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APPLYING HPLC - HILIC AS METHOD FOR ANALYSIS OF

PHARMACEUTICAL COMPOUNDS

Stefka Ivanova1, Lily Peikova

1, Petya Koleva

2, Dobrinka Tsvetkova

1,

Valentina Petkova1*

1Faculty of Pharmacy, Medical University – Sofia.

2Medical College, Medical University – Plovdiv.

ABSTRACT

HPLC separation method with high sensitivity and selectivity is used

as a detector with a large linear range and good reproducibility. It can

be applied in order to identify and quantify the main ingredient,

"related substances" and impurities. The identity of the target

component validation of HPLC required to prove the necessary

selectivity, while quantifying and determining the LOD and LOQ is

applied for an external certified reference material. Hydrophilic

Interaction Liquid Chromatography (HILIC) is the most successful

approach for the retention and separation of polar compounds. HILIC

has seen a substantial increase in popularity over the last decade,

driven by the increase in the development of polar drugs within the pharmaceutical industry

and also the growing field of metabolomics, which primarily involves the analysis of polar

molecules. This technique has been described as “reversed -reversed phase’” since in HILIC

the stationary phase is polar and the aqueous portion of the mobile phase acts as the stronger

solvent, which is the opposite of conventional reversed phase chromatography. There are

many stationary phases that can be used in a HILIC mode and many phases are generally

described as HILIC, independently of their chemistry.

KEYWORDS: HPLC, HILIC, LOD, LOQ, HILIC-MS/MS.

INTRODUCTION

The HILIC mode of separation is used extensively for separation of some bio

molecules,organic and some inogranic molecules by differences in polarity.

[1, 2] Its utility

has increased due to the simplified sample preparation for biological samples, when

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 5.210

Volume 5, Issue 1, 01-12 Review Article ISSN 2278 – 4357

Article Received on

29 Oct 2015,

Revised on 21 Nov 2015,

Accepted on 14 Dec 2015

*Correspondence for

Author

Prof. Dr. Valentina

Petkova

Faculty of Pharmacy,

Medical University –

Sofia.

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Petkova et al. World Journal of Pharmacy and Pharmaceutical Sciences

analyzing for metabolites, since the metabolic process generally results in the addition of

polar groups to enhance elimination from the cellular tissue. This separation technique is also

particularly suitable for glycosylation analysis and quality assurance of glycoproteines and

glycoforms in biologic medical products.[3]

For the detection of polar compounds with the

use of electrospray-ionization mass spectrometry as a chromatographic detector, HILIC can

offer a tenfold increase in sensitivity over reversed-phase chromatography because the

organic solvent is much more volatile. Raquel de Oliveira Vilhena and collaborators have

developed a new HILIC-MS/MS method for the simultaneous analysis of carbidopa,

levodopa and its metabolites in human plasma. Some samples from patients undergoing

treatment have been analyzed, and the results indicated that this new method is suitable for

therapeutic drug monitoring in Parkinson's disease patients.[4]

A group of scientists ( Eva

Cífková, Michal Holčapeka, ,

, Miroslav Lísa, David Vrána, Bohuslav Melichar, Vladimír

Študent) have made lipidomic differentiation between human kidney tumors and surrounding

normal tissues using HILIC-HPLC/ESI-MS and multivariate date analysis. The

characterization of differences among polar lipid classes in tumors and surrounding normal

tissues of 20 kidney cancer patients has been performed by hydrophilic interaction liquid

chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI–MS). A

notable increase of relative abundances of lipids containing four and more double bonds has

been detected in tumor compared to normal tissues.[5]

Mohammed Shahid Ali and fellow

workers used hydrophilic interaction liquid chromatographic (HILIC) procedure for the

simultaneous determination of pseudoephedrine hydrochloride (PSH), diphenhydramine

hydrochloride (DPH) and dextromethorphan hydrobromide (DXH) in cough-cold

formulations. Mobile phase consists of methanol:water (containing 6.0 g of ammonium

acetate and 10 mL of triethylamine per liter, pH adjusted to 5.2 with orthophosphoric acid),

95:5 (v/v). Column containing porous silica particles (Supelcosil LC-Si, 25 cm × 4.6 mm,

5 μm) has been used as stationary phase. Detection is carried out using a variable wavelength

UV–vis detector at 254 nm for PSH and DPH, and at 280 nm for DXH. Solutions were

injected into the chromatograph under isocratic condition at constant flow rate of

1.2 mL/min. Linearity range and percent recoveries for PSH, DPH and DXH were

determined to be 150–600, 62.5–250, 75–300 μg/mL and 100.7%, 100.1% and 100.8%,

respectively. The method’s stability has been determined and it has been established, that the

indicated excipients like saccharin sodium, sodium citrate, savor and sodium benzoate did

not interfere in the analysis.[6]

Tetsuya Nemoto and collaborators identified high-throughput

determination of nonsteroidal anti-inflammatory drugs in human plasma by HILIC-MS/MS.

