and its main metabolites by liquid chromatography coupled to

tandem mass spectrometry in pig plasma: Comparison of extraction methods

Cindy Ramiole, Benoit D‘Hayer, Vincent Boudy, Josette Legagneux, Pascal Houzé

ABSTRACT

A rapid, sensitive and specific liquid chromatography coupled to tandem mass spectrometry method

was developed for the simultaneous quantification pig plasma of ketamine and its two principal

metabolites, norketamine and dehydronorketamine. Three extraction procoles were assessed including

acetonitrile precipitation, Oase™ microplate extraction, and liquid–liquid extraction. Oase™

microplate extraction induced no significant matrix effect, important signal/noise ratio and good

recoveries, ranging from 82 to 87% for the considered compounds. Using this extraction procedure,

the assay was linear in the dynamic range 10–3000 ng/mL (R2 > 0.99) regardless of the analytes. Intra-

and inter-day accuracies were less than 12% for all compounds and intra- and inter-day precisions

expressed as RSD were within <9.9%. Samples were stable in different storage conditions. High

ketamine, norketamine and dehydronorketamine concentrations up to 15,000 ng/mL can be determined

with good precision using appropriate sample dilution. The assay was successfully applied to pig

plasma samples to determine the pharmacokinetics of ketamine and the consecutive metabolites after

buccal administration of a 4 mg/kg ketamine base solutions.

Ketamine metabolites with antidepressant effects: Fast, economical, and eco-friendly

enantioselective separation based on supercritical-fluid chromatography (SFC) and single

quadrupole MS detection

Georg M. Fassauer, Robert Hofstetter, Mahmoud Hasan, Stefan Oswald, Andreas Link

ABSTRACT

Increasing evidence accumulates that metabolites of the dissociative anesthetic ketamine contribute

considerably to the biological effects of this drug and could be developed as next generation

antidepressants, especially for acute treatment of patients with therapy-refractory major depression.

Analytical methods for the simultaneous determination of the plethora of hydroxylated,

dehydrogenated and/or demethylated compounds formed after administration of ketamine

hydrochloride are a prerequisite for future clinical investigations and a deeper understanding of the

individual role of the isomers of these metabolites. In this study, we present development and

validation of a method based on supercritical-fluid chromatography (SFC) coupled to single

quadrupole MS detection that allows the separation of ketamine as well as all of its relevant

metabolites detected in urine of healthy volunteers. Inherently to SFC methods, the run times of the

novel protocol are four times shorter than in a comparable HPLC method, the use of organic solvents

is reduced and we were able to demonstrate and validate the successful enantioselective separation and

quantification of R- and S-ketamine, R- and S-norketamine, R- and S-dehydronorketamine and

(2R,6R)- and (2S,6S)-hydroxynorketamine isomers differing in either constitution, stereochemistry, or

both, in one run. The developed method may be useful in investigating the antidepressant efficacy of

ketamine in clinical trials.

332

A new method based on supercritical fluid extraction for polyacetylenes and polyenes from

Echinacea pallida (Nutt.) Nutt. roots

Massimo Tacchini, Antonella Spagnoletti, Virginia Brighenti, Francesco Pio Prencipe, Federica Pellati

ABSTRACT

The genus Echinacea (Asteraceae) includes species traditionally used in phytotherapy. Among them,

Echinacea pallida (Nutt.) Nutt. root extracts are characterized by a representative antiproliferative

activity, due to the presence of acetylenic compounds. In this study, supercritical fluid extraction

(SFE) was applied and compared with conventional Soxhlet extraction (SE) in order to obtain a

bioactive extract highly rich in polyacetylenes and polyenes from E. pallida roots. The composition of

the extracts was monitored by means of HPLC–UV/DAD and HPLC–ESI–MSn by using an Ascentis

Express C18 column (150 mm × 3.0 mm I.D., 2.7 μm, Supelco, Bellefonte, PA, USA) with a mobile

phase composed of (A) water and (B) acetonitrile, under gradient elution. By keeping SFE time at the

threshold of 1 h (15 min static and 45 min dynamic for 1 cycle) with the oven temperature set at 40–45

