Post on 26-May-2020
Shu Y et al. Untargeted Metabolomics Profiling and … Planta Med Int Open 2020; 7: e45–e57
Original Paper Thieme
Untargeted Metabolomics Profiling and Global Semi-quantitation of a Prescription Chinese Herbal Medicine Formula Yinqiaosan Using UPLC-QTOF-MS with a Single Exogenous Reference Internal Standard
AuthorsYachun Shu1, 2 , Chandana Mannem2, Gang Xu2, Shashank Gorityala2 , Xiao Liu2, 3, Yan Xu2, 4
Affiliations1 AffiliatedHospitalofNanjingUniversityofChinese
Medicine,Nanjing,P.R.China2 DepartmentofChemistry,ClevelandStateUniversity,
Cleveland,OH,USA3 CollegeofPharmacy,NanjingUniversityofChinese
Medicine,Nanjing,P.R.China4 CaseComprehensiveCancerCenter,CaseWestern
ReserveUniversity,Cleveland,OH,USA
Key wordsYinqiaosan,Chineseherbalmedicineformula,untargetedmetabolomicsprofiling,globalsemiquantitativeanalysis,multivariatedataanalysis,UPLC-QTOF-MS
received 03.09.2019 revised 26.02.2020 accepted 10.03.2020
BibliographyDOI https://doi.org/10.1055/a-1141-0119Publishedonline:2020PlantaMedIntOpen2020;7:e45–e57©GeorgThiemeVerlagKGStuttgart·NewYork ISSN2509-9264
CorrespondenceYachunShuAffiliatedHospitalofNanjingUniversityofChineseMedicine155HanzhongRoad210029NanjingCityJiangsuProvinceP.R.China Tel.:+8625-86617141,Fax:+8625-86618555 yfy0093@njucm.edu.cn
YanXuDepartmentofChemistry,ClevelandStateUniversity2121EuclidAve.,SI333ClevelandOhio44115-2214USA Tel.:+12166873991,Fax:+12166879298 y.xu@csuohio.edu
Supporting Informationforthisarticleisavailableonlineat:http://www.thieme-connect.de/products.
AbstractYinqiaosanisaclassicChineseherbalmedicineformulathathasbeenusedtotreatvariousbacterialandviralinfectionsbyChi-nesemedicinedoctorsforovertwocenturies.Inthiswork,wedevelopedacomprehensivequalitativeandquantitativemeth-odforidentification,quantitation,andqualityassessmentofchemicalconstituentsofYinqiaosanformulainfourdifferentpreparationforms(i.e.,decoction,granule,pill,andtablet),whichemployedultra-performanceliquidchromatographyquadrupoletime-of-flightmassspectrometrywithasingleexogenousreferenceinternalstandardforuntargetedme-tabolomicsprofilingandglobalsemiquantitativeanalysis.Theuseofasingleexogenousreferenceinternalstandardpermit-tednotonlyqualitativeandquantitativeanalysesofmultipleherbalcomponentsinasingleinstrumentrun,butalsocross-comparisonofchemicalcontentsinbetweenallfourYinqia-osanpreparationforms.Theacquiredmasschromatogramswereanalyzed,quantitated,andcomparedusingmultivariatedataanalysisforsimilaritiesanddifferencesofchemicalcon-stituentsinfourYinqiaosanpreparationforms.Forthefirsttime,wewereabletoidentifyover100chemicalconstituentsfromeachpreparationformusingtheavailabledatabase.Amongthe49commonlyidentifiedcompoundsinthe4Yin-qiaosanpreparationforms,16havebeenreportedtohavepharmacologicalactivities,whichmaybeusedinanetworkpharmacologystudyofYinqiaosanforexploringtheunderlyingmechanismoftheherbalformula.
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Shu Y et al. Untargeted Metabolomics Profiling and … Planta Med Int Open 2020; 7: e45–e57
Original Papers Thieme
AbbrevIAtIonS
CHM ChineseherbalmedicineCV coefficientofvariationEBM evidence-basedmedicineESI electrosprayionizationIS internalstandardPCA principalcomponentanalysisPLS-DA partialleastsquaresdiscriminantanalysisQC qualitycontrolQ-marker qualitymarkerRCT randomizedcontrolledtrialSIM selectedionmonitoringTCM traditionalChinesemedicineTIC totalioncurrentUPLC-QTOF-MS ultra-performanceliquidchromatogra-
phy-quadrupoletime-of-flightmassspectrometry
IntroductionCHMformulasarethekeycomponentsofTCMinterventions,whichhavemanypreparationformsincludingdecoctions,granules,pills,tablets,capsules,powders,medicatedteas,medicatedwines,etc.[1].DespitethewidespreaduseofCHMformulasinAsiaandtheirgrowinguseintheWest,thefundamentalissuehinderingCHMin-tegrationintotheWesternmainstreamhealthcaresystemisthatrigorousscientificevidenceofCHMefficacyandsafetyobtainedthroughRCTsorsystematicreviewsofRCTshasbeenlimitedfromEBMperspectives[2].Thisismainlyattributedtotwomajorde-fectsintheRCTstodate:(1)improperdifferentiationorreportingofaTCMpatternorsyndromesincepatterndifferentiationiscru-cialinTCMforprescribingCHMtherapy,and(2)thelackofacom-prehensivequalityassessmentandcontrolofCHMformulassincethechemicalconstituentsinmedicinalplantsvarywithgeograph-icoriginandcultivar,partsinuse,timeofharvest,ecologicalenvi-ronmentandpotentialcontaminationwithheavymetals,pesti-cides,ormycotoxins,processinganddetoxificationmethodaswellasvariousformsofpreparations[1].
Themethodscurrentlyadoptedbytheinternationalpharma-copeialmonographs[3–5]foridentificationandqualityassessmentofmedicinalplantsandCHMformulasemphasizethequalitativeandquantitativedeterminationofagroupofsubjectivelyselectedQ-markerconstituentsthatmayormaynothaveatherapeuticef-fect.Thesemethodscanʼtdistinguishwhetheramarkerconstitu-entisendogenousorfalsifiedbecausenoinformationonqualita-tiveandquantitativechemicalprofilesofmedicinalplantsorCHMformulasisobtained.Therefore,comprehensivequalitativeandquantitativechemicalanalysismethodsaremuchmoredesirable,notonlyforphytochemicalprofilingandqualityassessmentofme-dicinalplantsandCHMformulasbutalsofordetectionofcounter-feits.UntargetedmetabolomicsprofilingusingLC-QTOF-MShasbeensuccessfullyappliedtofingerprintingherbalmedicines[6–8].Ifthistechniquecombineswiththeuseofasingleexogenousref-erenceISforsemiquantitativeanalysisandPCAforpatternrecog-
nition,itwouldprovideamoreadequatemethodforidentificationofchemicalconstituentsandqualityassessmentofmedicinalplantsandCHMformulas.Thisworkdemonstratessuchanat-tempt.