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A simple and sensitive method has been developed and validated for the analysis of thirteen

nonsteroidal anti-inflammatory drugs (NSAIDs) in human plasma samples by hydrophilic

interaction liquid chromatography (HILIC)–tandem mass spectrometry (MS/MS). The mass

spectra obtained by HILIC-MS has shown base peak ions due to [M + H]+ for indomethacin,

oxaprozin, ketoprofen, alminoprofen, zaltoprofen, tiaprofenic acid, pranoprofen, and

ketoprofen-d3 and due to [M − H]− for etodolac, ibuprofen, diclofenac, fenoprofen,

loxoprofen, naproxen, and ibuprofen-d3.[7]

A number of methods have been described in

literature and applied for simultaneous determination of atorvastatin calcium and olmesartan

medoxomil in a pharmaceutical formulation. By reversed phase high-performance liquid

chromatography, high-performance thin-layer chromatography, and UV spectrophotometric

methods. Soni H, Kothari C, Khatri D, Mehta P used validated RP-HPLC, HPTLC, and UV

spectrophotometric methods have been developed for the simultaneous determination of

atorvastatin calcium (ATV) and olmesartan medoxomil (OLM) in a pharmaceutical

formulation. The proposed methods were simple, precise, accurate, and applicable for the

simultaneous determination of ATV and OLM in a marketed formulation. The results

obtained by applying the proposed methods were statistically analyzed and were found

satisfactory.[8]

Abdelaleem EA and collaborators analyzed with HPTLC and RP-HPLC methods for

simultaneous determination of Paracetamol and Pamabrom in presence of their potential

impurities. Developed methods have been validated and proved to meet the requirements

delineated by ICH guidelines with respect to linearity, accuracy, precision, specificity and

robustness. The validated methods were successfully applied for determination of the

studied drugs in their pharmaceutical formulation. The results were statistically compared to

those obtained by the reported RPHPLC method where no significant difference was found;

indicating the ability of proposed methods to be used for routine quality control analysis of

these drugs.[9]

HPLC–MS/MS method for the quantitation and pharmacokinetic analysis of 3-

deazaneplanocin A in mice the scientists Cody J.Peer, Mahadev Rao,Shawn

D.Spencer,Shandiz Shahbazi,Patricia S.Steeq,David S.Schrump and William D.Figg found

that 3-Deazaneplanocin A (DZNep) has been shown to have anti-cancer activity in numerous

cancer types and its continued preclinical, and eventual clinical, drug development will

require rapid and sensitive bioanalytical methods in order to quantitate this drug for

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Petkova et al. World Journal of Pharmacy and Pharmaceutical Sciences

pharmacokinetic analyses. The ultra HPLC with positive thermospray tandem mass

spectrometric (LC–MS/MS) detection affords the most sensitive (limit of quantitation

5 ng/mL) and rapid (3 min run time) bioanalytical method to date for DZNep. Due to the

polar nature of this drug and the internal standard (tubercidin), a hydrophilic-interaction

column (HILIC) was used. The method was accurate, with less than 10% deviation from

nominal values, as well as precise, where both within-day and between-day precisions were

less than 15%. A liquid–liquid extraction procedure was able to recover ∼90% of drug from a

small volume (50 μL) of mouse plasma. This method was successfully applied to a

pharmacokinetic study in mice intravenously injected with DZNep.[10]

Literature search reveals a large number of published papers that describe various usage of

the HPLC-HILIC technique in many analytical fields. Hydrophilic interaction

chromatography (HILIC) in the analysis of antibiotics in different sample matrices including

pharmaceutical, plasma, serum, fermentation broths, environmental water, animal origin,

plant origin, etc. Specific applications of HILIC for analysis of aminoglycosides, β-lactams,

tetracyclines and other antibiotics are reviewed. HILIC can be used as a valuable alternative

LC mode for separating small polar compounds. Polar samples usually show good solubility

in the mobile phase containing some water used in HILIC, which overcomes the drawbacks

of the poor solubility often encountered in normal phase LC. HILIC is suitable for analyzing

compounds in complex systems that elute near the void in reversed-phase chromatography.