°C and 90 bar of pressure, an overall extraction yield of 1.18–1.21% (w/w) was obtained, with a high

selectivity for not oxidized lipophilic compounds. The biological activity of the extracts was evaluated

against human non-small lung A549 and breast carcinoma MCF-7 cancer cell lines. The cytotoxic

effect of the SFE extract was more pronounced towards the MCF-7 than the A549 cancer cells, with

IC50 values ranging from 21.01 ± 2.89 to 31.11 ± 2.l4 μg/mL; cell viability was affected mainly

between 24 and 48 h of exposure. The results show the possibility of a new ―green‖ approach to obtain

extracts highly rich in genuine polyacetylenes and polyenes from E. pallida roots. The bioactivity

evaluation confirmed the cytotoxicity of E. pallida extracts against the considered cancer cell lines,

especially against MCF-7 cells, thus suggesting to represent a valuable tool for applicative purposes in

cancer prevention.

Validated LC–MS/MS method for the simultaneous determination of rotigotine and its prodrug

in rat plasma and an application to pharmacokinetics and biological conversion in vitro

Chunjie Sha, Jiangbin Han, Fengjuan Zhao, Xin Shao, Youxin Li

ABSTRACT

Rotigotine behenate (RGTB), a long chain alkyl ester of the prodrug of rotigotine (RGT), has been

synthesized for use in a sustained delivery system. The aim of the present report was to develop and

validate a simple, sensitive and reliable LC–MS/MS method for the simultaneous determination of

RGT and its prodrug RGTB in rat plasma samples. Detection was performed on a 1290 Infinity UPLC

coupled Triple Quad 4500 mass spectrometer operated in positive MRM mode using an Eclipse XDB-

CN chromatography column (2.1 mm × 100 mm, 3.5 μm) by isocratic elution using a 0.2% formic acid

aqueous solution and acetonitrile, with stable isotope labeled RGT as an internal standard. The sample

preparation method employed 50 μL of a plasma sample and liquid-liquid extraction with a mixture of

diethyl ether–dichloromethane (3:2, v/v) as the extraction solvent. The proposed method was fully

validated by assessing its specificity, linearity, precision and accuracy, recovery, matrix effects and

stability. Good linearity was found within the range of 0.1–10.0 ng/mL for both analytes (r > 0.996).

This method was successfully applied to a pharmacokinetic study of a slow release RGTB formulation

in rats following a single intramuscular injection and biological conversion in vitro.

333

Determination of higenamine in dietary supplements by UHPLC/MS/MS method

A. Stajić, M. AnĎelković, N. Dikić, J. Rańić, B. Jančić-Stojanović

ABSTRACT

From 1st January 2017 higenamine was added on the WADA (World Anti-doping Agency) Prohibited

list under S3 group beta-2 agonists as at all times banned substance for the athletes. The main origine

of higenamine (or norcoclaurine) are different plants including Nandina domestica, Aconitum

carmichaelii, Asarum heterotropioides, Galium divaricatum, Annona squamosa, Nelumbo nucifera etc.

Higenamine main use is related to weight loss and it could be found (un)labeled in different dietary

supplements. The objective of this study was development of sensitive and reliable UHPLC/MS/MS

method for determination of higenamine in various dietary supplement samples. In order to obtain high

method sensitivity, hydrophilic interaction liquid chromatography (HILIC) mode was applied.

Separation was carried out on UHPLC Acquity BEH HILIC analytical column (2.1 mm × 100 mm, 1.7

μm particle size). Mobile phase consisted of 0.1% formic acid in water and acetonitrile, respectively,

was mixed in ratio of 30:70, v/v. Flow rate was set at 0.2 mL min−1. Quercetin was used as an internal

standard. ESI (+) source ionization mode using multi reaction monitoring (MRM) mode was utilized

and three ion transitions of higenamine were followed 272.08 → 107.01, 272.08 → 161.07 and 272.08

→ 77.08. Developed method was fully validated and applied for identification and quantification of

higenamine in different dietary supplements. According to the results, the most of investigated

supplements were free of higenamine, and on the other hand, presence of higenamine was confirmed

in some samples while it was not declared on the label.