YinqiaosanisaclassicCHMformuladevelopedbytheQingDy-nastyfamousChinesedoctorWuJutong(1758–1836).Itiscom-posedofnineherbsincludingJinyinhua(Flos lonicerae),Lianqiao(Fructus forsythiae),Bohe(Herba menthae),Jingjie(Herba schizone-petae),Niubangzhi(Fructus arctii),Jiegeng(Radix platycodonis),Dandouchi(Semen Sojae preparatum),Gancao(Radix glycyrrhizae),andDanzhuye(Herba lophatheri)[9].BasedonCHMclassification,Yinqiaosanhascoolenergy,anacridtaste,andpropertiesofreleas-ingexterior,clearingheatandremovingtoxicityfromhumanbod-ies[10].IthasbeenusedbyTCMdoctorsforovertwocenturiesastreatmentforvariousbacterialandviralinfectionssuchasinfluen-za[11],hand,foot,andmouthdisease[12],pharyngitis[13],pneu-monia[14],acutetonsillitis[15],measles[16],andmumps[17]. ArecentrandomizedclinicaltrialindicatedthatYinqiaosanplusan-otherCHMformula,Maxingshigan,wasaseffectiveasoseltamivir(alsoknownasTamiflu)forthetreatmentofH1N1influenzaAvirusinfections[18].
Yinqiaosanwasoriginallypreparedintheformofadecoction.Duetotheeaseofuse,carry,andstorage,Yinqiaosanhasbeenpre-paredinvariousformsoverthecourseofhistory,suchasYinqia-osanpeifangkeli(granule),Yinqiaojieduwan(pill),andYinqiao-jiedupian(tablet),whicharebeingusedinterchangeablyinclinicalinterventions.Currently,therearevariousformsofYinqiaosanpro-ducedbynumerousChinesemanufacturers[19].Thelackofcom-prehensivequalitativeandquantitativechemicalanalysismethodsforchemicalprofilingandquantitationnotonlyimpedecompletequalityassessmentofvariousYinqiaosanpreparationforms,butalsoconfusetheassessmentofoutcomesofclinicalstudies[2,20].Therefore,wehavedevelopedacomprehensivequalitativeandquantitativemethodthatcombinesuntargetedmetabolomicspro-filingandglobalsemiquantitativeanalysiswiththeuseofasingleexogenousIS,aswellaspatternrecognitionthroughPCAforiden-tification,quantitation,andqualityassessmentofYinqiaosaninfourpreparationforms(decoction,granule,pill,andtablet).
ResultsandDiscussionOneofthekeytechnicaladvancesofthemethoddevelopedistheimplementationofasingleexogenousreferenceIS(etoposide-d3),whichservesmultiplerolesincludingselectionofretentiontimeandmassshiftwindowsforUPLC-QTOF-MSanalysis,assessementandcorrectionofasamplematrixeffect,peaknormalizationformultivariatedataanalysis,andglobalsemiquantitativeanalysisofmulticomponentsineachYinqiaosanpreparationform,aswellascross-comparisonofchemicalcontentsinbetweenvariousYinqia-osanpreparationforms.
Ourexperimentaldata(notshown)indicatedthattherewerenochromatographicormassspectrometricinterferencestotheISintheblanksolutionsandsamplesofthefourYinqiaosanprepara-tionforms.ThematrixeffectofeachYinqiaosanpreparationformonthemassspectrometricdetectionoftheISexpressedasmatrixfactorwascalculatedbythemeanpeakareaoftheISspikedinsam-plesofeachYinqiaosanpreparationformoverthatoftheISspiked
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Shu Y et al. Untargeted Metabolomics Profiling and … Planta Med Int Open 2020; 7: e45–e57
intheblanksolutions.ThematrixfactorsoffourYinqiaosanprep-arationformsrangedfrom0.87–1.07(▶table 1),indicatingtherewasnosignificantsignalsuppressionorenhancementoftheISinanyofthesamplematrices.
Foruntargetedmetabolomicsprofiling,constituentidentifica-tion,andglobalsemiquantitationofchemicalconstituentsinYin-qiaosanformula,triplicatesampleswerepreparedforeachYinqia-osanpreparationformandthesolutionblank.Atotalof15Yinqia-osansampleswereanalyzedusingtheUPLC-QTOF-MSmethod
developed.Thechromatographicandspectra(MSandMS/MS)datawereacquiredbybothpositiveandnegativeESImodes.Therep-resentativeTICchromatogramsareshownin▶Fig. 1.ChemicalconstituentsofeachYinqiaosanpreparationformwereextractedaftersubtractingTICchromatogramsofthesolutionblanksfromthoseofYinqiaosansamplesolutions.
Thereareseveralnonvolatile,water-solubleQ-markersfromthenineherbsofYinqiaosan[3].Toillustratethistargetedfeatureofthemethoddeveloped,theseQ-markersweretargetedandex-
▶table 1 MatrixeffectsofYinqiaosansamplesonmassspectrometricdetectionoftheISa.
Sample matrix (n = 3) eSI mode PAISb in extracted sample matrix ± SDc PAIS in solution ± SD MFIS
d ± SD
Decoction + (4.75±0.06)×105 (5.4±0.2)×105 0.88±0.03
− (2.34±0.02)×106 (2.68±0.03)×106 0.87±0.01
Granule + (5.4±0.2)×105 (5.4±0.2)×105 1.00±0.05
− (2.52±0.03)×106 (2.68±0.03)×106 0.94±0.02
Tablet + (5.8±0.2)×105 (5.4±0.2)×105 1.07±0.05
− (2.37±0.04)×106 (2.68±0.03)×106 0.88±0.02
Pill + (4.83±0.04)×105 (5.4±0.2)×105 0.89±0.03
− (2.43±0.01)×106 (2.68±0.03)×106 0.91±0.01
aIS=1.69µM,bAIS=meanpeakareaofthespikedIS,cSD=standarddeviation,dMFIS=(PAISintheextractedsamplematrix)/(PAISinthesolution).
Coun
ts
Coun
ts
–TIC Blank
–TIC Decoction
–TIC Granules
–TIC Tablet
–TIC Pill
Retention Time (min)
f
g
h
i
j
x106
3210
x106
3
2
1
0
5 10 15 20 25 30 35 40 45 50 55
5 10 15 20 25 30 35 40 45 50 55
x106
3
2
1
05 10 15 20 25 30 35 40 45 50 55
x106
3
2
1
05 10 15 20 25 30 35 40 45 50 55
x106
3
2
1
05 10 15 20 25 30 35 40 45 50 55
x106+TIC Blank
+TIC Decoction
+TIC Granules
Retention Time (min)
+TIC Tablet
+TIC Pill
3
a
b
c
d
e
2
1
5 10 15 20 25 30 35 40 45 50 55
x106
3
2
1
0
x106
3
2
1
0
x106
3
2
1
0
x106
3
2
1
0
5 10 15 20 25 30 35 40 45 50 55
5 10 15 20 25 30 35 40 45 50 55
5 10 15 20 25 30 35 40 45 50 55
5 10 15 20 25 30 35 40 45 50 55
▶Fig. 1 RepresentativeTICchromatogramsofthesolutionblankandsamplesofvariousYinqiaosanpreparationformsbybothpositiveionizationmode(a–e)andnegativeionizationmode(f–j).
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Shu Y et al. Untargeted Metabolomics Profiling and … Planta Med Int Open 2020; 7: e45–e57
Original Papers Thieme
tracted.TherepresentativeSIMchromatogramsofsixQ-markers(i.e.,chlorogenicacid,luteolin7-glucoside,forsythiaside,liquiritin,forsythin,andarctiin)areprovidedin▶Fig. 2.
