Massspectrometryforthechromatographer??????LC/MSinDrugDevelopmentIdentifyunknownpeakstosupportmethoddevelopmentandvalidationCharacterizedegradationproductsinforceddegradationstudiesandstabilitysamplesElucidatedrugdegradationpathwaytosupportexcipientselectionandformulationdevelopmentConfirmpeakidentityforDSandrelatedsubstancesSupportQAissuesandNDASupportManufacturingprocessandfinisheddrugproductsFeaturesofLC/MSAdvantages:?Highsensitivity?Highselectivity?Speed?Combinedpowerofseparationandidentification?MolecularweightandstructureinformationDisadvantages:?Somecompoundsarenotionizable?Difficulttoidentifycompleteunknowns?LimitedstructureinformationcomparedtoNMRLC/MSAnalysis

WorkRequestLC/MScompatiblemethod

ConfirmChromatogramSelectIonizationModeESIvsAPCI,(+)vs(-)LC/MSincompatiblemethod

ModifyHPLCconditions ShowPeakEquivalencyDetermineMWMS/MS(n)StudyIsolation/PurificationProposedStructure NMRConfirmationReportNoConfirmation?YesIn-houseSynthesisLC-MSLC

InterfaceExtractionof Ionevaporation

MassAnalyzerDetectorTheanalytefromorionization.ThesolventFragmentationLC–SeparationofthemixtureofanalytesInterface–SeparationoftheanalytefromthesolventMA(massanalyzer)–separationoftheanalytemolecularionandfragmentsaccordingtotheirmasstochargeratioInfusionMSMicroTeeMSLC100μl/minpump

1μl/min

Samplesolution

?Sampleincontinuousflow

?Nosampleinjection

?NoLCseparation

?MSspectrumobtainedoverangiventime

?ImprovedS/Nduetotheaverageofmultiplespectra

?SelectLCflowtoassistionization??????ELECTROSPRAYFactorstoconsiderIonicstrengthSurfacetensionofthesolventVolatilityofthesolventCharacteroftheanalyteionsinsolution:solvated,ionpaired,etcMobilephasecompositionandamountofwaterpHofthemobilephaseIntensityIntensityConfirmationofmolecularweightESI(+)ESI(-)1.8x1071.6x1071.4x107294.057x1066x1065x106292.041.2x107 4x1061.0x1078.0x1066.0x1064.0x1062.0x1063x1062x1061x1060.0 100.00200.00300.00400.00500.00600.00700.00800.00

0100.00200.00300.00400.00500.00600.00700.00800.00m/zm/z1.EffectofinstrumentalparametersonESIresponse?Mobilephaseflowrate*?Capillary*/coronavoltage?Fragementor*/conevoltageAllplayimportantrolesinanalyteionsformationandionizationefficiencyEffectofcapillaryvoltageG.Valaskovic,J.Murphy,M.Lee,MilestonedevelopmentFormationofsmallmicronsizeddropletsisnotaproblemifflowrate,surfacetensionarelow.Anincreaseofthesemaymakeitdifficulttofortheelectricfieldtoproducethedesiredchargedaerosol.Theelectricfieldstrengthcanbeincreasedtotrytoovercomethoseeffects.HoweverifelectricfieldistoohighwillgiverisetoelectricaldischargeandthisisdetrimentaltoESsignal.Nebulizinggascanbeusedtohelpfocusthe#ofionstransportedintovacuumenvelopeofthemassspectrometer.00EffectofCone/FragmentorVoltageonfragmentationFB-25CV+1FB60CV+1TAE226FB-1807(8.225)1:ScanES+TAE226FB-2805(8.205)1:ScanES+100469.184.86e7100438.165.13e725VSingleQuad3:1DS60VSingleQuad469.183:1 3:1DS403.22%471.08937.44%372.20471.08100.19155.62214.09242.19255.11315.23348.40434.62506.91577.06652.27696.09721.16813.47831.58891.09941.49976.06 m/z99.94117.53193.08315.61359.54491.09534.26635.18720.40757.50870.45913.51937.57959.60m/z10020030040050060070080090010001002003004005006007008009001000Aspotentialdifference(deltaV)betweenendofcapillary(N)andfirstskimmer(S)isincreasedtheionsareacceleratedthroughthebackgroundgas,whichleadstomorecollisions.EffectofexperimentalparametersonESIresponseA.DependenceofionintensitiesonanalyteconcentrationConcentrationeffectoftetracyclineonthesignalintensityA.Kamel,P.R.Brown,B.Munson,AnalyticalChemistry,vol.71,968-977,1999EffectofSolutionpH(ESI)?VariationofsolutionpHchangestheacid-baseequilibriumofananalytespecies?Altersthedegreeofpositive/negativechargingviaprotonation/deprotonation?ShouldgetincreasedintensitywithdecreasingpHforbasiccompoundsinpositiveionmode?ShouldgetincreasedintensitywithincreasingpHforacidiccompoundsinnegativeionmode?Howeverion-pairing,ion-associationmayleadtoion-suppressionactuallycausingadecreaseinionizationefficiency?Thereisacriticalpointatwhichincreaseinprotonationceasestoincreaseionizationefficiencyforprotonatedbases.?Effectoforganicontheshiftofionizationequilibriainthemobilephase.Ibuprofen[M-H]s/n-

EffectofeluentpH600500400300200100 02468

pHEffectofpHon[M-H]-negative-ionresponseofIbuprofen.