Determination of genotoxic epoxide at trace level in drug substance by direct injection GC/MS

Liqin Chen, Wei Zhang, Steven Hu

ABSTRACT

A novel direct injection gas chromatography method coupled with selective ion monitoring mass

spectrometry (GC/SIM-MS) was developed for quantitation of trace levels of high boiling point (HBP)

epoxide genotoxic impurity (GTI) in drug substance. The injector temperature was optimized with the

aims to minimize matrix effects and enhance SIM signal response. The final injector temperature 160

°C was selected after balancing between these two factors. The column screening was conducted as

well and MN OPTIMA delta-3 silica capillary column was selected since it showed good peak

symmetry without column bleeding. The good linearity was established for the concentration in the

range from 0.0045 μg/mL to 0.5 μg/mL with a R2 = 0.9999. The limit of detection (LOD) and the limit

of quantitation (LOQ) were 0.0014 μg/mL and 0.0045 μg/mL, respectively. The recovery which

ranged from 95.0% to 112.5% could meet the ICH acceptance criteria. The validation results

demonstrated the good linearity, precision and accuracy of the method which can be further adopted as

an adequate quality control tool for quantitation of epoxide impurity at trace levels in drug substance

and drug product.

334

LC–MS/MS assay for the quantitation of the ribonucleotide reductase inhibitor triapine in

human plasma

Julia Matsumoto, Brian F. Kiesel, Robert A. Parise, Jianxia Guo, Jan H. Beumer

ABSTRACT

The ribonucleotide reductase inhibitor and radiosensitizer triapine (3-aminopyridine-2-carboxaldehyde

thiosemicarbazone (3-AP), NSC 663249) is clinically being evaluated via the intravenous (IV) route

for the treatment of cervical and vulvar cancer in combination with primary cisplatin chemoradiation.

The need for a 2-h infusion and frequent administration of triapine is logistically challenging,

prompting us to pursue oral (PO) administration. In support of the clinical trial investigating oral

triapine in combination with chemoradiation, we developed and validated a novel LC–MS/MS assay

for the quantification of triapine in 50 μL human plasma. After protein precipitation, chromatographic

separation of the supernatant was achieved with a Shodex ODP2 column and an isocratic acetonitrile-

water mobile phase with 10% ammonium acetate. Detection with an ABI 4000 mass spectrometer

utilized electrospray positive mode ionization. The assay was linear from 3 to 3,000 ng/mL and proved

to be accurate (97.1–103.1%) and precise (<7.4% CV), and met the U.S. FDA guidance for

bioanalytical method validation. This LC–MS/MS assay will be an essential tool to further define the

pharmacokinetics and oral bioavailability of triapine.

Development and validation of a liquid chromatography-tandem mass spectrometry method for

pharmacokinetic study of TM-53, a novel transcriptional coactivator with PDZ-binding motif

(TAZ) modulator

Ha-Yeon Lee, Nak Jeong Kim, Yong Moon Lee, Jin Sook Song, Sunjoo Ahn

ABSTRACT

Transcriptional coactivator with PDZ-binding motif (TAZ) is considered an attractive target for

osteoporosis, obesity, and muscle regeneration. TM-53, a promising TAZ modulator, was recently

introduced, and here, we developed a rapid, precise, and reliable analytical method for TM-53 and

characterized its pharmacokinetic properties in rat plasma. The hybrid triple quadrupole/linear ion trap

coupled to liquid chromatography method was developed and validated to quantify TM-53.

Additionally, TM-53 concentrations in plasma were analyzed, and its pharmacokinetic parameters

were calculated by non-compartmental analysis. Multiple reaction monitoring at m/z 569.4 → 207.1

showed the most sensitive signals for TM-53, and the linear scope of the standard curve was between

1.5 ng/mL and 500 ng/mL. The intra- and inter-day precisions of the quality control samples were

<15%, and their accuracies were ranged from 86.2% to 111.0%. Furthermore, the matrix effects,

extraction recoveries, and process efficiencies of this analytical method for evaluating TM-53 in rat

plasma were 99.1%, 99.9%, and 99.1% respectively. In short- and long-term stability studies, TM-53

showed good stability under frozen conditions, but TM-53 hydrolysis in the plasma matrix was

observed following storage at room temperature. This analytical method was successfully applied for

pharmacokinetic analysis of TM-53 in rat plasma and demonstrated excellent sensitivity, selectivity,

precision, and accuracy. These data indicated that this method can be applied for further preclinical

studies of TM-53.

