如何快速建立手性藥物分析LC及LCMSMS方法

ChiralMethodDevelopmentTechniquesforAnalytical&PrepHPLCAGENDA:MethoddevelopmenttechniquesfortheCHIROBIOTICseriesofchiralLCcolumnsTechniquesforchiralLC/MS/MSAdvancesinpreparativechiralLCAdvancedSeparationTechnologiesMarch2003

ProposedStructureofVancomycinp-acceptorionicsitehydrogenbonding&dipolestackingsitessugarmoietiesamineA,B,Careinclusionpockets(weak)

MultipleuseCovalentbondingBroadapplicabilityProposedStructuresofGlycopeptideCSPsTeicoplaninTeicoplaninAglyconeKeysitesProposedStructuresofGlycopeptideCSPsRistocetinAFeaturesofCHIROBIOTICPhasesReversedphaseLargestandmostversatilenumberofseparationsPolarOrganicphaseFastkinetics,secondmajorgroupofseparationsBestsuitedforLC/MSsystemsNormalphaseGenerallyprovidesca10-15%ofthetotalmethodsColumncouplingpossibleRapid,broadscreeningprotocolsEachcolumncanbeusedinall3modes

BroadSelectivityBasedontheSameStereogenicCenter:CHIROBIOTICT:AminoAlcoholsAlbuterolAtenololMetoprololMobilePhase:100/0.1/0.1:MeOH/HOAc/TEA@2.0mL/minuteOxprenololPropranololSotalol12.6413.846.817.4811.8212.557.087.838.509.478.719.66WhatisthePolarOrganicMode?ThePolarOrganicModewasdevelopedoriginallybyDrDWArmstrongandAstecin’85forusewithCYCLOBONDcyclodextrintechnology100%MeOHwithaddedacidandbase(typically0.1%,eachwitharangeof1.0to0.001%),orequivalentsaltsMethoddevelopmentisverysimpleandfastIonicWhatisthePolarOrganicMode?ThePolarOrganicModeterminologyisnowfrequentlyappliedtotheDaicelphases,butinthiscaseitmeans100%organiconly,usuallyMeOH,ACNorMeOH/EtOHThemechanism(predominantlyhydrogenbonding)forthePOMonDaicelisdifferentfromthatontheCHIROBIOTICphasesandtheoptimizationprocessisdifferentIonicWhatisthePolarOrganicMode?ThemechanismforthePOMontheCHIROBIOTICphasesispredominantlyionic,makingitessentialforacidandbasetobeadded.Thus,PolarIonicMode?IonicAdvantagesofPolarIonicMode?onCHIROBIOTICV,TandRNoproblemwithsaltcontainingcompoundsCompatiblewithcolumnswitchingfromC18columntochiralcolumnScale-upiseasyduetothevolatilityofmobilephasecomponentsComplementarytohexane/ethanolseparationsonpolysaccharideCSPsForLC/MS,canuseammoniumtrifluoroacetate,acetateorformate(0.001to0.5%w/vinmethanol)ApplicabletotheuseofgradientsformethoddevelopmentNormalphasevsPolarIonic?PhaseCHIROBIOTICcolumnsappeartobecomplementarytocellulosicandamylosicphasesManyseparationsthataredoneonChiracelinnormalphasecanalsobeaccomplishedinthepolarionic?modeonCHIROBIOTICcolumnsIfacompoundispolar,solubilitymaybebetterinthePIMratherthannormalphaseThePIMcanbeadvantageousandsaferforprepNOTE:IfusingCHIROBIOTICcolumnsintheNormalPhase,theyneedmuchhigherEtOHlevelsthanDaicelcolumnsformostchiralseparations–typically20-50%[EtOH]MetoprololCHIRACELODPeak1–11.9min.Peak2–18.2min.20/80/0.1:IPA/Hex/DEACHIROBIOTICTPeak1–6.8minPeak2–7.5min100/0.2/0.1:MeOH/HOAc/TEAAlprenololCHIRACELODPeak1-12.4minPeak2-16.4min20/80/0.1:IPA/Hex/TFACHIROBIOTICVPeak1–7.69min.Peak2–8.33min.100/0.01/0.01:MeOH/HOAc/TEACHIROBIOTICTAGSelectivityontheCHIROBIOTICTcanbeenhancedwithCHIROBIOTICTAG(aglyconefromteicoplanin)forcertainmoleculartypesEnhancedresolutionfor,,andcyclicaminoacids,neutrals,acids,sulfurcontainingmoleculesandcertainprimaryaminesCHIROBIOTICTAGMobilephasepossibilities:Reversedphase–methanolastheorganicmodifierPolarionic?phase–asfortheCHIROBIOTICTNeutralmoleculesusesinglesolvent:100%MeOH,EtOH,IPA,ACN.NoacidorbaserequiredNormalPhase–goodforlargenumberofchiralsulfoxides,coumarinsSelectivityofMethanolforNeutralMoleculesonCHIROBIOTICTAG5-Methyl-5-phenylhydantoin4-Methyl-5-phenyl-2-oxazolidinone100%MeOH@0.8mL/min100%MeOH@0.8mL/min5.08min9.62min5.35min8.21minChiralSulfoxidesonCHIROBIOTICTAGTeicoplanin TAGMobilephase:Hexane/EtOH,50/50Flowrate:2mL/minp-Flurophenylmethylsulfoxide77.65.99MethodDevelopmentTechniques