DesiredpHvalueswereachievedusingdifferentcombinations of0.1%formicacidand0.1%ammoniumhydroxideatconstant organiccomposition.Flowinjectionanalysis

Y.F.Cheng,RapidComminMassSpec.,2001;15;141-151ESIEfficiency

FullScanESIEfficiencyofAntidepressantsvsConcentrationofAcetonitrileinthemobilephase

4.00E+06 3.20E+06 2.40E+061.60E+068.00E+050.00E+00ImiDesiProClom020406080100PercentAcetonitrileMobilePhase:AnalyteConc:InfusionRate:

Aqueous//MeCN Aqueous=1mMolAmmoniumFormate,pH4.32withFormicAcid 100nM/mL20uL/minConclusionsTheelectrosprayionintensitycanbeenhancedby:?Concentrationofanalyteandinstrumentalparameters?SolutionphasepH?Typeandconcentrationofacidicmodifierorvolatilebuffer?Typeandconcentrationoforganicmodifier?GasphaseReactionsareimportant,feasibilitydependsonprotonaffinitiesofanalyteversusmodifierineluent?ESIisacomplexprocesscomprisesofmanydifferentvariables,nosetofparameterswillguaranteelargesignalintensityMSStructureelucidationBasicprinciple

NitrogenRuleIsotoperatio(Cl,Br,etc.)AdductionsLC/MSPostcolumnaddition(acidorbase)MS/MSorCID:fromfragmentstosubstructuresHydrogen-DeuteriumexchangeUtilizationofUVspectratosupportMSidentificationOthertechniquesforstructureelucidationExactMS:Elementalcomposition&chemicalformulaGC/MS:EImassspectrumNMRorLC/NMR:DetailstructureCalculatingMolecularWeightThreewaystocalculatemolecularweightAveragemass:Averageatomicweightforeachelement,(Merckindex,DSprofile)12.01115*60+1.00797*122+14.0067*20+…………..=1443.8857Monoisotopic(exact)mass:Exactmassofthemostabundantisotopeofeachelement12.0000*60+1.0078*122+14.0031*20+……………..=1442.8796Nominalmass:Integernominalmassofthemostabundantisotopeofeachelement12*60+1*122+14*20+………………….=1442Massdefect1444:1443:1442.Thisisasgoodasbeingamileoff!ALWAYSusethemonoisotopicmasswhencalculatingthemolecularweightinmassspectrometry!PositiveandNegativeAdductIons

Positiveiondetection?[M+H]+?[M+NH4]+?[M+Na]+?[M+K]+?[2M+H]+?[2M+NH4]+?[2M+Na]+Negativeiondetection?[M-H]-?[M-H+HOAC]-M+1M+18M+23M+392M+12M+182M+23M-1M+59MeCN=+41MeOH=+32?[M-H+TFA]-M+113Distinctmolecularionpatterncanbeusedtodeterminethemolecularweight.

H3C OOH3COOCH3OHCH3HNOCH3HOOHHOOOOOOCH3ESIfullscanmassspectrumofpaclitaxelwithmobilephasethatcontains2mMammoniumacetateandacetonitrileKerns,et.al,1994,AmericanChemicalSocietyP.R.Tiller,et.al,J.Chrom.A,794(1998)15-25:HydroxylationP.R.Tiller,et.al,J.Chrom.A,794(1998)15-25UsingLCMStodetermineartifactpeaksformedon-columnCF3CF3ONOCF3+HO+H HONOCF3OHC

FMW=503.4Acidicconditions

H CHOOH

FMW=521.1IntensityIntensityIntensityTICvs.EIC 8000000 6000000 4000000 2000000TIC24681012141618800000600000400000200000 0m/z=522,XIC24681012141618 0m/z=504,XIC24681012141618Time(min.)ExactMass-Q-TOFTrendsinAnalyticalChemistry,Vol.22,No.10,2003,ImmaFerrer,et.al.PostcolumnadditionMicroTeeMSLC1ml/min0.1ml/minwaste1%Formicacid

inIPA?Sampleinjection?LCseparation?NoimpactonLCchromatographicpattern?Improveionizationwitheitheracidorbase additionafterLCcolumn?SelectappropriatesolutiontoassistionizationNitrogenRuleThisruleisderivedfromthefactthat,perhapscoincidentally,forthemostcommonchemicalelementsinneutralorganiccompounds(hydrogen,carbon,nitrogen,oxygen,silicon,phosphorus,sulfur,andthehalogens),elementswithevennumberednominalmassesformevennumbersofcovalentbonds,whileelementswithoddnumberednominalmassesformoddnumbersofcovalentbonds,withtheexceptionofnitrogen,whichhasanominal(orinteger)massof14,buthasavalencyof3.Itshouldbenotedthatthenitrogenruleisonlytrueforneutralstructuresinwhichalloftheatomsinthemoleculehaveanumberofcovalentbondsequaltotheirstandardvalency(countingeachsigmabondandpibondasaseparatecovalentbondforthepurposesofthecalculation).Therefore,theruleistypicallyonlyappliedtothemolecularionsignalinthemassspectrum.Massspectrometrygenerallyoperatesbymeasuringthemassofions.Ifthemeasuredionisgeneratedbycreatingorbreakingasinglecovalentbond(suchasprotonat