335

Development of a sensitive LC–MS/MS method for quantification of coniferyl ferulate and its

metabolite coniferyl alcohol in rat plasma: Application to a pharmacokinetic study

Xinlun Dai, Li Pang, Zhen Zhang, Chunfeng Yang, Yumei Li

ABSTRACT

A rapid and simple LC–MS/MS method was developed and validated for the simultaneous

determination of coniferyl ferulate (CF) and its metabolite coniferyl alcohol (CA) using bavachromene

as an internal standard (IS). A TSQ Quantum Access mass spectrometer was operated under selected-

reaction monitoring mode using negative electrospray ionization. Extraction with ether was used in

sample preparation. The plasma samples were prepared and then chromatographed on a Phenomenex

Luna C18 column (2.1 mm × 50 mm, 1.7 μm; Torrance, USA) at 35 °C, using acetonitrile: water

(65:35, v/v) in an isocratic mode at a flow rate of 0.3 mL/min. Method validation was performed as per

the FDA guidelines and calibration curves showed good linearity over the concentration range of 2.5–

1000 ng/mL for both CF and CA. The intra- and inter-day precision and accuracy were within the

acceptable limits. The developed assay was successfully applied to a pharmacokinetic study of CA in

rats.

LC–MS/MS assay for the quantitation of the ATR kinase inhibitor VX-970 in human plasma

Brian F. Kiesel, Jonas Scemama, Robert A. Parise, Liza Villaruz, Jan H. Beumer

ABSTRACT

DNA damaging chemotherapy and radiation are widely used standard-of-care modalities for the

treatment of cancer. Nevertheless, the outcome for many patients remains poor and this may be

attributed, at least in part, to highly effective DNA repair mechanisms. Ataxia-telangiectasia mutated

and Rad3-related (ATR) is a key regulator of the DNA-damage response (DDR) that orchestrates the

repair of damaged replication forks. ATR is a serine/threonine protein kinase and ATR kinase

inhibitors potentiate chemotherapy and radiation. The ATR kinase inhibitor VX-970 (NSC 780162) is

in clinical development in combination with primary cytotoxic agents and as a monotherapy for tumors

harboring specific mutations. We have developed and validated an LC–MS/MS assay for the sensitive,

accurate and precise quantitation of VX-970 in human plasma. A dilute-and-shoot method was used to

precipitate proteins followed by chromatographic separation with a Phenomenex Polar-RP 80 Å (4 μm,

50 × 2 mm) column and a gradient acetonitrile-water mobile phase containing 0.1% formic acid from a

50 μL sample volume. Detection was achieved using an API 4000 mass spectrometer using

electrospray positive ionization mode. The assay was linear from 3 to 5,000 ng/mL, proved to be

accurate (94.6–104.2%) and precise (<8.4% CV), and fulfilled criteria from the FDA guidance for

bioanalytical method validation. This LC–MS/MS assay will be a crucial tool in defining the clinical

pharmacokinetics and pharmacology of VX-970 as it progresses through clinical development.

336

Quantitative determination of carfilzomib in mouse plasma by liquid chromatography–tandem

mass spectrometry and its application to a pharmacokinetic study

Jee Sun Min, Jiseon Kim, Jung Ho Kim, Doyun Kim, Soo Kyung Bae

ABSTRACT

A highly sensitive and rapid LC–MS/MS method was developed and validated to determine the levels

of carfilzomib in mice plasma by using chlorpropamide as an internal standard. Carfilzomib and

chlorpropamide were extracted from 5 μL of plasma after protein precipitation with acetonitrile.

Chromatographic separation was performed on Phenomenex Luna C18 column (50 × 2.0 mm id, 3

μm). The mobile phase consisted of 0.1% formic acid in acetonitrile −0.1% formic acid in water (1:1

v/v) and the flow rate was 0.3 mL/min. The total chromatographic run time was 2.5 min. Detection

was performed on a triple quadrupole mass spectrometer equipped with positive-ion electrospray

ionization by selected reaction monitoring of the transitions at m/z 720.20 > 100.15 (for carfilzomib)

and m/z 277.05 > 111.05 (for the internal standard). The lower limit of quantification was 0.075 ng/mL

and the linear range was 0.075–1250 ng/mL (r ≥ 0.9974). All validation data, including selectivity,

precision, accuracy, matrix effect, recovery, dilution integrity, stability, and incurred sample

reanalysis, were well within acceptance limits. This newly developed bioanalytical method was

simple, highly sensitive, required only a small volume of plasma, and was suitable for application in

pharmacokinetic studies in mice that used serial blood sampling.