Duetotheshortageofstandardreferencematerialandthecostconcern,determinationofmulticomponentsforqualityassessmentofCHMformulaiscurrentlynotrequiredbytheChineseregulato-ryagency.InthecaseofYinqiaosanproducts,onlyoneofthe Q-markers(i.e.,arctiin,chlorogenicacid,orforsythin)isregulatedbytheChinesePharmacopoeia[3].SinceYinqiaosanproductscon-tainmultiplebioactivecomponents,thecurrentmethodsforsin-gle-componentanalysiscanneitherassessthequalityofYinqia-osanproductsobjectivelynordetectcounterfeitseffectively.ThemethoddevelopedinthisworkprovidesamuchbettertechnicalsolutionforqualityassessmentofYinqiaosanproducts,whichnotonlyprofilesaproductbutalsoquantitatesmultipleQ-markerssi-multaneously.
IdentificationofchemicalconstituentsineachYinqiaosanprep-arationformwasaccomplishedbyfollowingtheproceduredetailed
in“Dataprocessing,constituentidentification,andstatisticalanal-ysis”inthe“MaterialsandMethods”section.Thedataobtainedwerethensubjectedtomolecularfeatureextractionsusingpossi-bleionadducts,isotopepatterns,andchargestatesaswellasapresetretentiontimeandmasswindow.Itisworthnotingthatthepost-processingfilterofthedataprocessingsoftwarewassetat3outof3toensureeachmolecularfeatureextractedpresentedinallthreemasschromatogramsofthetriplicatemeasurementsforeachYinqiaosanpreparationform.Thetotalnumberofconstitu-entsidentifiedwithchemicalnamesandthenumberofconstitu-entsidentifiedwithmolecularformulasineachpreparationformaresummarizedin▶table 2.Thenumberofchemicalconstituentsfoundindecoction,granule,pill,andtabletformsofYinqiaosanwere302,434,427,and388,respectively.Amongthe4prepara-tionforms,therewere104commonconstituents(▶Fig. 3),49identifiedwithbothchemicalnamesandformulas(▶table 3)and55identifiedonlywithchemicalformulas(▶table 1S,SupportingInformation).TheMS/MSspectraofthe49chemicalconstituents
▶Fig. 2 RepresentativeSIMchromatogramsofsixmarkerconstituentsofYinqiaosanalongwiththeISataconcentrationof1.69μM.
x103 1.801
1
1
1
1
1
1
1
Chlorogenic acid +ESI
+ESI
+ESI
+ESI
+ESI
+ESI
+ESI
–ESI
Luteolin 7-glucoside
Forsythiaside
Liquiritin
Forsythin
14.36
17.36
22.94
25.99
28.99
28.95
30.00
Arctiin
Etoposide d3 (IS)
Etoposide d3 (IS)
x104
x103
x104
x104
x104
x104
x104
6
4
2
0
6
4
4
3
2
2.52
1.5
0.50
5 10 15 20 25 30 35 40 45 50 55
1
1
0
2
0
6
1.51.25
10.75
0.50.25
8
2.5
Coun
ts
Retention time (min)
21.5
10.5
0
6420
0
4
2
0
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Shu Y et al. Untargeted Metabolomics Profiling and … Planta Med Int Open 2020; 7: e45–e57
commonlyidentifiedinallfourYinqiaosanpreparationformsareprovidedin(▶Fig. 1S,SupportingInformation).
SinceeachYinqiaosanpreparationformexertsasimilarthera-peuticeffectandisusedtotreatthesameillnesses,itisreasonabletothinkthatpharmacologicallyactiveconstituentsareamongthe49commonlyidentifiedchemicalconstituentsinallfourprepara-tionforms,whereastheuniquechemicalconstituentsineachprep-arationform(▶tables 2S– 9S,SupportingInformation)maycomefromtheherbsusedbythemanufacturers,wherechemicalcon-stituentsmayvarywithgeographicoriginandcultivar,timeofhar-vestandtheecologicalenvironmentoftheherbs,potentialagri-culturalpollutionsofpesticidesandherbicidesorindustrialcross-contaminationwithotherpharmaceuticalsaswellassidereactionsandby-productsassociatedwiththeuniquemanufacturingcondi-tions(e.g.,temperature,pressure,time,solvent,additives,etc.)ofeachYinqiaosanpreparationform.Thisstudyshowsthatproductprofilingisanindispensablepartofqualityassessment,andtheun-targetedmetabolomicsapproachnotonlyallowsustoprofilebutalsofingerprintanherbalproductintermsofQ-markers,originandcultivars,potentialcontaminations,andsignatureconstituentsas-sociatedwithaparticularpreparationormanufacturingprocess.
MultivariateanalysisoftheacquiredMSdata(i.e.,exactmasstoretentiontimepairandnormalizedpeakarea)wasfirstcarriedoutwithanunsupervisedPCAscoreplottoassessthesimilaritiesofchemicalconstituentsamongthefourYinqiaosanpreparationformsandthereproducibilityofreplicatesamplesofeachprepa-rationformbytheUPLC-QTOF-MSmethod,thenvisualizedbyasu-pervisedPLS-DAscoreplottoestablishrecognitionpatternsofthefourYinqiaosanpreparationforms.
AsshowninthePCAscoreplot(▶Fig. 4a),thedifferencesinchemicalconstituentsamongthefourYinqiaosanpreparationformswereapparent.Thefirsttwoprincipalcomponentsencom-passed72.3%ofthetotalvariance,andthetightgroupingoftrip-licatemeasurementsonthesamplesofeachpreparationformin-dicatedthattheUPLC-QTOF-MSmethoddevelopedhadexcellentreproducibility.ThePLS-DAscoreplot(▶Fig. 4b)wasinagreementwiththePCAscoreplot,andshoweddistinctivepatternsamongthefourYinqiaosanpreparationforms.Hence,thesepatternsmaybeusedforproductdifferentiationandrecognition.
Ahigh-throughputUPLC-QTOF-MSmethodforglobalsemi-quantitativeanalysisofchemicalconstituentsinCHMformulawasdemonstratedinthiswork.Asillustratedbythecontentsof49commonlyidentifiedchemicalconstituentsinfourYinqiaosanpreparationforms(▶table 4),theuseofasingleexogenousrefer-enceIS(etoposide-d3)inthemethodenabledustoperformnotonlyglobalsemiquantitativeanalysisofmulticomponentsineachYinqiaosanpreparationformbutalsocross-comparisonamongthefourpreparationforms.Therefore,themethoddevelopedprovidesanefficientandeconomicalsolutionforbothherbalcontentanal-ysisandproductcomparison.
ThereproducibilityofglobalsemiquantitativeanalysisbytheUPLC-QTOF-MSmethodwasinvestigatedbytriplicatemeasure-mentsofsampleofeachYinqiaosanpreparationform,andthepre-cisionofthesemiquantitativemeasurementswascalculatedintermsofCVofthechemicalcontents.Itwasshown(▶table 4)that91.8–98.0%ofthe49chemicalconstituentsofthe4preparationformshadCVvaluesthatfellwithintherecommendedvalues(CV≤30%)[21],whichindicatedthereliabilityoftheUPLC-QTOF-MSmethod.