Ion-pair reagents are not required in HILIC which makes it convenient to couple with MS

hence its increased popularity in recent years. In this review, the retention mechanism in

HILIC is briefly discussed and a list of important applications is provided including main

experimental conditions and a brief summary of the results. The references provide a

comprehensive overview and insight into the application of HILIC in antibiotics analysis.[11]

According to Donald S. Rislev and colleagues this method can be applied for analyzing

magnesium from magnesium stearate in pharmaceutical tablets using hydrophilic interaction

liquid chromatography with nano quantity analyte detection. Common validation parameters

were evaluated to assess the methods quantitative potential for magnesium (from magnesium

stearate) including: linearity, accuracy, specificity, solution stability, repeatability, and

intermediate precision. Overall, the method described in this report proved to be very robust

and represents a novel technique to conveniently separate and detect magnesium from

magnesium stearate in pharmaceutical preparations both quickly and accurately.[12]

A group

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of scientists (Tijana Rakić, Biljana Jančić Stojanović, Anđelija Malenović, Darko

Ivanović, Mirjana Medenica ) have found improved chromatographic response function in

HILIC analysis mixture of antidepressants. As a model mixture six antidepressants were

chosen: selegiline, mianserine, sertraline, moclobemide, fluoxetine and maprotiline. Due to

complexity of retention mechanisms in HILIC system, detailed examination of experimental

space assessing the influence of important factors (acetonitrile content in the mobile phase,

buffer concentration and pH of the mobile phase) and their interactions was done by applying

3 3 experimental design. N CRF * is developed and designed to be the only output of the

system which simultaneously measures the separation of all the examined substances, the

chromatographic run duration and the quality of the obtained peaks shape. It allowed

objective estimation of overall chromatogram quality and excluded the arbitrary judgment in

ambiguous situations. The applied function highlighted the influence of investigated factors

on entire mixture and enabled identification of experimental regions where the

chromatographic behavior was satisfactory. Applied experimental design strategy combined

with N CRF * proved to be valuable assistance in HILIC separation of complex mixtures.[13]

M. Gratacós-Cubarsí and collaborators are developed analysis of vitamin B1 in dry-cured

sausages by hydrophilic interaction liquid chromatography (HILIC) and diode array

detection. A method based on hydrophilic interaction liquid chromatography (HILIC) and

diode array detection (DAD) was developed to quantify thiamine (vitamin B1) concentration

in Spanish dry-cured sausages (“chorizo,” “fuet,” and “salchichón”). Samples were extracted

with diluted acid (HCl 0.1 M) followed by an enzymatic hydrolysis to release vitamin B1

vitamers from food matrix. Crude extracts were purified on a weak cation exchange SPE

cartridge and total thiamine concentration was determined by LC-HILIC-DAD with a limit of

detection better than 0.01 mg/100 g. The proposed conditions, that do not require the

derivatization of the extracts nor the use of fluorescence or MS detectors, are suitable to

provide chromatographic separation and identification of vitamin B1 within 8 min.

Selectivity, repeatability and accuracy of the method were evaluated with both spiked

samples and the reference material Pig Liver BCR®

487. Quantification of vitamin B1 was

also carried out for different kinds of commercial samples of Spanish dry-cured products.[15]

L. Matysová, L. Havlíková, , R. Hájková, A. Krivda, P. Solich used application of HILIC

stationary phase to determination of dimethinden emaleate in topical gel. A novel high

performance liquid chromatography method for the determination of dimethindene maleate in

pharmaceutical gel using hydrophilic interaction liquid chromatography (HILIC) with UV

detection was developed and validated.Following optimal conditions for the analysis of

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dimethindene maleate were used: analytical column SeQuant ZIC®–HILIC

(50 mm × 2.1 mm, 5 μm), and mobile phase consisted of a mixture of acetonitrile and

aqueous solution of acetic acid (25 mM) and ammonium acetate (2.5 mM) (87.5:12.5, v:v).

