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LiquidChromatographyCoupledtoTandemMass Spectrometry
Processes NeededtoGeneratea Mass Spectrum1)Generatedesolvatedions
(Sources)2)Transferionsintomass spectrometervacuum
(InterfaceRegion)3)Massanalyzeions
(Quadrupoles)4)Detections
(Detector)5)Processthedata
(Software) LiquidChromatography Process1TheSourceTheSource2TheSourceTheSource3IonizationSourcesTurboIonSpray-IonSpray(pneumaticallyassistedElectrospray)optionwhichutilizesaheatedauxiliarygasflow.HeatedNebulizer-AtmosphericPressureChemicalIonization(APCI)sourcewhichionizescompoundsinthegasphase.IonizationSourcesTurboIonSpr4+-+--+IonEvaporationand Formation
++- +-++++- +--+ ++
-+++ ++++ -+- ++ ++ + ++-+--+IonEvaporationand 5IonSpray(ESI)IonSpray:-Electrosprayionization(ESI)-Gentlestionizationtechnique-ApplicabletopolarandionicsubstancesIonSpray(ESI)IonSpray:-Elect6Intensity,cps15+MultiplyChargedIons+Q1:frommyoglobina
16+etc.1060.51131.01.56e6cps1.0e6
998.0 942.5893.0
14+1212.0
13+1305.05.0e5
848.5809.0
12+1414.0
11+1542.510+
1696.08001000120014001600
m/z,amuMoleculeswhichcanbemultiplyprotonatedordeprotonatedcanexistinmorethanonechargestatePeaksat[M±nH]n±(n=numberofcharges)Intensity,cps15+MultiplyCharg7NebulizerCoolZonePrimaryFlashZoneSecondaryFlashZoneNebulizerCoolZonePrimaryFlas8HeatedNebulizer(APCI)HeatedNebulizer:-AtmosphericPressureChemicalIonization(APCI)-coronadischarge-polartonon-polarthermallystablecompoundsHeatedNebulizer(APCI)HeatedN9AtmosphericPressureChemicalIonization(APCI)CoronaDischargeChemistry–PositiveIons
1)EIonatmospherecausese-removalfromN2&O2formingN2+?&O2+?(primaryions)2)InacomplexseriesofreactionsN2+?&
O2+?reactwithH2O&CH3OHforming H3O+&CH3OH2+asreagentionsforCI.3)H3O+&CH3OH2+donateprotonstoanalyteforming[M+H]+AtmosphericPressureChemicalI10AnalytePolarity
GC-MSvs.LC-MSIonic
IonSpray APCINon-IonicGC-MS101102
103MolecularWeight104105AnalytePolarity GC-MSvs.LC-M11IonProductionAPIChemistryandExampleSpectraIonProductionAPIChemistryand12IncreasingAcidity??+?
GASPHASEAcid-BaseScaleforPositiveIons&NeutralMoleculesNeutralMolecules
Strongacid
formicacid aceticacid water methanol phenol formaldehyde N2O methane ethane benzene acetonitrile ethylacetate ammonia triethylamine
WeakacidReagentIonsWeakBaseHCOOH2+CH3COOH2+H3O+CH3OH2+C6H6OH2+HCHOH+N2OH+CH5+C2H5C6H6H+CH3CNH+C2H5OCOC2H6+NH4+C6H15NH+StrongBaseAdditionofaceticacidorformicacidwillincreaseintensityinpositiveionmodebyhigherconcentrationofH+.Trifluoroaceticacid(TFA)workswellinpositiveionmodeataconcentrationupto0.05%,butlingersforalongtimeinnegativeionmode.(TFAionsareatm/z113andm/z227—dimer)Changingfromacetonitriletomethanolmayincreasesignalintensity.IncreasingAcidity??+? GASPHAS13IncreasingBacicity???