Development and implementation of a pass/fail field-friendly method for detecting sildenafil in

suspect pharmaceutical tablets using a handheld Raman spectrometer and silver colloids

Adam Lanzarotta, Lisa Lorenz, JaCinta S. Batson, Cheryl Flurer

ABSTRACT

A simple, fast, sensitive and effective pass/fail field-friendly method has been developed for detecting

sildenafil in suspect Viagra and unapproved tablets using handheld Raman spectrometers and silver

colloids. The method involves dissolving a portion of a tablet in water followed by filtration and

addition of silver colloids, resulting in a solution that can be measured directly through a glass vial.

Over one hundred counterfeit Viagra and unapproved tablets were examined on three different devices

during the method development phase of the study. While the pass/fail approach was found to be

92.6% effective on average, the efficacy increased to 97.4% on average when coupled with the

software‘s ―Discover Mode‖ feature that allows the user to compare a suspect spectrum to that of a

stored sildenafil spectrum. The lowest concentration of sildenafil in a water/colloid solution that

yielded a ―Pass‖ was found to be 7.6 μg/mL or 7.6 parts per million (ppm). For the analysis of suspect

tablets, this value was found to be as low as 10 μg/mL and as high as 625 μg/mL. This variability was

likely related to the tablet formulation, e.g., concentration of sildenafil, presence and concentration of

water-soluble and/or water-insoluble ingredients. However, since most counterfeit Viagra and

unapproved tablets contain >50 mg sildenafil per tablet, such low concentrations will not be

encountered often. Limited in-lab and in-field validation studies were conducted in which

analysts/field agents followed the procedure outlined in this study for small sample sets. These

individuals were provided with written instructions, a ∼20 min demonstration regarding how to

perform the procedure and use the instrument, and a kit with field-friendly supplies (purified bottled

water from a local grocery store, disposable plastic pipettes, eye-dropper with a silver colloid solution,

etc.). The method proved to be 98.3% and 91.7% effective for the in-lab and in-field validation studies,

respectively, which demonstrated the ruggedness, simplicity and practicality of the method.

337

Multi-residue determination of 47 organic compounds in water, soil, sediment and fish—Turia

River as case study

Eric Carmona, Vicente Andreu, Yolanda Picó

ABSTRACT

A sensitive and reliable method based on solid-liquid extraction (SLE) using McIlvaine-Na2EDTA

buffer (pH = 4.5)-methanol and solid-phase extraction (SPE) clean up prior to ultra-high-performance

liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS) was applied to

determine 47 organic contaminants in fish, soil and sediments. The SPE procedure to clean-up the

extracts was also used as extraction method to determine these compounds in water. Recoveries ranged

from 38 to 104% for all matrices with RSDs < 30%. Limits of Quantification for the target compounds

were in the range of 10–50 ng/g for soil, 2–40 ng/g for sediment, 5–30 ng/g for fish and 0.3–26 ng/L

for water. Furthermore, the proposed method was compared to QuEChERS (widely used for

environmental matrices) that involves extraction with buffered acetonitrile (pH 5.5) and dispersive

SPE clean-up. The results obtained (recoveries>50% for 36 compounds in front of 9, matrix effect <

20% for 31 compounds against 21, and LOQs <25 ng g−1 for 38 compounds against 22) indicates that

the proposed method is more efficient than QuEChERS, The method was applied to monitoring these

compounds along the Turia River. In river waters, Paracetamol (175 ng L−1), ibuprofen (153 ng L−1)

and bisphenol A (41 ng L−1) were the compounds most frequently detected while in sediments were

vildagliptin (7 ng g−1) and metoprolol (31 ng g−1) and in fish, bisphenol A (33 ng g−1) or

sulfamethoxazole (13 ng g−1).

Separation of bioactive chamazulene from chamomile extract using metal-organic framework

Reda M. Abdelhameed, Hassan Abdel-Gawad, Mohamed Taha, Bahira Hegazi

ABSTRACT

Isolation of bioactive compounds from extracts of pharmaceutical plant is very important. In this work,

copper benzene-1,3,5-tricarboxylate metal organic framework (Cu-BTC MOF) has been synthesized.

