Chiral capillary electrophoresis–electrospray mass spectrometry

Chiralcapillaryelectrophoresis–electrospraymassspectrometry

couplingusingvancomycinaschiralselector

SalvatoreFanalia,ClaudiaDesiderioa,GeorgSchulteb,StefanHeitmeierb,

bb,1b,DirkStrickmann,BezhanChankvetadze,GottfriedBlaschke*

aIstitutodiCromatografiadelC.N.R.,AreadellaRicercadiRoma,P.O.Box10,00016MonterotondoScalo,Rome,Italy

b¨¨InstituteofPharmaceuticalChemistry,UniversityofMunster,Hittorfstrasse58-62,48149Munster,Germany

Abstract

Capillaryelectrophoresis–electrospraymassspectrometrycoupling(CE–ESI-MS)usingvancomycin(VC)asachiral

selectorisdescribedinthisstudy.TheselfelectrophoreticmobilityofVC,asapositivelychargedchiralselectoratlowpH,allowsustoavoidthecontaminationoftheionsourceoftheESI-MSwiththechiralselectorinasimpleway.Theusefulnessofthistechniqueisillustratedfortheanalysisofchiralanioniccompoundsofpharmaceuticalimportance.Additionally,examplesofastereospecificdeterminationofibuprofenanditsphaseImetabolitesaswellasetodolacanditsmetabolitesinurinesamplesaredescribed.TheadvantagesofMSforpeakidentification,peakpuritytestingandforselectivemonitoringofoverlappingpeaksaredemonstrated.©1998ElsevierScienceB.V.

Keywords:Enantiomerseparation;Chiralselectors;ChiralCE–ESI-MS;Vancomycin;Arylpropionicacids;Etodolac;Ibuprofen

1.Introduction

Theseparationofenantiomericcompoundscanbeeasilyachievedbycapillaryelectrophoresis(CE)usingthedirectseparationmethod,e.g.,byadditionofthechiralselectortothebackgroundelectrolyte(BGE).

AwidenumberofchiralselectorshavebeenusedinCE[1–3]suchascyclodextrinsandtheirderiva-tives,chiralsurfactants,proteins,metal–aminoacidcomplexes,crownethers,andrecentlyantibiotics[4–11].

ChiralCE–massspectrometry(MS)couplingisdevelopingwithinthelastfewyears[12–14].MSis

*Correspondingauthor.1Permanentaddress:DepartmentofChemistry,TbilisiStateUniversity,ChavchavadzeAve1,380028,Tbilisi,Georgia.sensitive,specificandatthesametimeauniversaldetectionsystem.Additionally,MSprovidesimpor-tantinformationaboutthestructureofanalyteswhichisespeciallyusefulfortheidentificationofmetabolitesinclinicalandbiopharmaceuticalanaly-ses.On-lineMScouplingisadvantageousinCEbecausefractioncollectionforfurtheranalysisisverytime-consumingandmoredifficultinthistechniquecomparedwithhigh-performanceliquidchromatography(HPLC).

InchiralCE,thepresenceofchiralselectorsintheBGEmaycausesomeproblemsduetotheinterfer-encewithanalytes(UV)orcontaminationofthedetector(MS).TheappearanceofthechiralselectorintheionsourceoftheMSmaydecreasethesensitivityduetothecompetitionwithananalyteforavailablechargeaswellasduetotheincreaseofthebackgroundnoise[12,13].Toavoidtheappearance

0021-9673/98/$19.00©1998ElsevierScienceB.V.Allrightsreserved.PIIS0021-9673(97)00978-3

70S.Fanalietal./J.Chromatogr.A800(1998)69–76ofchiralselectorintheionsourcetheuseofcolumn-couplingandmultiplevoltageswitchingtechniquehasbeenproposedrecently[13].Analternativeapproachcanbebasedontheoppositemigrationdirectionofananalyteandachiralselector.ThisprinciplewasproposedforUV-adsorbingchiralselectorsbyValtchevaetal.[15]andinvolvedfillingonlypartoftheseparationcapillarywiththechiralselector.Aslightlymodifiedversionofthistech-niqueallowingthefillingofthewholecapillarywithachiralselectorwasdescribedinRef.[16].TheusefulnessofthepartialfillingtechniqueinchiralCEwithUVdetectionhasbeendemonstratedinanumberofstudies[8,9,11,17].Thetechniquepro-posedinRef.[16]hasbeenrecentlyusedalsoinchiralCE–electrospray(ESI)-MScouplinginordertoavoidtheappearanceofchargedcyclodextrinsintheionsource[14].