GASPHASEAcid-BaseScaleforNegativeIons&NeutralMoleculesNeutralmolecules
WeakAcid
NH3
Water Toluene Methanol Ethanol Acetonitrile Acetone CH3SH CH3NO2
HCN Phenol Aceticacid Benzoicacid HCl
StrongAcidReagentIonsStrongBaseNH2-OH-C6H5CH2-CH3O-C2H5O-CH2CN-CH3COCH2-CH3S-CH2NO2-CN-C6H5O-CH3COO-C6H5COO-Cl-WeakBaseForR-COOHcompoundsuseNH4acbuffer(pH=6.4).TryadditionofNH3incaseofneutralanalytesinnegativeionmode.Stayawayfromtriethylamine,itcollectsinthesource!IncreasingBacicity??? GASPHAS14IonizationSuppressionCompoundsThatCauseSensitivitySuppression–Saltscaninterferewithionizationandcanclusterto complicatespectrum(butalsoaidinidentification)–Strongbasesorquaternaryaminescaninterferewith
positivemodeanalytes,e.g.Triethylamine(TEA)–Acids-Sulfuric/SulfonicacidsandTFAinterfereinnegativemodeexperiments–Phosphatebufferandnon-volatileionpairingagents(e.g. SDS)cancauseseveresuppressionandcomplexspectraDimerization([2M+H]+)canoccurathighconcentration,leadingtonon-linearityduringquantitation–DimerSignalatm/z=(MW*2)+1–Cancausenon-linearityathighconcentrationsIonizationSuppressionCompound15ProgesteroneinPositiveIonESI-MS[M+Na]+ProgesteroneinPositiveIonE16Intensity,cpsIntensity,cps6e7EffectofPhosphateBuffer–Q1MCA(10scans):fromcckq1-venano
254.43424.538.78e7cps8e77e7
CCKdissolvedinPhosphateBuffer
566.445e74e73e7
733.932e7
835.931e7898.221000.4211421164.6130040050060070080090010001100m/z,amu–Q1MCA(10scans):fromCCKPURIFIEDQ1SACN16.74.0e73.5e7570.444.44e7cps3.0e72.5e72.0e71.5e7PhosphateBufferremovedfromCCKsample1142.031.0e7292.82377.02439.03497.03700.63798.63879.235.0e630040050060070080090010001100m/z,amuIntensity,cpsIntensity,cps6e7E17++++++++++++++----+++++----------Adducts&ClustersformedinLC/MS
Adduct*/AdductIonFormedCauseIonicAPCIESI+/-m/zofIonLithium[M+Li]Ammonia[M+NH4]Water[M+H3O]Sodium[M+Na]LithiumSaltsAmmonia/NH4OHWater/AcidsSodiumsalts
IonicAdductAdduct Ionic[M+H+6][M+H+17][M+H+18][M+H+22]Methanol[M+H+CH3OH]Chloride[M+Cl]Potassium[M+K]Acetonitrile[M+H+CH3CN)FormicAcid[M-H+HCO2H]AceticAcid[M-H+CH3CO2H]TFA[M-H+CF3CO2H]MethanolinSolventChlorinatedsolventPotassiumsaltsAcetonitrileinsolventFormicAcidAceticAcidTFAAdduct Ionic IonicAdductAdductAdductAdduct+/-+/-[M+H+32][M-H+32][M+35(&37)][M+H+38][M+H+41][M-H+41][M-H+46][M-H+60][M-H+114]*AdductscanoftenbebrokenupbyusingahigherDPvoltage.++++++++++++++----+++++-------18IonTransferIonTransfer19