UsingColumnCouplingGenericscreeningmethodsforfastchiralmethoddevelopmentColumncouplingutilizedtoenablesimultaneousmulti-columnscreeningMethodDevelopmentTechniquesUsingCoupledColumns3solventssystems,plus3differentCHIROBIOTICcolumns(closecoupledkit)provides9screensin105minutesSCREENINGMOBILEPHASES:Polarionicmode?(MeOH/AcOH/TEA,100/0.02/0.01,2.0mL/min)Reversedphasemode(MeOH/TEAA(0.1%,pH6.0),25/75,1.0mL/min)Normalphasemode

(Hex/EtOH,40/60,1.5mL/min)OptimizationinthePolarIonicMode?

fromCoupledColumnScreenforLC/MSTrimipraminet1: 11.27t2: 11.54t1: 20.7t2: 22.8ScreenOptimisedR+V+T(10cm)MeOH/HOAc/TEA:100/0.02/0.012mL/min.V(25cm)100/0.02v/w:MeOH/AFTA0.8mL/min.CASEHISTORY:GenericMethodDevelopmentScreen:4Unknowns

PolarIonicMode,Run1Columns: CHIROBIOTICR+V+T(100x4.6mmeach)MobilePhase: 100/0.1/0.05,MeOH/AcOH/TEAResults:

SampleA

SampleBSampleCSampleDRun2(onSampleBandC):ReversedPhaseScreenColumns: CHIROBIOTICR+V+T(100x4.6mmeach)MobilePhase: 20/80:MeOH/0.1%TEAA,pH6.0Results:

SampleBSampleCMobilePhase: 40/60:MeOH/0.1%TEAA,pH6.0Results: Noselectivityobserved16.01SampleCRun3(onSampleC):NormalPhaseColumn: CHIROBIOTICR+V+T(100x4.6mmeach)MobilePhase: 1.40/60:EtOH/Hexane 2.5/95:EtOH/HexaneResults: Noselectivity(retention)observed 1.502.24OptimizedResults:SampleAColumn: CHIROBIOTICVMobilePhase: 100/0.2/0.1:MeOH/AcOH/TEAFlowRate: 0.7mL/min12.3913.53OptimizedResults:SampleBColumn: CHIROBIOTICTMobilePhase: 40/60:MeOH/0.1%TEAA,pH4.1FlowRate: 0.6mL/min16.5417.71OptimizedResults:SampleDColumn: CHIROBIOTICTMobilePhase: 100/0.2/0.1:MeOH/AcOH/TEAFlowRate: 1.0mL/min22.4824.96UsingCHIROBIOTICPhasesforSFCArecent(subcritical)study:TAG,T>Rforseriesof110compoundsincludingneutralketones,sulfoxides,underivatizedaminoacids,carboxylicacidsandb-blockersBaselineseparationachievedwithin15minutesonatleastoneCHIROBIOTICCSPRef:YLiu&DWArmstrong,InpressUsingCHIROBIOTICPhasesforSFCSulfoxideD6Mobilephases:7%MeOHinCO2A:CHIROBIOTICTB:CHIROBIOTICTAGC:CHIROBIOTICRABCChiralLC/MS/MS