Thepharmacologicalactivitiesofthe49commonlyidentifiedchemicalconstituentsinall4Yinqiaosanpreparationformswerein-vestigatedthroughtextmininganddatabasesearching.Sixteenoftheforty-ninechemicalconstituentswerefoundtohavevarious
▶table 2 SummaryofchemicalconstituentsfoundinfourYinqiaosanpreparationforms.
Yinqiaosan Constituents Common constituents
Forms eSI mode Identified Unidentified total eSI mode Identified Unidentified total
Decoction+ 158 37
302
+ 46 21
104
− 21 86
Granule+ 79 177
434− 36 142
Tablet+ 91 163
388
- 3 34− 24 110
Pill + 67 186427
− 38 136
Pill (P)
Granules (G) Tablet (T)
G
P 226
155
13
27
104
3
24245
1
7
162
5
196
35
D
T
Decoction (D)
▶Fig. 3 VenndiagramofthechemicalconstituentsfoundineachpreparationformofYinqiaosan.
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Original Papers Thieme▶
tabl
e 3
Common
chemicalcon
stitu
entsdetectedandidentifi
edinallfourYinqiaosanpreparationform
s.
no.
Form
ula
nam
etr
(min
)o
bser
ved
mas
sD
atab
ase
mas
sPr
ecur
sor
ion,
m/z
1C 1
6H18O9
Chlorogenicacid
*
1.80
354.09
6135
4.09
5135
5.10
26[M
+H]+
2C 1
1H12N
2O2
D-Tryptop
han
3.01
204.09
1420
4.08
9120
5.09
86[M
+H]+
3C 3
4H34O18
Isorientin4'-O
-glucoside2''-O-p-hydroxybenzoagte
4.88
730.17
6473
0.17
7773
1.18
41[M
+H]+
4C 2
4H24O
11Echioidinin2'-(6''-acetylglucoside)
6.36
488.13
3248
8.13
3748
9.13
93[M
+H]+
5C 1
9H25N5O
6Asn-Trp-Thr
8.11
419.18
2041
9.18
1742
0.18
95[M
+H]+
6C 2
0H26O9
BruceineD
8.40
410.15
7341
0.15
8341
1.16
44[M
+H]+
7C 1
7H18N
4O5
202-79
18.49
358.12
8635
8.12
8138
1.11
77[M
+Na]
+ ;3
59.138
2[M
+H]+
8C 2
1H28O8
Vernofl
exuo
side
8.49
408.17
8540
8.17
9340
9.18
59[M
+H]+
9C 2
1H24O
12Ca
techin-4-ol3-O
-BeTa-D-galactopyrano
side
9.59
468.12
7446
8.12
7946
9.13
45[M
+H]+
10C 2
8H34O
12Ca
ohuo
sideD
9.79
562.20
5656
2.20
6656
3.21
30[M
+H]+
11C 1
1H14O5
Genipin
11.14
226.08
5322
6.08
5522
7.09
25[M
+H]+
12C 2
0H18N
4O6
Phe-His-O
H11
.14
410.12
1441
0.12
2641
1.12
87[M
+H]+
13C 1
3H22N
4O8S
2Cysteineglutathion
edisulfide
11.19
426.08
8542
6.08
7121
4.05
06[M
+2H]2+
14C 2
0H24N
4O6
Asp-Pro-Trp
11.69
416.16
8141
6.16
7741
7.17
54[M
+H]+
15C 1
8H26O7
Prop
ofolglucuronide
12.04
354.16
7635
4.16
7235
5.17
44[M
+H]+
16C 2
3H20O9
Pong
amosideA
12.25
440.11
1144
0.11
1244
1.11
85[M
+H]+
17C 1
5H12O
4Liqu
iritig
enin
13.37
256.07
4825
6.07
5225
7.08
20[M
+H]+
18C 2
1H21ClN
2O8
Dem
eclocycline
13.43
464.09
7746
4.09
9048
7.08
72[M
+Na]
+ ;4
65.105
2[M
+H]+
19C 3
2H31N
3O5
KT572
013
.78
537.22
3653
7.22
5053
8.23
08[M
+H]+
20C 2
2H22O7
Artoindo
nesianinR
14.13
398.13
7139
8.13
6939
9.14
37[M
+H]+
21C 2
1H20O
11Luteolin7-glucoside
*
14.42
448.10
8744
8.10
3844
9.10
98[M
+H]+
22C 2
2H24O7
Dihydroanhydrop
odorhizol
15.60
400.15
2040
0.15
3140
1.15
94[M
+H]+
23C 2
5H23ClN
2O7
TyrM
e-Phe4Cl-O
H17
.02
498.11
8849
8.12
0149
9.12
58[M
+H]+
24C 2
9H36O15
Forsythiaside *
17
.32
624.20
3562
4.20
5464
7.19
94[M
+Na]
+
25C 2
8H30O
11IkarisosideD
17.98
542.18
1154
2.18
0954
3.18
75[M
+H]+
26C 2
9H32O
12Am
orph
igeninO-glucoside
19.41
572.19
0257
2.19
0157
3.19
77[M
+H]+
27C 2
3H28O
11Albiflo
rin20
.08
480.16
3448
0.16
4048
1.17
06[M
+H]+
28C 2
1H19ClN
2O8
Oxazepamglucuronide
20.73
462.08
3146
2.08
2546
3.09
03[M
+H]+
29C 3
4H30N
2O9
Atalanine
21.18
610.19
2461
0.19
3161
1.19
97[M
+H]+
30C 2
1H22O9
Liqu
iritin
*
22.87
418.12
9341
8.12
8241
9.13
66[M
+H]+
31C 2
8H50N
2O19P 2
N,N'-D
iacetylchitobiosyldiph
osph
odolicho
l23
.52
780.24
8678
0.25
0278
1.25
77[M
+H]+
32C 3
2H42O15
3,4,7-Trihydroxy-5-m
etho
xy-8-prenylflavan4-O
-(beta-D-xylop
yranosyl-
(1-6)-beta-D-glucopyrano
side)
24.24
666.25
0866
6.25
0866
7.25
81[M
+H]+
33C 2
7H34O
11Forsythin
* 25
.88
534.20
8653
4.21
0155
7.20
15[M
+Na]
+
34C 2
3H36O8
3''-H
ydroxypravastatin
26.01
440.24
0544
0.24
1844
1.24
77[M
+H]+
35C 2
1H25ClN
2O3
Cetirizine
27.06
388.15
4838
8.15
4741
1.14
41[M
+Na]
+
36C 2
7H34O
11Arctiin
*
29.02
534.21
0753
4.21
0155
7.20
17[M
+Na]
+
e50
Shu Y et al. Untargeted Metabolomics Profiling and … Planta Med Int Open 2020; 7: e45–e57
pharmacologicalactivities(▶table 5),rangingfromantibacterial,antiviral,anti-inflammatory,andantifungaltoantioxidant,anti-cancer,andtreatmentofallergicrhinitis[22–52].Thesepharma-cologicalactivecompoundsmayservenotonlyasleadcompoundsinnewdrugdevelopment,butalsoasbaitfortheretrievalofpro-teintargetsinanetworkpharmacologystudy[53,54].ThelattermayhelpustounderstandtheunderlyingactionmechanismofYinqiaosanformula.