IonPath“ElectronicIonSkiSlope”O(jiān)RSKIQ1Q0STRO1ST2RO2(LINAC)ST3RO3EXITCEMDFOR=45V0V,SKatgroundMoveswithC.E.Q0=-10VRO1=-11VRO3,moveswithRO2(-2-3V)Lens(IQn)androd(ROn)voltagesarelinkedKeepionsmovingatcorrectspeedthroughsystemForbestsensitivity,propermassfiltering IonPathORSKIQ1Q0STRO1ST2RO220MS&MS/MS(tandem)MS&MS/MS(tandem)21QuadrupoleTheoryQuadrupoleactsasamassfilter
–Separatesionsbasedonm/zratioQuad.madeof4rods–“A”pole-verticalrods;“B”pole-
horizontalrods(byconvention)DC&RFvoltagesareimposed:–U=(DC)A-(DC)B(FDC)–V=RFvoltage(RFp-p)–V=Const.?M?r02?ν2Note:MαV?ν=RFfrequency–r0=?distancebetweenrodsQuadrupoleTheoryQuadrupoleac22U(DC)OperatingLineStabilityDiagramDC/RF(throughtheapices)→HighUnitLowV(RF)U(DC)OperatingLineStabilityD23MSOperationScanModesMSOperationScanModes24Q1MSScansStart/StopModeQ1MSScansStart/StopMode25Q3Single-StageMSScansStart/StopModeQ3Single-StageMSScansStart/26MS/MSScanModesAvailableScanModes–ProductIonProvidesstructuralinformation–PrecursorIonProvidesstructuralinformationcomplementarytoProductIon–NeutralLossProvidescompoundclassspecificity–MRMUsedforQuantitationInallMS/MSmodes,Q3isamassfilter
–Q3mayscanorbefixedatagivenm/z
–Q2is“source”ofproductionsenteringQ3MS/MSScanModesAvailableScan27MS/MS–ProductIonScanm1+fixedm3+scannedMS/MS–ProductIonScanm1+fi28MS/MS–PrecursorIonScanm1+scannedm3+fixedMS/MS–PrecursorIonScanm1+29MS/MS–NeutralLossScanm1+scannedδmm3+scannedMS/MS–NeutralLossScanm1+s30
MS/MS–MRM(MultipleReactionMonitoring)Precursorion fixedFragmentation (CAD)Production fixed MS/MS–MRMPrecursorionFragm31
FragmentationWhenanionfragmentsthefollowingformulasapply:–Positivemode:–Negativemode:[Precursor]+[Precursor]-→Product+→Product-+Neutral+NeutralTwomodesoffragmentationonSciexInstruments:
–CAD=CollisionallyActivatedDissociation
EnergycontrolledbyCE;occursinQ2(collisioncell) IonscollidewithN2molecules(CADgas),collisionenergyis convertedintovibrationalenergy,andbondsbreak
–CID=Collision-InducedDissociation
EnergycontrolledbyDP;occursbetweenorificeandskimmer IonscollidewithN2(fromcurtaingas)andsolventmolecules, collisionenergyisconverted… Fragmentation–Positivemode32IonDetectionIonDetection33Detector&SignalHandling
Deflector IonPath SignalOutputCEM1M?1000pF 10kVSignalHandling BoardCEMHornVoltage(-)
CEMBiasVoltage(+)SystemController (inSystem ElectronicsBox)Detector&SignalHandlingCEM134
LiquidChromatographyCoupledtoTandemMass Spectrometry
Processes NeededtoGeneratea Mass Spectrum1)Generatedesolvatedions
(Sources)2)Transferionsintomass spectrometervacuum
(InterfaceRegion)3)Massanalyzeions