inBioanalysisChallengesforChiralLC/MS/MS

inBioanalysisAdaptabilityofspectrophotometricmethodstoLC/MS/MSwithminimizationofionsuppressionLimiteduseofinorganicbuffersSeparationsinhigherorganic,loweraqueousmobilephasespreferredMethodsusingflammablesolventssuchashexanemayrequireadditionalsafetymeasures(eg,N2and/orpostcolumndilutionwithorganic/aq)PossibleadaptabilitytocassettedosingandmultiplexingLC/MS/MStechniquesConsiderationsforUsingCHIROBIOTIC

ChiralLCColumnsinLC/MS/MSHighenantioselectivityin100%MeOH(polarionicmode?)withaddedacid/baseorsaltsLesstoxicandlowerboilingpointthanhexaneNon-corrosiveWorksespeciallywellwithESIColumnsarealsocompatiblewithammoniumacetateorformate(aqueousmode)andwithallprinciplesolventsusedinAPCI(fromalcoholstohydrocarbons,DMSO,DMF)HighThroughputBioanalyticalChiralbyLC/APCI/MS/MS:ExamplesCompoundColumnColumnsizeMobilephaseFlowratetRenantiomersRitalinicacid1CHIROBIOTICT50x4.6mm100MeOH/0.03%ATFA1.2ml/min5.0,10.4minMethylphenidate2(2500samples)CHIROBIOTICV150x4.6mm100MeOH/0.03%ATFA1.0ml/min6.1,7.2minFluoxetine1CHIROBIOTICV50x4.6mm100MeOH/0.09%ATFA1.0ml/min6.2,6.8minNicardipine1CHIROBIOTICV50x4.6mm100MeOH/0.05%ATFA1.0ml/min1.1,1.6minMetoprolol1CHIROBIOTICT150x4.6mm100MeOH/0.025%ATFA1.2ml/min7.0,7.8minSalbutamol3(4000samples)CHIROBIOTICT250x4.6mm100/0.5/0.1MeOH/AcOH/NH4OH2.0ml/min3.2,3.7min(metabat2.0)1.RBakhtiar&FLSLee,RapidCommuninMS,14,1128-1135(2000)2.LRamos,RBakhtiar,TMajumdar,MHayes&FTse,RapidCommunMS,13,2054-2062(1999)3.KJoyce,AEJones,RJScott,RABiddlecombe&mSPleasance,RapidCommunMS,12,1899-1910(1998)ApplicationforChiralLC/APCI/MS/MSinPharmacokinetics:Salbutamolandits

4-O-sulfateMetabolite*

*KarinaB.Joyce,AnneE.Jones,RebeccaJ.Scott,RobertA.Biddlecombe,StephenPleasance,DrugMetabolismandBioanalysis,GlaxoWellcomeR&D,Ware,UK,RapidCommunicationsMS,12,1899-1910(1998)CHIROBIOTICT(Teicoplanin),250x4.6mmMeOH/AcOH/NH4OH:100/0.5/0.1@2mL/min3minuteassay/96wellSPE100pg/mLLOQforparentcompound,5ng/mLforsulphatemetabolism; 25pg/mLLOQfor80mLinjection4000sampleclinicalstudyChiralmetaboliteNeatsolutionat10ng/mLof:Atenolol(m/z267.2145.0),Mianserin(m/z265.2208.2),Terbutaline(m/z226.0152.2),Propranolol(m/z260.2116.2).[Ref:RBakhtiar,inpress]UsingtheCHIROBIOTICR,V,TKitforDrugMetabolism:LC/APCI/MRMChromatogramsLC/MS–ConclusionsTheuseofthepolarionicmode?

