API3000LCMSMS操作培训手册课件

API3000OperatorTrainingCourse

Analyst-NT

FosterCityCAPALLabAPI3000OperatorTrainingCouWhatisLC/MSLC:LiquidChromatography

MS:MassSpectrometer

aninstrumentthatproducesionsandseparatestheminthegasphaseaccordingtotheirmass-to-chargeratio(m/z)WhatisLC/MSLC:LiquidChromaElementMassAveragemassofallnaturalisotopesCarbon AbundanceIntegerMass12C=12.0000Da98.91% 1213C=13.0034 1.09% 13(12.0000)(0.9891)+(13.0034)(.011)=12.011ElementMassAveragemassofalNaturalisotopesAbundanceElement

Abundance ExactMass

X+1Factor

X+2Factor

1H 99.99 1.00782H 0.01 2.0141

10B 49.9 10.012954.03NB11B 50.1 11.0093

12C 98.91 12.000013C 1.09 13.00341.1NC0.0060(NC*NC)14N 99.63 14.003115N 0.37 15.00010.37NN35Cl 75.77 34.968937Cl 24.23 36.965932.5NCl79Br 50.7 78.9183

81Br 49.3 80.916398.0NBr

127I 100 126.9045

NaturalisotopesAbundanceElemNaturalIsotopesAbundanceElement

Abundance ExactMass

X+1Factor

X+2Factor

16O 99.76 15.994917O 0.04 16.99910.04NO18O 0.20 17.99920.02NO19F 100 18.998428Si 92.2 27.976929Si 4.7 28.97655.1Nsi30Si 2.2 29.97383.4NSi31P 100 30.973832S 95.0 31.972133S0.75 32.97150.8NS

34S 4.22 33.9679 4.4NsNaturalIsotopesAbundanceElemDefiningthemassesIntegerMass-SumofProtonsandNeutronsofanelementNominalMass-SumoftheintegermassesofthemostabundantisotopesoftheelementsinacompoundAverageMass-SumoftheatomicweightsoftheelementsinacompoundExactmass-Sumofthemostabundantisotopesofelementsinacompound-TheoreticalcalculationonlyAccuratemass-experimentallydeterminedmassMonoisotopicmass-sumoftheexactmassesofthemostabundantisotopesofelementsinacompoundDefiningthemassesIntegerMasExamplesofMassesHaloperidol:C21H23NO2FClNominalMass: 375AverageMass: 375.8737ExactMass: 375.1401Monoisotopicmass 375.1401m/z365376386378100755025%Intensity

C21H23NO2FClExamplesofMassesHaloperidol:MultiplyChargedIonsMultiplyChargedIons:Signal=(MW+N)/N(N=#ofcharges)SincetheQuadseparatesionsbasedonm/zratioandionscanhavemultiplechargesinIonSpray.Aseriesofmultiplychargedionsisusuallyproducedforlargermolecules.MultiplyChargedIonsMultiplyAPI3000LCMSMS操作培训手册CommonadductsPOSITIVEMODENa22Da.higherthanM+HK38Da.higherthanM+HLi9Da.higherthanM+HNH418Da.higherthanM+HACN41Da.higherthanM+HNEGATIVEMODETFA114Da.higherthenM-H(113and227background)Acetate60Da.higherthenM-HFormic46Da.higherthenM-HCommonadductsGASPHASEAcid-BaseScale

forPositiveIon&NeutralMoleculesGASPHASEAcid-BaseSGASPHASEAcid-BaseScaleforNegativeIons&NeutralMoleculesGASPHASEAcid-BaseScaleforIonizationSuppressionCompoundsThatCauseSensitivitySuppressionSaltscaninterferewithionizationandcanclustertocomplicatespectrum(butalsoaidinidentification)Strongbasesorquaternaryaminescaninterferewithpositivemodeanalytes,e.g.Triethylamine(TFA)Acid-Sulfuric/SulfonicacidsandTFAinterfereinnegativemodePhosphatebufferandnon-volatileionpairingagents(e.g.SDS)cancauseseveresuppressionandcomplexspectrumDimerization([2M+H]+)canoccurathighconcentration,leadingtonon-linearityduringquantitationDimerSignalatm/z=(MW*2)+1Cancausenon-linearityathighconcentrationsIonizationSuppressionCompoundBasicstepsofLC/MS

analysisSampleIntroduction：inliquidstatesbysyringepumporLCpumpIonization：IonSpray，APCIVacuumsystem:preventcollisionsofionswithresidualgasmoleculesintheanalyzerduringtheflightfromionsourcetothedetectorInterface:

preventexcessivegasloadMassanalysis:QuadrupoleMassfilter,TimeofFlightDataprocesssystem:computerbased