It is used in separating of chamazulene from chamomile extract. The Cu-BTC MOF not only shows

good chamazulene adsorption but also maintains good desorption properties. However, the research on

this field is still new and the maturation of novel MOFs or the enhancements of known ones are

required.The chamomile extract obtained after each stage of the treatments was carefully characterized

by thin-layer chromatography (TLC), Fourier-transform infrared spectroscopy (FTIR), UV–vis

spectrometry and gas chromatography-mass spectrometry (GC–MS). The morphology and the

crystallinity of Cu-BTC MOF were investigated using scanning electron microscopy (SEM) and

powder X-ray diffraction (PXRD), respectively. Breakthrough experiments in a column was

investigated and the data was fitted with Bohart-Adams model. Monte Carlo simulation was conducted

to investigate the preferential adsorption sites of Cu-BTC for chamazulene molecules.

338

1H NMR based pharmacometabolomics analysis of urine identifies metabolic phenotype of

clopidogrel high on treatment platelets reactivity in coronary artery disease patients

Arwa M. Amin, Lim Sheau Chin, Chin-Hoe Teh, Hamza Mostafa, Baharudin Ibrahim

ABSTRACT

Clopidogrel high on treatment platelets reactivity (HTPR) has burdened achieving optimum

therapeutic outcome. Although there are known genetic and non-genetic factors associated with

clopidogrel HTPR, which explain in part clopidogrel HTPR, yet, great portion remains unknown, often

hindering personalizing antiplatelet therapy. Nuclear magnetic resonance (1H NMR)

pharmacometabolomics analysis is useful technique to phenotype drug response. We investigated

using 1H NMR analysis to phenotype clopidogrel HTPR in urine. Urine samples were collected from

71 coronary artery disease (CAD) patients who were planned for interventional angiographic

procedure prior to taking 600 mg clopidogrel loading dose (LD) and 6 h post LD. Patients' platelets

function testing was assessed with the VerifyNow® P2Y12 assay at 6 h after LD. Urine samples were

analysed using 1H NMR. Multivariate statistical analysis was used to identify metabolites associated

with clopidogrel HTPR. In pre-dose samples, 16 metabolites were associated with clopidogrel HTPR.

However, 18 metabolites were associated with clopidogrel HTPR in post-dose samples. The pathway

analysis of the identified biomarkers reflected that multifactorial conditions are associated with

clopidogrel HTPR. It also revealed the implicated role of gut microbiota in clopidogrel HTPR.

Pharmacometabolomics not only discovered novel biomarkers of clopidogrel HTPR but also revealed

implicated pathways and conditions.

Permeability through the Caco-2 cell monolayer of 42 bioactive compounds in the TCM formula

Gegen-Qinlian Decoction by liquid chromatography tandem mass spectrometry analysis

Qi Wang, Yi Kuang, Wei Song, Yi Qian, Min Ye

ABSTRACT

Caco-2 cell monolayer model was used to evaluate the intestinal permeability of 42 bioactive

compounds in the famous traditional Chinese medicine (TCM) formula Gegen-Qinlian Decoction

(GQD). These compounds include alkaloids, flavonoids and glycosides, triterpenoid saponins, and

coumarins. Their transportations across the cell monolayers in the forms of herb extract and formula

extract were monitored by liquid chromatography coupled with tandem mass spectrometry

(LC/MS/MS) analysis. Most alkaloids from Huang-Lian; flavonoid C-glycosides from Ge-Gen and

Huang-Qin; O-glycosides from Ge-Gen, Huang-Qin and Gan-Cao; O-glucuronides from Huang-Qin;

and coumarins from Gan-Cao exhibited favorable permeability. Their PAB values were > 1.05 × 10−5

cm/s, and efflux ratios (ER, PBA/PAB) were ≤ 1.0. In contrast, triterpenoid saponins showed poor

permeability (PAB ≤ 1.50 × 10−6 cm/s, ER ≤ 1.5), indicating a paracellular diffusion mechanism.

Furthermore, GQD could remarkably improve the intestinal transport of alkaloids in Huang-Lian,

flavonoid C-glycosides in Ge-Gen, as well as coumarins and flavonoid O-glycosides in Gan-Cao.