(Quadrupoles)4)Detections
(Detector)5)Processthedata
(Software) LiquidChromatography Process35TheSourceTheSource36TheSourceTheSource37IonizationSourcesTurboIonSpray-IonSpray(pneumaticallyassistedElectrospray)optionwhichutilizesaheatedauxiliarygasflow.HeatedNebulizer-AtmosphericPressureChemicalIonization(APCI)sourcewhichionizescompoundsinthegasphase.IonizationSourcesTurboIonSpr38+-+--+IonEvaporationand Formation
++- +-++++- +--+ ++
-+++ ++++ -+- ++ ++ + ++-+--+IonEvaporationand 39IonSpray(ESI)IonSpray:-Electrosprayionization(ESI)-Gentlestionizationtechnique-ApplicabletopolarandionicsubstancesIonSpray(ESI)IonSpray:-Elect40Intensity,cps15+MultiplyChargedIons+Q1:frommyoglobina
16+etc.1060.51131.01.56e6cps1.0e6
998.0 942.5893.0
14+1212.0
13+1305.05.0e5
848.5809.0
12+1414.0
11+1542.510+
1696.08001000120014001600
m/z,amuMoleculeswhichcanbemultiplyprotonatedordeprotonatedcanexistinmorethanonechargestatePeaksat[M±nH]n±(n=numberofcharges)Intensity,cps15+MultiplyCharg41NebulizerCoolZonePrimaryFlashZoneSecondaryFlashZoneNebulizerCoolZonePrimaryFlas42HeatedNebulizer(APCI)HeatedNebulizer:-AtmosphericPressureChemicalIonization(APCI)-coronadischarge-polartonon-polarthermallystablecompoundsHeatedNebulizer(APCI)HeatedN43AtmosphericPressureChemicalIonization(APCI)CoronaDischargeChemistry–PositiveIons
1)EIonatmospherecausese-removalfromN2&O2formingN2+?&O2+?(primaryions)2)InacomplexseriesofreactionsN2+?&
O2+?reactwithH2O&CH3OHforming H3O+&CH3OH2+asreagentionsforCI.3)H3O+&CH3OH2+donateprotonstoanalyteforming[M+H]+AtmosphericPressureChemicalI44AnalytePolarity
GC-MSvs.LC-MSIonic
IonSpray APCINon-IonicGC-MS101102
103MolecularWeight104105AnalytePolarity GC-MSvs.LC-M45IonProductionAPIChemistryandExampleSpectraIonProductionAPIChemistryand46IncreasingAcidity??+?
GASPHASEAcid-BaseScaleforPositiveIons&NeutralMoleculesNeutralMolecules
Strongacid
formicacid aceticacid water methanol phenol formaldehyde N2O methane ethane benzene acetonitrile ethylacetate ammonia triethylamine
WeakacidReagentIonsWeakBaseHCOOH2+CH3COOH2+H3O+CH3OH2+C6H6OH2+HCHOH+N2OH+CH5+C2H5C6H6H+CH3CNH+C2H5OCOC2H6+NH4+C6H15NH+StrongBaseAdditionofaceticacidorformicacidwillincreaseintensityinpositiveionmodebyhigherconcentrationofH+.Trifluoroaceticacid(TFA)workswellinpositiveionmodeataconcentrationupto0.05%,butlingersforalongtimeinnegativeionmode.(TFAionsareatm/z113andm/z227—dimer)Changingfromacetonitriletomethanolmayincreasesignalintensity.IncreasingAcidity??+? GASPHAS47IncreasingBacicity???
GASPHASEAcid-BaseScaleforNegativeIons&NeutralMoleculesNeutralmolecules
WeakAcid
NH3
Water Toluene Methanol Ethanol Acetonitrile Acetone CH3SH CH3NO2
HCN Phenol Aceticacid Benzoicacid HCl
StrongAcidReagentIonsStrongBaseNH2-OH-C6H5CH2-CH3O-C2H5O-CH2CN-CH3COCH2-CH3S-CH2NO2-CN-C6H5O-CH3COO-C6H5COO-Cl-WeakBaseForR-COOHcompoundsuseNH4acbuffer(pH=6.4).TryadditionofNH3incaseofneutralanalytesinnegativeionmode.Stayawayfromtriethylamine,itcollectsinthesource!IncreasingBacicity??? GASPHAS48IonizationSuppressionCompoundsThatCauseSensitivitySuppression–Saltscaninterferewithionizationandcanclusterto complicatespectrum(butalsoaidinidentification)–Strongbasesorquaternaryaminescaninterferewith
positivemodeanalytes,e.g.Triethylamine(TEA)–Acids-Sulfuric/SulfonicacidsandTFAinterfereinnegativemodeexperiments–Phosphatebufferandnon-volatileionpairingagents(e.g. SDS)cancauseseveresuppressionandcomplexspectraDimerization([2M+H]+)canoccurathighconcentration,leadingtonon-linearityduringquantitation–DimerSignalatm/z=(MW*2)+1–Cancausenon-linearityathighconcentrationsIonizationSuppressionCompound49ProgesteroneinPositiveIonESI-MS[M+Na]+ProgesteroneinPositiveIonE50Intensity,cpsIntensity,cps6e7EffectofPhosphateBuffer–Q1MCA(10scans):fromcckq1-venano
254.43424.538.78e7cps8e77e7
CCKdissolvedinPhosphateBuffer
566.445e74e73e7
733.932e7
835.931e7898.221000.4211421164.6130040050060070080090010001100m/z,amu–Q1MCA(10scans):fromCCKPURIFIEDQ1SACN16.74.0e73.5e7570.444.44e7cps3.0e72.5e72.0e71.5e7PhosphateBufferremovedfromCCKsample1142.031.0e7292.82377.02439.03497.03700.63798.63879.235.0e630040050060070080090010001100m/z,amuIntensity,cpsIntensity,cps6e7E51++++++++++++++----+++++----------Adducts&ClustersformedinLC/MS
Adduct*/AdductIonFormedCauseIonicAPCIESI+/-m/zofIonLithium[M+Li]Ammonia[M+NH4]Water[M+H3O]Sodium[M+Na]LithiumSaltsAmmonia/NH4OHWater/AcidsSodiumsalts
IonicAdductAdduct Ionic[M+H+6][M+H+17][M+H+18][M+H+22]Methanol[M+H+CH3OH]Chloride[M+Cl]Potassium[M+K]Acetonitrile[M+H+CH3CN)FormicAcid[M-H+HCO2H]AceticAcid[M-H+CH3CO2H]TFA[M-H+CF3CO2H]MethanolinSolventChlorinatedsolventPotassiumsaltsAcetonitrileinsolventFormicAcidAceticAcidTFAAdduct Ionic IonicAdductAdductAdductAdduct+/-+/-[M+H+32][M-H+32][M+35(&37)][M+H+38][M+H+41][M-H+41][M-H+46][M-H+60][M-H+114]*AdductscanoftenbebrokenupbyusingahigherDPvoltage.++++++++++++++----+++++-------52IonTransferIonTransfer53
IonPath“ElectronicIonSkiSlope”O(jiān)RSKIQ1Q0STRO1ST2RO2(LINAC)ST3RO3EXITCEMDFOR=45V0V,SKatgroundMoveswithC.E.Q0=-10VRO1=-11VRO3,moveswithRO2(-2-3V)Lens(IQn)androd(ROn)voltagesarelinkedKeepionsmovingatcorrectspeedthroughsystemForbestsensitivity,propermassfiltering IonPathORSKIQ1Q0STRO1ST2RO254MS&MS/MS(tandem)MS&MS/MS(tandem)55QuadrupoleTheoryQuadrupoleactsasamassfilter
–Separatesionsbasedonm/zratioQuad.madeof4rods–“A”pole-verticalrods;“B”pole-
horizontalrods(byconvention)DC&RFvoltagesareimposed:–U=(DC)A-(DC)B(FDC)–V=RFvoltage(RFp-p)–V=Const.?M?r02?ν2Note:MαV?ν=RFfrequency–r0=?distancebetweenrodsQuadrupoleTheoryQuadrupoleac56U(DC)OperatingLineStabilityDiagramDC/RF(throughtheapices)→HighUnitLowV(RF)U(DC)OperatingLineStabilityD57MSOperationScanModesMSOperationScanModes58Q1MSScansSt
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