BasicstepsofLC/MSanalysisSAPIAnalyticalDomainsNeutral101102103104105MolecularWeightIonicAnalytePolarityIonSprayAPCIGC/MSAPIAnalyticalDomainsNeutral1API3000LCMSMS操作培训手册QuadrupoleTheoryThreetypesofvoltagespresentonaQuadrupole:RFp-p–Fixedfrequency(1MHz)oscillatingvoltage(VACMass);establishesharmoniciontrajectories.FilteringDC(FDC)–DCvoltagedifferencebetweenpoles(lineartoRFp-p)thatdefinestheresolution.RodOffsetVoltage–LowDCvoltagethatdefinesaxialionenergy(velocitythroughquadrupole).“RFonly”modemeans:NoFDCbetweenpoles;onlyRFp-p,RodOffsetpresent.Quad.isin“TotalIonMode”(transmittingallions,notfiltering).QuadrupoleTheoryThreetypesoAPI3000LCMSMS操作培训手册API3000LCMSMS操作培训手册API3000LCMSMS操作培训手册API3000LCMSMS操作培训手册API3000LCMSMS操作培训手册API3000LCMSMS操作培训手册API3000LCMSMS操作培训手册API3000LCMSMS操作培训手册API3000LCMSMS操作培训手册API3000LCMSMS操作培训手册QuadrupoleTheory(cont.)Quadrupolesanalyzersoperateinconstantpeakwidthmode(SameResolutionoverMassrange)APIResolutionSpecification

Unit=0.7(±0.1amuFWHH)High=.5(±0.1amuFWHH)Low=~1.1(±0.1amuFWHH)Usercansethigherresolutiondependingonproblemtobesolved(i.e.,settingsingly-chargedresolutionto0.5–0.6amuFWHHaidsindetectingdoubly-chargedions)ResolutionoffsetssettheDC/RFratioatvariouspointsalongmassaxisResolutionAtUnit,HighandLowautomaticallytunedItisalwaysrecommendedtocalibratemassaxisafterresolutionisadjustedforaquadrupoleQuadrupoleTheory(cont.)QuadrResolution=0.7amuFWHHLC2Tune1.4b11.pictLC2Tune1.4b11.pictResolution=0.7amuFWHHLC2TuResolution=0.6amuFWHHResolution=0.6amuFWHHResolution=0.5amuFWHHResolution=0.5amuFWHHQuadrupoleTheory&IonEnergyIonenergy

EffectsResolutionandPeakShapeLowerenergy(slower)=morecycles,betterresolutionHigherenergy(faster)=lesscycles,lowerresolution(typically0.5–2eV)isgivenbythevoltagedifferencebetweentheions“startingpotential”(Q0)andtheQ1rodoffset(RO1)(timesthenumberofcharges)Inc.IEalsoinc.Eofionstodistortpeakshape.AlwaysatradeoffbetweenresolutionandsensitivityinallquadrupoleanalyzersQuadrupoleTheory&IonEnergyQuadrupoleTheory&IonEnergyQ1Scan=n(Q0-RO1);Q1IE=0.5-2eVQ3Scan=n(RO2-RO3);Q3IE=(1-6eV)CollisionEnergyforMS/MS:CADenergy=n(Q0-RO2);n=#chargesProductionenergy=n(RO2-RO3);n=#chargesthisaffectsresolutionofproductions(lowerenergyyieldsbetterresolution)QuadrupoleTheory&IonEnergyIonEnergiesExamplesofanIonEnergyof.5eVinQ1LowerVelocitythroughQuadsallowsforgreaterfilteringandbetterresolutionSensitivityisofcourselostasresolutionincreasesIonEnergiesExamplesofanIIonEnergiesExamplesofanIonEnergyof1.5eVinQ1HighervelocitythroughQuadsallowsforlessfilteringandworseresolutionSensitivityisofcoursegainedasresolutiondecreasesIonEnergiesExamplesofanIResolutiondefinitionforMagneticSectorinstrumentsResolutiondefinitionfor: Quadrupole TOFTheResolutionRe-evolutionResolutiondefinitionResolutiAPI3000HardwareOverviewAPI3000LCMSMS操作培训手册API3000