Inconclusion,aUPLC-QTOF-MSmethodwiththeuseofasingleexogenousreferenceIShasbeendevelopedforuntargetedmetab-olomicsprofilingandglobalsemiquantitationoftheprescriptionChineseherbalmedicineformulaYinqiaosan.Thechemicalprofilesof4Yinqiaosanpreparationforms(i.e.,decoction,granule,pill,and▶
tabl
e 3
Common
chemicalcon
stitu
entsdetectedandidentifi
edinallfourYinqiaosanpreparationform
s.
37C 3
1H42O15
Erub
erinC
33.29
654.25
0665
4.25
1065
5.25
77[M
+H]+
38C 2
0H30O5
19(R)-Hydroxy-PGA2
34.33
350.20
9335
0.20
9135
1.21
67[M
+H]+
39C 2
0H32O5
Tuberonicacid
35.72
352.22
4735
2.22
4535
3.23
22[M
+H]+
40C 1
5H19NO
Pron
etalol
38.36
229.14
8322
9.14
7523
0.15
57[M
+H]+
41C 1
8H14O
3OvaliteninA
39.93
278.09
3527
8.09
3227
9.10
06[M
+H]+
42C 1
9H26O
6S2-Metho
xyestradiol-1
7β3-sulfate
40.02
382.14
4338
2.14
4140
5.13
36[M
+Na]
+ ;3
83.151
2[M
+H]+
43C 2
3H28O
417
-Phenyltrin
orprostagland
inA2
47.86
368.20
1136
8.20
0536
9.20
85[M
+H]+
44C 2
4H40O
63α
,6α,7β,12α
-Tetrahydroxy-5α
-cho
lan-24
-oicacid
48.05
424.28
2442
4.28
3642
5.28
98[M
+H]+
45C 2
4H41NO
4Ca
ssaidine
48.75
407.30
4440
7.30
3440
8.31
16[M
+H]+
46C 2
4H38O
43α
,12α
-Dihydroxy-5β-chol-6-en-24
-oicacid
51.34
390.27
8739
0.27
8941
3.26
76[M
+Na]
+ ;3
91.286
1[M
+H]+
47C 3
6H64N8O
17Glycopeptide
7.86
880.44
1788
0.44
1687
9.43
33[M
-H]-
48C 3
1H39NO
10S
Labrifo
rmin
16.28
617.23
3261
7.22
7861
6.22
59[M
-H]-
49C 4
9H76O
20LanatosideC
21.15
984.49
0698
4.49
3549
1.23
83[M
-2H
]2- ;983
.485
1[M
-H]-
* Markercon
stitu
ent.
Con
tinued
▶Fig. 4 Visualizationofmultivariatedataanalysis.aThePCAscoreplot,andbthePLS-DAscoreplotoffourYinqiaosanpreparationforms.
10
5
5
0
– 5
– 10
0
– 5
– 20 – 10
DecoctionGranulesPillTablet
DecoctionGranulesPillTablet
0
Decoction
Decoction
Pill
Tablet
Granules
Granules
Tablet
Pill
PC1 (54.8 %)
a
b
PLS1 (54.8 %)
PC2
(17.
5%
)PL
S2 (1
6.6
%)
10 20
– 20 – 10 0 10 20
e51
Shu Y et al. Untargeted Metabolomics Profiling and … Planta Med Int Open 2020; 7: e45–e57
Original Papers Thieme▶
tabl
e 4
Semiquantitativeanalysisof49common
lyidentifi
edcom
poundsin4Yinqiaosanpreparationform
s(n=3).
no.
Form
ula
nam
eD
± S
D (µ
g/g)
Cv (
%)
G ±
SD (µ
g/g)
Cv (
%)
t ± SD
(µg/
g)Cv
( %
)P
± SD
(µg/
g)Cv
( %
)
1C 1
6H18O9
Chlorogenicacid
*
2.6±0.4
1314
.5±0.3
23.9±0.2
41.4±0.1
9
2C 1
1H12N
2O2
D-Tryptop
han
18±2
1261
±2
43.4±0.1
41.8±0.2
13
3C 3
4H34O18
Isorientin4'-O
-glucosid
e2''-O
-p-hydroxybenzoagte
22±5
2243
±10
2485
±17
2042
±2
5
4C 2
4H24O
11Echioidinin2'-(6''-acetylglucoside)
13±5
3916
±1
45.0±0.2
32.7±0.1
5
5C 1
9H25N5O
6Asn-Trp-Thr
3.9±0.8
2022
±3
1415
±3
205±2
43
6C 2
0H26O9
BruceineD
7±2
2742
±16
3824
±3
147.5±0.5
6
7C 1
7H18N
4O5
202-79
113
±2
1611
3±16
1414
±2
137.5±0.5
7
8C 2
1H28O8
Vernofl
exuo
side
6±2
3049
±7
1321
±1
610
±1
9
9C 2
1H24O
12Ca
techin-4-ol3-O
-BeTa-D-galactopyrano
side
4.1±0.5
1230
.6±0.7
230
±1
53.3±0.2
5
10C 2
8H34O
12Ca
ohuo
sideD
2.8±0.7
2416
±3
205±1
284±1
35
11C 1
1H14O5
Genipin
12.5±0.5
412
2±1
111
±3
2818
±3
15
12C 2
0H18N
4O6
Phe-His-O
H72
±2
360
1±10
210
5±24
2313
8±50
38
13C 1
3H22N
4O8S
2Cysteineglutathion
edisulfide
18.3±0.2
110
9±12
1119
±4
2115
±2
12
14C 2
0H24N
4O6
Asp-Pro-Trp
3.2±0.6
1727
.5±0.7
26.8±0.7
113.3±0.4
13
15C 1
8H26O7
Prop
ofolglucuronide
13±2
1310
5±44
4253
±10
1917
±2
9
16C 2
3H20O9
Pong
amosideA
34±3
1029
2±20
711
9±3
269
±8
12
17C 1
5H12O
4Liqu
iritig
enin
11±1
1188
±3
417
±2
1224
±2
6
18C 2
1H21ClN
2O8
Dem
eclocycline
4.1±0.5
1330
.2±0.8
322
±2
113.5±0.4
12
19C 3
2H31N
3O5
KT572
026
±3
1213
2±5
417
.0±0.8
523
±3
11
20C 2
2H22O7
Artoindo
nesianinR
4.7±0.9
1914
±3
184.1±0.4
94.7±0.4
9
21C 2
1H20O
11Luteolin7-glucoside
*
9±1
1265
±3
512
.2±0.2
22.1±0.2
8
22C 2
2H24O7
Dihydroanhydrop
odorhizol
4.0±0.4
922
.2±0.2
18.4±0.2
34.6±0.4
10
23C 2
5H23ClN
2O7
TyrM
e-Phe4Cl-O
H48
±11
2323
8±61
2627
5±13
512
1±19
16
24C 2
9H36O15
Forsythiaside *
7±2
2319
6±38
2033
6±23
793
±7
7
25C 2
8H30O
11IkarisosideD
27±3
1145
4±15
366
6±26
414
5±14
10
26C 2
9H32O
12Am
orph
igeninO-glucoside
12±2
1488
±2
238
±3
714
.0±0.7
5
27C 2
3H28O
11Albiflo
rin4±1
2940
±6
144±1
242.7±0.7
26
28C 2
1H19ClN
2O8
Oxazepamglucuronide
5±1
2519
±6
327.5±0.4
63.8±0.5
12
29C 3
4H30N
2O9
Atalanine
5.9±0.9
1539
.0±0.6
216
.5±0.7
44.9±0.2
4
30C 2
1H22O9
Liqu
iritin
*
12±3
2619
7±10
530
±3
117.6±0.4
5
31C 2
8H50N
2O19P 2
N,N'-D
iacetylchitobiosyldiph
osph
odolicho
l22