InthispaperachiralCE–ESI-MScouplingfortheseparationofseveralracemicarylpropionicacids

(APAs)employingvancomycin(VC)asachiralselectorisreported.

2.Experimental

2.1.Chemicalsandreagents

Aceticacid,ammoniumacetate,ammoniumhy-droxideandmethanolwerepurchasedfromMerck(Darmstadt,Germany).Racemicflurbiprofen(FP),ketoprofen(KP)andcarprofen(CP)werefromSigma(Daisenhofen,Germany).(1)-and(2)-Nap-roxen(NP)werekindlyprovidedbyDr.CeciliaBartolucci,IstitutodiStrutturisticaChimica,C.N.R.(Montelibretti,Rome,Italy).

Racemicetodolac(ET)anditsphaseImetabo-lites,7-hydroxyetodolac(7-HET)and8-(19-hydroxy-ethyl)etodolac(8-HET)weregiftsfromWyeth

¨Pharma(Munster,Germany).Racemicibuprofen

Fig.1.Chemicalstructuresofthecompounds.

S.Fanalietal./J.Chromatogr.A800(1998)69–7671

(IB)anditsphaseImetabolites,2-hydroxyibuprofen(HIB)andcarboxyibuprofen(CIB)weregiftsfrom

¨Kanoldt(Hochstadt/Donau,Germany).Thestruc-turesofthechiralcompoundsusedinthisstudyare

showninFig.1.

VC,methacrylicacid3-trimethoxysilylpropylester,ethylenediaminetetraaceticacid,acrylamide,sodiumpersulfateandN,N,N9,N9-tetramethyl-ethylenediaminewereobtainedfromFluka(Buchs,Switzerland).

Stocksolutionsofracemicsamples(1mg/ml)werepreparedinmethanol,storedat48Canddilutedto50–100mg/mlinacetatebufferpH4.8beforetheanalysis.50mMAceticacid–ammoniumacetate,pH4.8,containingvariousamountsofVCwastheBGEusedfortheenantioseparationofthestudiedAPAs,ETortheirmetabolites;theBGEscontainingthechiralselectorwerefreshlyprepareddaily.

operatedinaconstantvoltagemodeandtheelectro-phoreticrunswereperformedinapolyacrylamidecoatedcapillary[18]of44cm350mmI.D.filledeitherwith50mMaceticacid–ammoniumacetatebufferpH4.8orthesamebuffersupplementedwiththeappropriateamountofVC.Hydrostaticinjection(10cm)for5–10satthecathodicendofthecapillarywasusedinallexperiments.Theotherendoftheseparationcapillarywasprotrudedtotheionspraytipwhereavoltageof2.6kVwasmaintained.ALCQiontrapmassspectrometer(Finnigan,Bran-ford,CT,USA)equippedwithanelectrosprayinterfacewasusedinthenegativeionmodeforthedetectionofanalytes[12–14].Thesheathliquidconsistingofmethanol–water–ammonia(50:48:2)wasdeliveredataflow-rateof6ml/minusingasyringepump.

2.2.Apparatus

AGromcapillaryelectrophoresissystem100highvoltagepowersupply(Herrenberg,Germany)was

3.Resultsanddiscussion

VCisoneofthemostpowerfulchiralselectorsfortheseparationofcompoundscontainingfreecar-

Fig.2.Schemeofthecounterflowelectrophoreticseparationoftheanionicanalytesusingvancomycinasthechiralselector.

72S.Fanalietal./J.Chromatogr.A800(1998)69–76Fig.3.CE–ESI-MSelectropherogramsofracemicnon-steroidal-antiinflammatorydrugs:(a)(6)-carprofen(CP),(b)(6)-flurbiprofen(FP),(c)(6)-ketoprofen(KP)and(d)(6)-naproxen(NP)inthepresenceof5mMofvancomycinintheBGE.Capillary(polyacrylamidecoated)44cm350mmI.D.;appliedvoltage,20kV;BGE,50mMaceticacid–ammoniumacetatepH4.8;injection,hydrostatic(10cm)310satthecathodicendofthecapillary.

S.Fanalietal./J.Chromatogr.A800(1998)69–7673

boxylicgroups[4,8–11].However,thehighab-sorbanceintheUVrangemaycausesignalinterfer-encewiththeanalyteandconsequentlysomesen-sitivityrelatedproblemsinCEwithUVdetection.Recently,thepartialfillingtechniquehasbeenproposedasthesolutiontothisproblem[8–11,17].AninterferencebetweenthesignalsofVCandtheanalytesisnottheprobleminCEwithMSdetection.However,theappearanceofthechiralselectorintheMSionsourcemaycauseadecreaseofdetectionsensitivitybycompetitionwiththeanalyteforanavailablechargeaswellasbyanincreaseofthebaselinenoiseduetothecontaminationoftheionsource[12,13].Therefore,itisdesirabletoavoidthepenetrationofnonvolatilebuffercomponentsintheMS.

AsrepresentedinFig.2thetechniquesimilartopartialfillingtechniquemayalsobeusefulinchiralCEwithMSdetection[14].Thistechniqueallowsustoavoidinasimplewayacontaminationoftheionsourcewiththechiralselectorandconsequentlyimprovesthesensitivityandstabilityoftheresponse.ThishasbeendemonstratedrecentlyforchiralCE–ESI-MScouplingusingchargedcyclodextrins[14].Aceticacid–ammoniumacetatebufferatpH4.8wasselectedfortheexperiments.ThisbuffermeetsseveralimportantrequirementsforchiralCE–MScoupling,namely:(i)itisvolatileandthuscompat-iblewiththeMSdetector,(ii)thestudiedanalytesmoveasanionsinarelativelyshorttimeand(iii)VC,whichispositivelychargedatthispH,migratesintheoppositedirectionofanalytesawayfromtheESI-MSsource.

InordertoconfirmthatthechiralselectordoesnotappearintheMSinterfacethefollowingexperimentwasperformed.TheseparationcapillarywasfilledwiththeBGEcontaining12.5mMVCandhighvoltagewasappliedsothattheanodewasontheMSsideofthecapillary.Thedetectionwasperformedalternativelyinbothinthepositiveandinthenegativeionmode.Onlytheminorpeakswithm/zrangingfrom700.6to760.5weredetectedinbothmodes.ItseemsworthnotingthatalthoughVCispositivelychargedintheseparationcapillarythesheathliquidusedfortheMSdetectionoftheanionicanalytesisalkalinewhichmaytransformthechiralselectortoitsanionicform.Thus,thisexperi-mentconfirmedundoubtedlythatthechiralselectordoesnotcontinuouslyentertheMS.ThetracesfoundareprobablyduetothepenetrationintotheMSduringfillingtheseparationcapillarywiththeBGEcontainingVC.

TheCE–ESI-MSelectropherogramsofracemicCP,FP,KPandNPusingtheBGEwith5mMVC

Fig.4.CE–ESI-MSelectropherogramsofracemic(a)etodolac(ET)anditsmetabolites,(b)7-hydroxyetodolac(7-HET)and(c)8-(19-hydroxyethyl)etodolac(8-HET)inthepresenceofvancomycinintheBGE.Experimentalconditions:5mMvancomycininaandband12mMinc;appliedvoltage15kVinaandband12kVinc.ForotherexperimentalconditionsseeFig.3.

74S.Fanalietal./J.Chromatogr.A800(1998)69–76areshowninFig.3.Verygoodenantiomericres-olutionwasachievedforallarylpropionicacids.Similarresultshavebeenobtainedfortheenantio-mericseparationofracemicETandtwoofitsphaseImetabolites(Fig.4).Theanalysistimeoftheseparatedenantiomerswasrelativelyshort(lessthan15min)andthepeakefficiencyhigh(90000–110000plates/m).Onlytwoamongfourpossible8-HETstereoisomerswereresolved.

Themasstrackscorrespondingtomolecularmass-esofIB(m/z204.5–205.5),anditsphaseImetabo-lites,2-HIB(m/z220.5–221.5)andCIBs(m/z234.5–235.5),fromthefull-scanCE–ESI-MSspec-traoftheirmixtureareshowninFig.5.Theadditionof5mMofVCintheBGEallowedtheenantio-separationofIB,2-HIBandallfourstereoisomersofCIB.However,peakoverlappinghasbeenobservedforIBand2-HIB.Inspiteofthesevereoverlappingofthemigrationzonestheselectionofcorrespondingmasstracksallowsustoperformpeakidentificationofthecompoundpossessingdifferentmolecularmasses.ThisisoneoftheimportantadvantagesofMSasadetectionsystemwhensamplesofcomplexoriginareanalyzed.Anadditionaladvantageofthis

Fig.5.CE–ESI-MSelectropherograms(selectedmasstracks)andfullscanESI-MSspectra(b)ofselectedpeaksofracemicibuprofen(IB),2-hydroxyibuprofen(2-HIB)andcarboxyibuprofens(CIB).ExperimentalconditionsasinFig.3.

S.Fanalietal./J.Chromatogr.A800(1998)69–7675

detectionprincipleistheinformationaboutpeakpurity.ThiscanbedonebyscanningthefullMSspectraofeachpeak.AsindicatedbytheMSspectra(Fig.5b)thepeakofIBenantiomerwiththemigrationtime12.63containstheimpurityofHIB(m/z5221)andthefirstpeakofthelatterone(t512.69)ismarkedlycontaminatedwithIB(m/z5205).Thesecondpeaksofbothcompounds(t513.36and13.15)seemtoberelativelypure.

AnotheradvantageofCE–MScouplingistheon-lineidentificationofresolvedpeakswithoutanysamplecollection.Forinstance,itisobviousthatallfourpeakswithm/z5234.5–235.5belongtothestereoisomersofCIB.

Becker-ScharfenkampandBlaschke[19]studiedthephaseIandthephaseIImetabolismofthenonsteroidalantiinflammatorydrugET.EvidenceforastereoselectiveeliminationofET-acylglucuronideand7-hydroxyetodolac-acylglucuronide(7-HETG)werefoundbyindirectdeterminationofthediffer-encesoftheconcentrationsofETand7-HETbeforeandafteralkalinehydrolysisoftheconjugatesusingHPLC.Later,BerendesandBlaschke[20]developedaHPLCmethodwhichinvolvestheacetylationofhydroxygroupsandmethylationofcarboxygroupsinordertoincreasethelipophilicityofthepolaracylglucuronidesofETand7-HET.Bothofthesetechniquesinvolveatediousandtime-consumingsampleextractionandpretreatment.Therefore,thedirectanalysisoftheurinesamplewithoutanysampleextractionandpretreatmentwasattempted.AsshowninthechiralCE–ESI-MSelec-tropherogramofaurinesampleofavolunteerreceivingasingle400mgdoseofracemicET(Fig.6)thesinglepeaksofETG(m/z61.9),oneoftheisomericHETs(m/z5302.1)andoneoftheglucuro-

Fig.6.CE–ESI-MSelectropherograms(selectedmass-tracks)(a)andfullscanESI-MSspectra(b)correspondingtoeachpeakofaurinesampleofavolunteerreceivinga400mgsingleoraldoseofracemicetodolac.Thecumulativeurinesamplewascollectedin3–7hafterdrugadministrationandwasdirectlyintroducedintotheseparationcapillarybyhydrodynamicinjection(10cm)for10s.Theconcentrationofvancomycinwas12.5mM;theappliedvoltage15kV;otherexperimentalconditionswereasinFig.3.

76S.Fanalietal./J.Chromatogr.A800(1998)69–76nidesoftheisomericHETs(m/z77.9)couldbeobservedintheurinetogetherwithendogenoushippuricacid(m/z5178.2).Accordingtopreviousresults[19,20]thepeakwithm/z5302.1mostlikelybelongstooneoftheenantiomersof7-HETandthepeakwithm/z77.9tothecorrespondingglucuro-nide.Furtherstudiesonthissubjectareinprogressnow.

Thus,asshowninthispreliminarystudychiralCE–ESI-MScouplingrepresentsaveryeffectivealternativeforexpensiveandtime-consumingHPLCmethodsfortheseparation,identificationandproba-blyalsoforthequantificationofchiraldrugsandtheirphaseIandphaseIImetabolitesinbiologicalmatrices.Insomecasesthebiologicalfluidscanbedirectlyinjectedintotheseparationcapillarywithoutanysamplepretreatment.

Acknowledgements

B.C.thankstheRectorateoftheUniversityofMunster¨forthefinancialsupportofhisstayasavisitingprofessorattheInstituteofPharmaceuticalChemistryofthisuniversity.Theshort-termstayofS.F.attheInstituteofPharmaceuticalChemistry,

UniversityofMunster,¨wasfinanciallysupportedby

DAAD(GermanSocietyforAcademicExchange,Bonn,Germany).

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