The analysis time was less than 3 min at a flow rate of 0.3 ml min−1

. UV detection was

performed at 258 nm. The method was validated and system suitability parameters were

evaluated. The method is suitable for application for routine determination of dimethindene

maleate in topical pharmaceutical preparation.[16]

Scientists (Romain Simon, Quentin

Enjalbert, Jordane Biarc, Jérôme Lemoine, Arnaud Salvador) made evaluation of hydrophilic

interaction chromatography (HILIC) versus C18 reversed-phase chromatography for targeted

quantification of peptides by mass spectrometry. Hydrophilic-interaction liquid

chromatography (HILIC) is a widely used technique for small polar molecule analysis and

offers the advantage of improved sensitivity in mass spectrometry. Although HILIC is today

frequently employed as an orthogonal fractionation method for peptides during the proteomic

discovery phase, it is still seldom considered for quantification. In this study, the

performances in terms of peak capacity and sensitivity of 3 HILIC columns were compared to

traditional reversed phase liquid C18 column in the context of targeted quantification of

proteotypic peptides using selected reaction monitoring mode (SRM). The results showed

that the maximum sensitivity in HILIC chromatography was achieved by using an amide

column without salt buffer and that the signal increased compared to classic reversed phase

chromatography. However, the intensity improvement is quite low compared to the one

obtained for small molecules.[26-30]

This is due on one hand to a higher matrix effect in HILIC

and on the other hand to a change of charge states of peptides in organic solvent (doubly

charged to monocharged).[31-36]

The doubly charged ions can be more readily dissociated than

singly charged ions, making them ideal for SRM peptide quantification. As a result

“supercharging” reagents are added to the mobile phase to shift from predominant singly

charged ions to the more favorable doubly charged species.[14]

Using such optimized

conditions, peptide signal is improved by a factor of between two and ten for 88% of the

peptides of the 81 peptides investigated.[17-20]

The sensitivity of evaporative universal

detectors and LC/MS in the HILIC and the reversed-phase HPLC modes can be compared.

Scientists (Clifford R. Mitchell , Ye Bao, Nancy J. Benz, Shuhong Zhang ) developed and

offered the idea that HILIC mobile phases are mostly composed of polar organic solvent and

are more volatile than reversed-phase mobile phases. Therefore the more easily evaporated

HILIC mobile phases should produce greater sensitivity for those detectors that remove

mobile phase by evaporation.[37-42]

The responses of 12 compounds were measured in the

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reversed-phase mode and the HILIC mode with three detectors: evaporative light scattering

detector (ELSD), corona charged aerosol detector (cCAD), and electrospray mass

spectrometry (ESI-MS). The compounds studied were very polar compounds that were

retained in the HILIC mode. Generally, the HILIC mode was able to achieve greater

sensitivity than the reversed-phase mode for these compounds.[18-25]

Alpert, A.J. and collaborators studied hydrophilic-interaction chromatography of complex

carbohydrates. Some carbohydrates exhibited anomer separation, which made it possible to

determine the orientation of the reducing end relative to the stationary phase. Amide sugars

were consistently good contact regions. Relative to amide sugars, sialic acids and neutral

hexoses were better contact regions at lower levels of organic solvents than at higher levels.

Both asialo- and sialylated glycans could be resolved using the same set of conditions. With

derivatized carbohydrates, detection was possible at the picomole level by UV detection or

on-line electrospray mass spectrometry. Selectivity compared favorably with that of other

modes of HPLC. HILIC is promising for a variety of analytical and preparative

applications.[43-46]

CONCLUTION

Hydrophilic interaction chromatography (HILIC) is a liquid chromatography technique when

a polar stationary phases - silica or polar bonded phases and aqueous mobile phase containing

amount of a less polar solvent (often acetonitrile) is applied. HILIC is important for the

separation of highly polar substances including biologically active compounds, such as drugs,

amino acids, peptides, proteins, nucleosides, neurotransmitters, etc. HILIC is also appropriate

method for analysis of plant extract polar components such as carbohydrates, amino acids,

peptides, phenolic acids, flavonoids and some alkaloids. Plant extracts are usually

multicomponent mixtures of compounds of wide polarity range, which often cannot be

analyzed by use of a single separation and detection method because of the high chemical

diversity. Good results are obtained by use of coupled method, e.g., reversed-phase liquid

chromatography (RP-LC) and HILIC mode separation. The elution order in HILIC is usually

opposite to that in RP separations. This orthogonality determines one of the advantages of

HILIC - the possibility of creating multidimensional separations. An important feature of

HILIC is the improved sensitivity with electrospray mass spectrometry. This is significant for

the analysis of components existing in small concentration in multicomponent mixtures. The

high acetonitrile content also gives additional advantage of HILIC - faster separations due to

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the lower viscosity of HILIC eluents compared to standard RP ones. The presented review

deals with the optimization of HILIC separations and application of the method for analysis

of plant extracts components.

ACKNOWLEDGEMENT

Project DP-13/2015 Medical university – Plovdiv.

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