PERFORMANCE:PerformanceSpecificationsQ1MassRange:5to3000m/zQ3MassRange:5to3000m/zDynamicRange:1to4e6cpsMassAccuracy:0.01%overtheentiremassrangeScanSpeed:2400amu/sAPI3000PERFORMANCE:PerformancAPI-3000IonPathAPI-3000IonPathSystemOverviewSampleIntroductionMethodsSampleInletsandIonizationTypesVacuumInterfaceCurtaingasinterfaceDifferentiallypumpedinterfaceVacuumChamber&AnalyzerRegionQuadrupoles,collisioncell&ionopticsDetectorRegionSystemOverviewSampleIntroducAPI3000GasFunctionandSettingAPI3000GasFunctionandSetti10portvalcovalveconfiguration10portvalcovalveconfigurat10portvalcovalveconfiguration10portvalcovalveconfigurat10portvalcovalveconfiguration10portvalcovalveconfiguratSampleIntroductionMethodsContinuousInfusion-Providesaccurate,lowflowrates.Allowscontinuoussampleintroductionfortuningandoptimization.FlowInjectionAnalysis(FIA)-CombiChem.andSourceOptimizationLiquidChromatograph-Allowsseparationsofmixtureswithmicroborecolumnsatlowflowsorconventionalcolumnsathighflows.SampleIntroductionMethodsConIonizationSourcesTurboIonSpray-IonSprayoptionwhichutilizesaheatedauxiliarygasflowtoprovidebettersensitivityathigherflowrates(2µL/min—1mL/min)HeatedNebulizer-Sampleinletforflowratesof0.2-2.0mL/min.Usesheat(400–500°C)andnebulizergastovaporizeHPLCeluentand“steamdistill”sampleintothegasphaseforAPCI.CoronaDischarge-HighvoltageneedletoionizecompoundsinthegasphasebyAtmosphericPressureChemicalIonization(APCI).IonizationSourcesIonizationSources:

TurboIonSpray1stpictureshowsaturboionspraywithand“hairdryer”portiononthebottom.2ndpictureshowsthesourcefromtherearwithadjustmentoptions3rdpictureshowsthegasinputs,heatercablesandinterlocksIonizationSources:

TurboIonSpTurboIonSpraySource:ISVoltage-DeclustersandgeneratesionsIS-VerypHdependentforchargingofmoleculeNebulizer&TurboGas-Declustersandde-solvatesIons~10,000,000ionsoncolumn4,000,000-40,000ionsOperatorImpactArea~1000ionsHighVoltageSampleNebulizerGaschargeddroplets++++–++–++–++++++++++++++IonSource(atmosphere)IonSprayinletIonsToQ01TOR-3VacuumInterfaceTurboGasTurboIonSpraySource:ISVoltagTurboIonSpray-IonEvaporationRayleighLimit=10cm2/VTurboIonSpray-IonEvaporatioTurboIonSpray(cont.)TurboIonSpray(cont.)TurboIonSpray(cont)OptimalConditionsTIS:Curtaingas(CG)SETASHIGHASPOSSIBLE(Min.=8)Caninc.CGforlowMWwithoutsensitivityloss.Highersprayerpressuremayrequirehighercur.gasSprayerAlignmentDONOTSPRAYDOWNORIFICE!GenerallynotcompounddependentSprayfurtherawayfromorificeforhigherflowrate.TurboIonSpray(cont)OptimalCoTurboIonSpray(cont)Needlevoltage(+)ion;1500to6000VDC(-)ionwithairasneb;-3000to-4500VDCOxygenreducescoronaeffectinNeg.Mode(-)ionwithN2asneb;-2000to-3500VDNebulizerFlowHigherliq.flowratemayrequirehigherneb.flowFlowrateandsolventcompositionBestsensitivityatlowerflowrate(2-400µL/min.)Canoperateatupto100%water,butbettersensitivitywithsomeorganiccontentTurboIonSpray(cont)NeedlevolTurboIonSpray(cont)ModifiersOrganicacids(e.g.formic,acetic)promoteionizationofbasiccompounds(sp3N-containing)AdductFormation-Neutralcompoundscontainingnucleophiliclonepairs(sp2N,sp3O)canbeionizedbycationizationwithalkalimetalorammoniumions.RecommendedBuffers-Ammoniumformateoracetateareat(2-10mMoptimum,canseesuppressioneffectsover20mM),.1%FormicAcid,(.1%TFAOK)TurboIonSpray(cont)ModifiersTurboIonSpray(cont)CompoundsThatCauseSensitivitySuppressionSaltscaninterferewithionizationandcanclustertocomplicatespectrum(butalsoaidinidentification)StrongbasesorquaternaryaminescaninterferewithpositivemodeanalytesAcids-SulfuricandTFAinterfereinnegativemodeexperiments.DONOTUSEPHOSPHATEBUFFERSNon-CovalentDimmersinIonSprayDimmerSignal=(MW*2)+1onecauseofnon-linearityathighconcentrationsTurboIonSpray(cont)CompoundsHeatedNebulizer(cont.)1stpictureinsideofAPCI/HN,withcoronaneedleoffsetbetweenorificeandquartztube2ndpicturebackofAPCI/HNgungas1andauxconnections,heatercontrolandheaterpowerconnection,backlatteradjustment.HeatedNebulizer(cont.)1stpiHeatedNebulizer(cont.)Apositionsettingof2-4onthehousingunitisusuallysufficienttoavoidsprayingdowntheorifice.HeatedNebulizer(cont.)AposiHeatedNebulizerInletAPCIFactors:APCIisahighflow(0.2-2.0mL/min.)inletSuitableforpolar,thermallystablecmpdsUsually,MW<1300amuProbeisheatedtofacilitatevaporizationRequiresnebulizingandauxiliarygasRequirescoronadischargeneedletoproduceionization(APCI)HeatedNebulizerInletAPCIFacAtmosphericPressureChemicalIonization(APCI)APCIutilizescoronadischarge