providesthefastestmethodsforchiralLC/MS/MSwithwidestapplicabilityThesamecolumncanbeusedforreversedphaseseparationsinhighorganicwithNH4AcbufferSimultaneouschiralseparationsofferthepossibilityofparallelloading,multiplexingandcassettedosingColumncouplingprovidesfast,simplegenericscreeningBiocatalysisCourtesyofDSMFineChemicalsDeterminationoftheconversionandenantiomericexcessofsubstrate/reactionproductsinaD-hydantoinase/D-carbamoylasereaction

Column:CHIROBIOTICT(250x4.6mm,5

m)Eluent:80/2015mMNH4AcpH4.1/MeOHFlow: 1.0mL/min

3,4:Product6,7:Startingmaterial1,5:Intermediate2:ImpurityBiocatalysisCourtesyofDSMFineChemicalsDeterminationoftheenantiomericexcessofcis-andtrans-diolreactionproductsfromanepoxyhydrolasereaction

Column: CHIROBIOTICR(250x4.6mm,5

m)Eluent: 0.1%ammonia,pH4.1withformic acid/MeOH(50/50%v/v)Flow: 1.0mL/min

ABABUsingCHIROBIOTICCSPsforPreparativeLCApplicationsCASESTUDY1:NicardipineColumn: CHIROBIOTICV (250x4.6mm,5)Mobilephase: 100/0.2/0.1,MeOH/AcOH/TEAUV: 230nmFlowrate: 1mL/minPeak1:4.66Peak2:5.57

=1.50CASESTUDY1:NicardipineOptimizationforprep100/0.1w%,MeOH/NH4TFA100/0.1w%,MeOH/NH4OAcPeak1:4.07Peak2:4.69Peak1:3.41Peak2:3.47

=1.50

=1.09Column: CHIROBIOTICVFlowrate: 1mL/minCASESTUDY1:Nicardipine

Column: CHIROBIOTICV (250x22.1mm),5m)Load: 20mgin4mLMobilephase: 100/0.1w%,MeOH/NH4TFAUV: 230nmFlowrate: 12mL/minThroughput: 1.2mg/gCSP/hrPeak1:8.26Peak2:9.51FractionCollections123456Purity Peak1:99.67% Peak2:99.46%CASESTUDY2:PolarIonicMode?

Column: CHIROBIOTICV,5mm 250x4.6mmMobilephase: 100/0.2/0.1, MeOH/AcOH/TEAFlowrate: 0.9mL/minUV: 254nmInj: 2mLPeak1:10.51minPeak2:11.53mina=1.14BASICCOMPOUNDCASESTUDY2:PolarIonicMode?

Column: CHIROBIOTICV5mm (Modified)250x4.6mmMobilephase: 100/0.5/0.5, MeOH/AcOH/TEAFlowrate: 1mL/minUV: 254nmInj: 100mgBASICCOMPOUNDPeak1:8.83minPeak2:11.82mina=1.50CASESTUDY2:PolarIonicMode?BASICCOMPOUND

Column: CHIROBIOTICV,5mm (Modified)250x22.1mmMobilephase: 100/0.5/0.5, MeOH/AcOH/TEAFlowrate: 15mL/minUV: 254nmInj. 140mg(in2mlMeOH)Peak1:10.22Peak2:12.54CASESTUDY3:N-AcetylTyptophanColumn: CHIROBIOTICTAG(250x4.6mm,5m)UV: 254nmFlowrate: 1mL/minMobilephase:100/0.1w%,MeOH/NH4OAcMobilephase: