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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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