±1
672
±2
29.2±0.3
43.2±0.4
12
32C 3
2H42O15
3,4,7-Trihydroxy-5-m
ethoxy-8-prenylflavan4-O-(beta-
D-xylopyranosyl-(1-6)-beta-D-glucopyranosid
e)3.7±0.6
1515
.9±0.5
31.81
±0.08
44.3±0.4
9
33C 2
7H34O
11Forsythin
* 1.4±0.1
1095
±3
313
8±7
513
±1
11
34C 2
3H36O8
3''-H
ydroxypravastatin
3.7±0.6
175.9±0.7
124.9±0.3
61.4±0.2
12
35C 2
1H25ClN
2O3
Cetirizine
2.03
±0.09
47.0±0.9
1228
±3
1110
.0±0.3
3
36C 2
7H34O
11Arctiin
*
2.1±0.3
1431
8±9
351
±5
116.8±0.6
8
37C 3
1H42O15
Erub
erinC
2.9±0.3
1091
±10
1112
.6±0.3
23.9±0.3
7
e52
Shu Y et al. Untargeted Metabolomics Profiling and … Planta Med Int Open 2020; 7: e45–e57
tablet)wereobtained,andthe49commonchemicalconstituentsand16pharmacologicalactivecompoundswereidentified.Simul-taneoussemiquantitativeanalysisofthemulticomponentsineachYinqiaosanpreparationformwascarriedoutalongwithuntarget-edmetabolomicsprofiling,andcross-comparisonofthechemicalcontentsinbetweenfourpreparationformswasaccomplished.PCAandPLS-DAanalysesshowedthattheUPLC-QTOF-MSmethoddevelopedwasreproducibleandthechemicalconstituentsfoundinthefourYinqiaosanpreparationformsdisplayeduniquepatternsforproductdifferentiationandrecognition.Themethoddevelopedisusefulfortheidentification,quantitation,andcross-comparisonofchemicalconstituentsnotonlyinYinqiaosanproducts,butalsoinotherCHMformulas.
MaterialsandMethods
Chemicals and Chinese herbal medicine formulasAmmoniumhydroxideandformicacidwerepurchasedfromSig-ma-Aldrich.OptimaLC/MSgradeacetonitrileandmethanol,andHPLCgradewaterwerefromFisherScientific.Deionizedwaterwasobtainedfromanin-houseBarnsteadNanopureewaterpurifica-tionsystem(ThermoScientific)witharesistivitymeterreadingof18.2MΩ-cm.Etoposide-d3(purity,97.8%)waspurchasedfromTorontoResearchChemicalsandusedasthesingleexogenousref-erenceinternalstandardinthiswork.
TheYinqiaosandecoctionwaspreparedbyJiangsuProvincialHospitalofTraditionalChineseMedicine(Nanjing,Jiangsu,China)withninekindsofprocessedCHMs(Jinyinhua,BatchNo.150320;Lianqiao,BatchNo.150718;Bohe,BatchNo.150701;Jingjie,BatchNo.150313;Niubangzhi,BatchNo.150601;Jiegeng,BatchNo.150601;Dandouchi,BatchNo.150401;Gancao,BatchNo.150701;andDanzhuye,BatchNo.150302).Yinqiaosan peifangkeli(granules)weremanufacturedbyJiangyinTianjiangPharmaceuticalCo.(Ji-angyin,Jiangsu,PRC)asindividualpackagesforeachCHM[i.e.,Jiny-inhua(BatchNo.1503112),Lianqiao(BatchNo.1501092),Bohe(BatchNo.1412140),Jingjie(BatchNo.1501094),Niubangzhi(BatchNo.1411156),Jiegeng(BatchNo.1503133),Dandouchi(BatchNo.1410023),Gancao(BatchNo.1502100),andDanzhuye(BatchNo.1501021)].Yinqiaojieduwan(pills)(BatchNo.14033352)wasproducedbyBeijingTongrentang(FengtaiDistrict,Beijing,PRC).Yinqiaojiedupian(tablets)(BatchNo.140695)wasmadebytheYunnanTengyaoPharmaceuticalCo.(Tengyue,Yunnan,PRC).
Preparation of the internal standard solutionThestocksolutionofISwaspreparedbydissolving1.00mgetopo-side-d3powderin1.00mLofmethanoltoaconcentrationof1.00mg/mL.TheworkingsolutionoftheISwaspreparedbya1/10di-lutionofthestocksolutioninmethanoltoaconcentrationof100μg/mL(or169μM).
Preparation of various forms of Yinqiaosan sample solutionsYinqiaosandecoctionsamplesolutionTheYinqiaosandecoctionwaspreparedusingthefollowingproce-dure:First,sevenofthenineprocessedCHMs,Jinyinhua(FlosLoni-cerae,9.00g),Lianqiao(FructusForsythiae,9.00g),Niubangzhi▶
tabl
e 4
Semiquantitativeanalysisof49common
lyidentifi
edcom
poundsin4Yinqiaosanpreparationform
s(n=3).
38C 2
0H30O5
19(R)-Hydroxy-PGA2
8±1
1217
±1
616
.1±0.8
56.2±0.8
13
39C 2
0H32O5
Tuberonicacid
5.6±0.4
721
±3
1618
±3
146±1
17
40C 1
5H19NO
Pron
etalol
3.9±0.6
1523
±2
810
±1
129.4±0.7
7
41C 1
8H14O
3OvaliteninA
1.3±0.3
217±2
282.0±0.5
242.3±0.2
9
42C 1
9H26O
6S2-Metho
xyestradiol-1
7β3-sulfate
4.3±0.6
1426
.4±0.4
22.98
±0.02
18±1
19
43C 2
3H28O
417
-Phenyltrin
orprostagland
inA2
1.4±0.2
127.4±0.7
106.3±0.3
53.5±0.4
11
44C 2
4H40O
63α
,6α,7β,12α
-Tetrahydroxy-5α
-cho
lan-24
-oicacid
1.46
±0.01
111
±1
107.7±0.6
84±2
41
45C 2
4H41NO
4Ca
ssaidine
0.54
±0.06
125.0±0.2
48.0±0.6
72.4±0.4
17
46C 2
4H38O
43α
,12α
-Dihydroxy-5β-chol-6-en-24
-oicacid
4±1
3112
±5
437±2
223.7±0.8
21
47C 3
6H64N8O
17Glycopeptide
12±1
1069
±7
109.4±0.2
38.6±0.1
2
48C 3
1H39NO
10S
Labrifo
rmin
28.7±0.8
318
2±13
712
±4
3313
±1
11
49C 4
9H76O
20LanatosideC
8±2
2428
4±57
2017
±5
2983
±10
13
* Markercon
stitu
ents.D
=decoctio
n,G=granu
le,T=tablet;P=pill.