APCIisa“three”stepprocess:1)Needleathighvoltageionizesnebulizinggas(airornitrogen)formingprimaryions2)Primaryionsreactimmediatelywithsolventmoleculesformingreagentions3)Reagentionsreact(byprotontransfer)withanalytemoleculesforming(M+H)+inpositiveionmodeor(M-H)-innegativeionmodeAtmosphericPressureChemicalAtmosphericPressureChemicalIonization(APCI)Coronadischargeexample-positiveion1)EIonatmospherecausee-removalfromN2,O2formingN2+•,O2+•2)InacomplexseriesofreactionsN2+•,O2+•reactwithH2O,CH3OHformingH3O+andCH3OH2+asreagentionsforCI.3)H3O+,CH3OH2+donateprotonstoanalyteforming[M+H]+AtmosphericPressureChemicalHeatedNebulizer-APCIAPCIOptimalConditions:ProbeAlignmentDONOTSPRAYDOWNORIFICE!NotcompounddependentOptimizeontuningortestcompoundProbeTemperature

DistillationLimitsactualtemperatureexperiencedbyAnalyte~150DegreesCMinimumtemperaturesetbysolventandflowrate.Maycausethermaldegradationofanalyte.FlowrateandsolventcompositionWorksfrom0.2to2.0mL/min.HeatedNebulizer-APCIAPCIOpHeatedNebulizer-APCIBuffers:Buffers/modifiersnotrequiredforionizationVolatilebufferstoleratedupto50mMVerypolarmodifiersmayreducesensitivitytolesspolaranalyteCoronadischargeneedleposition:Slightlyoff-axistoprobe;alignwithorificeCurtainGas:SETASHIGHASPOSSIBLE(withnosens.loss)Minimumvalueis9(MW<1000)HeatedNebulizer-APCIBuffersVacuumInterface:CurtainGas&DifferentiallyPumpedInterfacesSkimmerdiameteris2.6mm,LargerthenotherinstrumentsforbettersensitivityOrificediameterisunchanged(254µmdiameter)Orifice-ring-skimmerspacingisunchangedOrifice-skimmerpressureis1TorrVacuumInterface:CurtainGas&VacuumInterface:CurtainGas&DifferentiallyPumpedInterfacesVacuumInterface:CurtainGasVacuumSystemSystemcontrollermonitorsvacuumsystem“transparently”Safetyoverridesdisableelectronicsandshutdownturbosif:BAGpressurehigherthan1x10-4torrCGpressurelessthan20psiRotarypumpsareneverdisabledbysystem,theyareturnedon/offmanuallyVacuumSystemSystemcontrollerVacuumInterface:CurtainGas&DifferentiallyPumpedInterfacesCurtainGas(CG)-Ultra-HighPuritynitrogenkeepsnon-ionizedspeciesfrombeingsuckedintovacuumchamberCGaidsiniondeclustering(withCIDpotentials)Twostagetransitionfromatmospheretolowpressureregionofanalyzer(1x10-5torr)Ionsaredrawnindueto:Pressuredifferential(bothions&CG)Electricfieldgradients(ionsonly)VacuumInterface:CurtainGasAnalyzer-Q0RegionAtmos.tovac.flowisafree-jetexpansionOR–SKregionis“highpressure”(1torr),CIDfragmentationmayoccurhere(alsoinSK–Q0region,6x10-3torr)IonsarefocusedintoSKbytheRNG,confinedbyQ0,andattractedtowardIQ1NeutralspeciesarepumpedawayQ0uses“collisionalfocusing”ofionsAnalyzer-Q0RegionAtmos.toAPI-3000IonPath6mTorr1TorrVarian550S25BPumpLeybold3611e-5torrBackedbyD10EDP/ORFP/RNGEP/Q0IQ1STRO1IQ2RO2(LINAC)ST3RO3DFCEMDifferentialPumpingZones(Skimmer,QO&MainQ0=firstQuad.Skimmer,IQ#=Inter-Quad.Lens,ST=StubbiesQuad.,RO#=Quad.,RO2LINAC,DF=Deflector,CEMIonenergies