These results indicate herb–herb interactions in GQD.

339

Simultaneous quantification of arginine, alanine, methionine and cysteine amino acids in

supplements using a novel bioelectro-nanosensor based on CdSe quantum dot/modified carbon

nanotube hollow fiber pencil graphite electrode via Taguchi method

Sara Hooshmand, Zarrin Es‘haghi

ABSTRACT

A number of four amino acids have been simultaneously determined at CdSe quantum dot-

modified/multi-walled carbon nanotube hollow fiber pencil graphite electrode in different

bodybuilding supplements. CdSe quantum dots were synthesized and applied to construct a modified

carbon nanotube hollow fiber pencil graphite electrode. FT-IR, TEM, XRD and EDAX methods were

applied for characterization of the synthesized CdSe QDs. The electro-oxidation of arginine (Arg),

alanine (Ala), methionine (Met) and cysteine (Cys) at the surface of the modified electrode was

studied. Then the Taguchi‘s method was applied using MINITAB 17 software to find out the optimum

conditions for the amino acids determination. Under the optimized conditions, the differential pulse

(DP) voltammetric peak currents of Arg, Ala, Met and Cys increased linearly with their concentrations

in the ranges of 0.287–33670 μM and detection limits of 0.081, 0.158, 0.094 and 0.116 μM were

obtained for them, respectively. Satisfactory results were achieved for calibration and validation sets.

The prepared modified electrode represents a very good resolution between the voltammetric peaks of

the four amino acids which makes it suitable for the detection of each in presence of others in real

samples.

Simultaneous optimization of pH and binary organic composition by grid form modeling of the

retention behavior in reversed-phase ultra high-performance liquid chromatography

Tsukasa Sasaki, Kenichiro Todoroki, Toshimasa Toyo‘oka

ABSTRACT

A novel computer-assisted methodology for the simultaneous optimization of aqueous pH and binary

organic eluent composition through a broad range of analytical conditions of reversed-phase ultra

high-performance liquid chromatography is proposed. Two of nonlinear prediction models were

employed to fit into the retention time (tR) on a linear gradient elution with a predefined slope. One

model was derived from Bernoulli-type probability distribution to predict the value of tR against the

pH value of the aqueous eluent. This sigmoid-shaped model was successfully fitted for tR value shift

in the presence of three levels of organic eluent compositions (volumetric mixing of

acetonitrile/methanol ratios 1:0, 1:1, and 0:1). The resultant pH versus tR value models were

subsequently combined into grid form by quadratic multiple regression models based on the solubility

parameter theory and their binary organic composition axes. The predicted tR values afforded from

grid models were highly accurate for 13 different acidic non-steroidal anti-inflammatory drugs [root

mean square error (RMSE) ≤0.030] and 16 basic histamine H1-receptor blockers (RMSE ≤0.067) in a

pH ranging from 2.5 to 9.0 and an acetonitrile/methanol volumetric mixing ratio ranging from 1:0 to

0:1. Each compatibility score was defined as the indicator of the peak separation. Scores were

calculated for all combinations of aqueous pH values and binary organic compositions via the

predicted tR values. A colored map generated from the calculated scores was greatly effective in

determining optimal combinations of both mobile phase conditions. By employing this predictive data,

all analytes in both acidic and basic sample mixtures were finally separated at their respective

optimized conditions.

340

On-Line two dimensional liquid chromatography based on skeleton type molecularly imprinted

column for selective determination of sulfonylurea additive in Chinese patent medicines or

functional foods

Pengqi Guo, Xinya Xu, Guoning Chen, Kamran Bashir, Qiang Fu

ABSTRACT

Substandard and counterfeit anti-diabetic medicines directly influence the health and impose a great

danger to individual patients and to public health. Counterfeiting has become a serious and

underreported problem in the pharmaceutical industry. There are a large number of counterfeit

medicines flooded in anti-diabetic markets which effect human health directly and indirectly.