Con
tinued
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Shu Y et al. Untargeted Metabolomics Profiling and … Planta Med Int Open 2020; 7: e45–e57
Original Papers Thieme
(FructusArctii,9.00g),Jiegeng(RadixPlatycodonis,6.00g),Dan-douchi(SemenSojaePreparatum,5.00g),Gancao(RadixGlycyr-rhizae,5.00g),andDanzhuye(HerbaLophatheri,4.00g)werefirstsoakedin696mLofdeionizedwaterinsideaclaypotfor30min;next,thepotwasheatedoveragasrangewithahighflame.Oncethewaterinsidethepotstartedtoboil,thegasflamewasadjustedtolowandthepotcontinuedtobeheatedforanother10min.Atthispoint,theremainingtwoprocessedCHMs,Bohe(HerbaMen-thae,6.00g)andJingjie(HerbaSchizonepetae,5.00g),wereadded,andboilingwascontinuedforanother5min.Attheendofheating,thehotdecoction(<500mL)waspouredintoa500-mLglassbeakerandcooledtoroomtemperature.Thecooleddecoc-tionwastransferredintoa500-mLvolumetricflask,andthebeak-erwasrinsedthreetimeswithdeionizedwater.Therinsesolutionwasthencombinedwiththedecoctionintheflask,andadditionaldeionizedwaterwasaddedtothe500-mLmarkoftheflask.Thesolutionwasvortexedandallowedtosettleonabenchtopfor30min;then,3.00mLofthesupernatantwerepipettedandtrans-ferredintoaborosilicateglasstube(16×100mm;FisherScientif-ic)followedbytheadditionof6.90mLofmethanoland0.100mLoftheISworkingsolution(169μM).Themixturewasvortexedfor30susingaMaxiMixIIVortexMixer(ThermoScientific).Next,1.00mLofthesolutionwastransferredintoa1.5-mLmicrocentrifugetube(VWR),andcentrifugedat18000×gfor10minusingaSor-vallST40Rcentrifuge(ThermoFisherScientific).Aftercentrifuga-tion,500µLofthesupernatantwastransferredintoan8-mmclearglassscrewthreadautosamplervialfromThermoFisherScientificandsubjectedtoUPLC-QTOF-MSanalysis.
Yinqiaosangranule,pill,andtabletsamplesolutionsFirst,1/12.5ofthedailymaximumdoseofeachpreparationformofYinqiaosanwasweighedoutprecisely,andeachsamplewas
transferredintoa50.0-mLcentrifugetube(Corning).Then,deion-izedwaterwasaddedtothe40.0-mLmark.Eachsamplewassoakedindeionizedwaterfor90min,followedbyvortexmixingfor3min,thenplacedinaThermoFisherScientificFS-28ultrasonicbathfor30min.Aftersettlingonabenchtopfor30min,3.00mLofthesu-pernatantwerepipettedandtransferredintoaborosilicateglasstube(16×100mm),andthen6.90mLofmethanoland0.100mLoftheISworkingsolution(169μM)wereadded.Themixturewasvortexedfor30s.Next,1.00mLofthesolutionwastransferredintoa1.5-mLmicrocentrifugetubeandcentrifugedat18000×gfor10min.Aftercentrifugation,500µLofthesupernatantweretrans-ferredintoan8-mmclearglassscrewthreadautosamplervialandsubjectedtoUPLC-QTOF-MSanalysis.
Preparation of QC sampleTheQCsample(800µL)couldbepreparedbymixing200µLofeachofthefourYinqiaosansamplesolutions(see“Preparationofvari-ousformsofYinqiaosansamplesolutions”),andusedwitheachbatchanalysisbymonitoringtheselectivityandreproducibilityofthemethodoverthecourseofanalysisonthe49commonlyiden-tifiedcompounds.
UPLC-QTOF-MS systemTheUPLC-QTOF-MSsystemusedinthisworkconsistedofanAgi-lent1290InfinityUPLCsystemcoupledwithanAgilent6540QTOFmassspectrometer.TheUPLCsystemincludedasolventreservoir,adegasser,aG4220Abinarypump,aG1330Bthermostat,aG4226Aautosampler,aG1316Cthermostattedcolumncompart-ment,andaG4212Adiodearraydetector.ThemassspectrometerwasequippedwithanAgilentjetstreamESIprobe.
LiquidchromatographicseparationwasachievedusinggradientelutiononaWatersACQUITYUPLCBEHC18(2.1mmi.d.×100mm,
▶table 5 PharmacologicalactiveconstituentsfoundinallfourYinqiaosanpreparationforms.
no. name PubChem CID CAS reported pharmacological activity
1 Chlorogenicacid * 1794427 327-97-9 antibacterial,antiviral[26–27]
2 Luteolin7-glucoside * 5280637 5373-11-5 antibacterial,antifungal,antioxidant,anti-inflammatory[28,29],antidiabetic[30]
3 Forsythiaside * 5281773 79916-77-1 antibacterial,antiviral,anti-inflammatory[31–32]
4 Forsythin * 101712 487-41-2 antioxidant,anti-inflammatory[33]
5 Liquiritin * 503737 551-15-5 antioxidant[34],antioxidative,anticancer,andneuroprotective[35]
6 Arctiin * 100528 20362-31-6 antiviral,anti-inflammatory[36–39]
7 D-Tryptophan 9060 153-94-6 antibacterial[40]
8 BruceineD 441788 21499-66-1 anticancer:inhibitshepatocellularcarcinomagrowth[41]
9 202-791 122114 101342-80-7 acalciumchannelagonist[42]
10 Genipin 442424 6902-77-8 anticancer:suppressescolorectalcancercells[43–45]
11 Liquiritigenin 114829 578-86-9 restoresosteoblastdamage[46],attenuatescardiacinjury[47],preventspalmitate-inducedbeta-cellapoptosis[48]
12 Demeclocycline 54680690 127-33-3 forthetreatmentofhyponatremiasecondarytoSIADH[49]
13 KT5720 3844 108068-98-0 reversesmultidrugresistance,achemosensitizer[50–51]
14 Albiflorin 51346141 39011-90-0 amelioratesobesity[52]
15 Cetirizine 2678 83881-51-0 forthetreatmentofallergicrhinitis[53–54]
16 LanatosideC 656630 17575-22-3 anticancer:inducesapoptosisinhumangastriccancercells[55]andhumanhepatocellularcarcinomacells[56]
* Markerconstituents.
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1.7µm,130Å)columnwithaninlineVHPfilter(0.5µm,stainlesssteel)fromUpchurchScientific.ThemobilephaseforthepositiveESI-QTOF-MSconsistedofA)0.1%formicacidaqueoussolution(v/v)andB)acetonitrile.ThemobilephaseforthenegativeESI-QTOF-MSconsistedofA)0.05%ammoniumhydroxideaqueoussolution(v/v)andB)acetonitrile.Thegradientelutionprofilewasasfollows:0–4min,5%B;4–7min,5–10%B;7–20min,10–15%B;20–30min,15–22%B;30–35min,22–35%B;35–40min,35–50%B;40–45min,50–70%B;45–50min,70–90%B;50–52min,5%B;52–60min,5%B.Theflowratewas0.4mL/min.Thecolumntemperaturewasat60℃.Thesampleinjectionvolumewas5.00µL.Priortosampleanalyses,thecolumnwasequilibratedwiththemobilephaseforatleast30minataflowrateof0.4mL/min.
TheQTOF-MSwasoperatedinbothpositiveandnegativeESImodes.Thechromatographicandspectradata(.d)wereacquiredforeachESImodeusingAgilentMassHunterDataAcquisitionsoft-ware(VersionB.05.01).Theoperationparametersofthemassspectrometerwereasfollows:dryinggas(N2)temperature,350°C;dryinggasflowrate,10.0L/min;nebulizergas(N2)pressure,35psi;sheathgasflowrate,11.0L/min;sheathgas(N2)temperature,325°C;capillaryvoltage,4000V;nozzlevoltage,500V;fragmen-torvoltage,100V;skimmervoltage,65V;octopoleradio-frequen-cyvoltage,750V;collisionenergy,10,20,and40eV.ThedatawereacquiredbyautoMS/MSmodewithanextendeddynamicrange (2GHz).TheMSscanrangewas50–1000m/zatascanrate5spectra/s.TheMS/MSscanrangewas50–1000m/zatascanrateof3spectra/swithanisolationwidthatnarrow(~1.3m/z).Tomaintainthemassaccuracy,themassspectrometerwascalibrat-edandtunedbeforeanalysis,andinternalreferencemassesfromthereferencemasssolutionwereusedforreal-timemasscorrec-tionatm/z121.0508and922.0098forthepositiveionmode,andm/z112.9885and1033.9881forthenegativeionmodethrough-outtheacquisitionprocess.Thereferencemasssolutionwaspre-paredusinganAgilentAPI-TOFreferencemasssolutionkit(PartNo:G1969-85001).
Method validationThespecificityandreproducibilityoftheUPLC-QTOF-MSmethodwereassessedbyreplicatemeasurementsofsamplesoffourYin-qiaosanpreparationforms.PCAscoreplotswereconstructedusingtheacquiredMSdata,andtheCVswerecalculatedontheconcen-trationsofreplicatemeasurementsof49commonlyidentifiedcompoundsthroughglobalsemiquantitativeanalysis.
Data processing, constituent identification, and statistical analysisThedatafiles(.d)ofreplicatemeasurementsforfourYinqiaosanpreparationformsacquiredbyAgilentMassHunterDataAcquisi-tionsoftwareatthesameionpolarity(i.e.,eitherpositiveornega-tiveESI)werefirstassessedusingAgilentMassHunterQualitativeAnalysissoftware(Version:B.06.00)forthesignal/noiselevel,andretentiontimeandmassshiftswithrespecttothespikedIS,whichwerethenprocessedusingAgilentProfindersoftware(VersionB.06.00)forbatchrecursiveanalysis.Thedatafilesweregroupedintofourpreparationformsandsubjectedtomolecularfeatureex-tractionbyselectingapeakheightthresholdof1000counts,pos-
sibleionadducts,isotopemodelofcommonorganicmolecules,chargestatesuptotwo,aretentiontimewindowof0.10%+0.60min,amasswindowof20.00ppm+2.00mDa(foralignmentoftheISinallrunswiththesamepolarity),andapost-processingfil-terat3outof3replicatemeasurementsforeachYinqiaosanprep-arationform.
Eachmolecularfeatureextracteddatafile(.cef)byProfindersoftwarealongwiththecorrespondingdatafile(.d)byMassHunt-erDataAcquisitionsoftwarewasimportedintoAgilentMassHunt-erQualitativeAnalysissoftware.Using“FindbyFormula”,MS/MSdataalongwithitsMSandretentiontimedatawereextracted.Thenewdatafiles(.cef)inthesameionpolarityofreplicatemeasure-mentsofeachYinqiaosanpreparationformwerethenimportedintoAgilentMassProfilerProfessionalsoftware(Version:B.13.1.1)foranMETLINAMdatabasesearchandmolecularformulagenera-tion.Theselectionofelementsandlimitsformolecularformulagenerationwereasfollows:C(3–156),H(0–180),O(0–40),N(0–20),S(0–14),Cl(0–12),F(0–12),Br(0–10),P(0–9),andSi(0–15)[55].Thetopfiveconstituentswiththehighestscoreswereanno-tated,andcross-checkedwiththeTraditionalChineseMedicineIn-tegratedDatabase[56]andTraditionalChineseMedicineSystemsPharmacologyDatabaseandAnalysisPlatform[57]forfinalnameassignments.Thecombineddatafile(.cef)ofreplicatemeasure-mentsofeachYinqiaosanpreparationformwiththenamesofchemicalconstituentswassavedforglobalsemiquantitativeanal-ysis.
Formultivariatedataanalysis,themolecularfeatureextracteddatafiles(.csv)bytheProfindersoftwarewereimportedintoMe-taboAnalyst4.0[58]intermsofmass,retentiontime,andpeakarea.Themasstoleranceandtheretentiontimetoleranceweresetat0.025and30s,respectively.Samplenormalizationwasper-formedbytheISreferencefeatures(i.e.,mass,retentiontime,andpeakarea),andthedatawerelogtransformedandautoscaled.PCAandPLS-DAwereperformedusingtheISfornormalization.PCAandPLS-DAscoreplotswereconstructedforsimilaritycomparisonofthechemicalconstituentsandreproducibilityassessmentoftheUPLC-QTOF-MSmethodaswellaspatternrecognitionamongthefourYinqiaosanpreparationforms.
Global semiquantitative analysisThedatafiles(.d)withthesameionpolarityofreplicatemeasure-mentsofeachYinqiaosanpreparationformbytheMassHunterDataAcquisitionsoftwareandtheircorrespondingcombineddatafile(.cef)withthenamesofchemicalconstituentsbytheMassProfilerProfessionalsoftwarewereimportedintoAgilentMassHunterQuantitativeAnalysissoftware(Version:B.06.00).Inthemethodsetuptask,theretentiontimewindowwassetat0.6min,etopo-side-d3ammoniumadductwaschosenastheISandflagged,otheridentifiedchemicalconstituentswerechosenastargetsrelativetotheIS,andproperionpolaritywaschosen.Aftervalidatingthemethodsetup,globalsemiquantitativeanalysiswasperformedbasedonthepeakarearatioofeachindividualtargettotheIS,andthequantitationdatawereexportedasexcelfileforreporting.
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Original Papers Thieme
Supporting InformationTheMS/MSspectraofthe49chemicalconstituentscommonlyidentifiedinallfourYinqiaosanpreparationforms(▶Fig. 1S),thecommonchemicalconstituentsdetectedbutunidentifiedinallfourYinqiaosanpreparationforms(▶table 1S),andtheuniquecon-stituentsfoundindecoction,granule,pill,andtabletformsofYin-qiaosan(▶tables 2S– 9S)areavailableasSupportingInformation.
AcknowledgementsYachunShuwouldliketoacknowledgethefundingsupportoftheChinaNaturalScienceFoundation(81503216)andJiangsuProv-ince(LGY2016017).
ConflictofInterest
Theauthorsdeclarethattheyhavenoconflictofinterest.
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