Therefore, some novel analytical techniques are necessary to be established for detecting these

counterfeit drugs. In this study, a novel skeleton type molecularly imprinted column was successfully

prepared. Based on the column, a simple, fast and reliable two-dimensional chromatography analytical

system was established for selective determination of the illegal sulfonylurea additive in traditional

Chinese patent medicines and functional foods. The developed method was validated. The linearitiesof

the method were tested with calibration curves using ten calibration points in the concentration range

of 0.25–12.5 μg/g. The LODs were 0.0125 μg/g and 0.01 μg/g for tolbutamide and glibenclamide

respectively. The five batches of Chinese patent medicines and dietary supplements obtained from

different markets and online websites were tested by the validated method. With good retention time

and spectral confirmation, chemical anti-diabetic substances were identified and quantified in

traditional Chinese medicine and in dietary supplements.

Impact of chemotherapy on metabolic reprogramming: Characterization of the metabolic profile

of breast cancer MDA-MB-231 cells using 1H HR-MAS NMR spectroscopy

Roberta M. Maria, Wanessa F. Altei, Heloisa S. Selistre-de-Araujo, Luiz A. Colnago

ABSTRACT

Doxorubicin, cisplatin, and tamoxifen are part of many chemotherapeutic regimens. However, studies

investigating the effect of chemotherapy on the metabolism of breast cancer cells are still limited. We

used 1H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy to study the metabolic

profile of human breast cancer MDA-MB-231 cells either untreated (control) or treated with

tamoxifen, cisplatin, and doxorubicin. 1H HR-MAS NMR single pulse spectra evidenced signals from

all mobile cell compounds, including fatty acids (membranes), water-soluble proteins, and metabolites.

NMR spectra showed that phosphocholine (i.e., a biomarker of breast cancer malignant

transformation) signals were stronger in control than in treated cells, but significantly decreased upon

treatment with tamoxifen/cisplatin. NMR spectra acquired with Carr-Purcell-Meiboom-Gill (CPMG)

pulse sequence were interpreted only qualitatively because signal areas were attenuated according to

their transverse relaxation times (T2). The CPMG method was used to identify soluble metabolites

such as organic acids, amino acids, choline and derivatives, taurine, guanidine acetate, tyrosine, and

phenylalanine. The fatty acid variations observed by single pulse as well as the lactate, acetate,

glycine, and phosphocholine variations observed through CPMG 1H HR-MAS NMR have potential to

characterize both responder and non-responder tumors in a molecular level. Additionally, we

emphasized that comparable tumors (i.e., with the same origin, in this case breast cancer) may respond

totally differently to chemotherapy. Our observations reinforce the theory that alterations in cellular

metabolism may contribute to the development of a malignant phenotype and cell resistance.

341

An integrated strategy for rapid discovery and identification of the sequential piperine

metabolites in rats using ultra high-performance liquid chromatography/high resolution mass

spectrometery

Zhanpeng Shang, Wei Cai, Yanfeng Cao, Fei Wang, Jiayu Zhang

ABSTRACT

Piperine, one of the major bioactive constituents isolated from natural flavorings and medicinal-

culinary herbs, possesses various biological activities. In the present study, an integrated strategy based

on ultra high-performance liquid chromatography/high resolution mass spectrometry was established

to reveal piperine metabolism in rats. First of all, post-acquisition data-mining methods, including high

resolution extracted ion chromatograms (HREICs) and multiple mass defect filtering (MMDF), were

used to screen piperine metabolite candidates in a full-scan HRMS1 level. Then, parent ion list-

dynamic exclusion coupled with data-dependent data-acquisition method was utilized to acquire MSn

datasets. In addition, the established reverse molecular assembly (RMA) approach based on paired

diagnostic product ions (pDPIs) coupled with neutral loss fragments (NLFs) was used to ascertain and

identify the major-to-trace piperine metabolites efficiently. And then, the calculated ClogP values were

utilized to distinguish the positional isomers. As a result, a total of 148 piperine metabolites were

detected and characterized tentatively. The results demonstrated that piperine mainly underwent

hydrogenation, dehydrogenation, hydroxylation, glucuronide conjugation, sulfate conjugation, ring-

cleavage, and their composite reactions. Our results not only provided novel and useful data to better

understand the safety, toxicity and efficacy of this potential therapeutic agent, but also indicated that

the proposed strategy was reliable for a rapid discovery and identification drug-related constituents in

vivo.

***

Indexing & AbstractingRobust HPLC–MS/MS method for levofloxacin and ciprofloxacin determination in...

Documents

Transcript of Indexing & AbstractingRobust HPLC–MS/MS method for levofloxacin and ciprofloxacin determination in...