Q1=Q0-RO1,CE=Q0-RO2(CollisionEnergy),Q3=RO2-RO3(eV)TunablePotentials(MassOrCmpd.Dependent)

OR,RNG,RO2,(ST3unlinked)EPfixed(IQ1,STRO1LinkedEP),IQ2Fixed,(ST3,R03linkedRO2)API-3000IonPath6mTorr1TorrAnalyzer-IQ1,ST&RO1IonspassingthroughIQ1arefocusedbystubbies(pre-filter)intoQ1(massfilter)Q1separatesionsbasedonmass/chargeratioStubbieshaveRFvoltageandDC“offset”Typicalvoltagesfor(+)ion:EP/Q0 Fixed @ -10VIQ1 Fixed @ -11VST Fixed @ -15-17VRO1 Fixed @ -10.5-11V(Individualinstrumentsmayvary)CEP&CXP-CollisioncellpotentialCEP=Collisioncellentrances=Q0-IQ2Fixedon3000CXP=Collisioncellexitpotential=RO2-ST3(massdependent)Massdependenton3000Analyzer-IQ1,ST&RO1IonspAPI-3000IonPath(cont)GriddedexitlenshardwiredtoIQ3Stubbies2andIQ2linkedasIQ2IQ3hardwired1voltmoreattractivethanQ2(RO2)API-3000IonPath(cont)GriddAPI-3000MassFilterRailStubbies2and3heldinQ2endcapsStubbies2coupledtoQ1RFandstubbies3coupledtoQ3RFAPI-3000MassFilterRailStubbAPI-3000CollisionCell-LinacQ2Linac(linearaccelerator)eliminatescross-talkandallowsfasterMS/MSscanningwithoutsensitivitylossesQ2rodsaretiltedandseparateDCpotentialsareappliedtoeachpairofrodstocreateanaxialelectricfieldAPI-3000CollisionCell-LinaCollisionalFocusingCollisionalfocusingisapatentedtechnique,ownedbyPE-Sciex,whichimprovesiontransmissioninQ0andQ2.

Highpressure (8mtorr) Lowpressure (<8mtorr)CollisionalFocusingCollisionaPE-Sciex’sLINACTechnologyPE-Sciex’sLINACTechnologyGenerationoftheLINACAxialFieldGradientFieldgradientestablishedbyanglingrods,eachpairhavingadifferentDCpotentialEntranceofcollisioncellExitofcollisioncell+4v+4v+6v+6v+4v+4v+6v+6vfieldoncenterline≈5.75vfieldoncenterline≈4.25v•entranceexit•GenerationoftheLINACAxialIonPathLinac“Off”Injectionofselegilinetodeterminethelevelofcross-talkintheamphetamineMRMtransitionIonPathLinac“Off”InjectionIonPathwithLinacInjectionofselegilinetodeterminethelevelofcross-talkintheamphetamineMRMtransitionwithaLinacQ2IonPathwithLinacInjectionSelegilineresponsewithdifferentMRMdwelltimesSelegilineresponsewithdiffeEffectofdwelltimeonresolutionprecursorionscanmodeEffectofdwelltimeonresolu

Q1fullscanQ3fullscanSelectedionmonitoring(SIM)ProductionscanPrecursorionscanConstantneutrallossscanMultiplereactionmonitoring(MRM)APITripleQuadBasicScanModesQ1fullscanAPITripleQuadBSingleMSOperationQ1FullScan(Start–Stop)Q1alwaysusedassingleMSanalyzerUsedprimarilyforident.ofprecursorionIntheAPI-2000,Q3operatesinRF-onlymodeAPI-2000operatesverysimilartoanAPI-150EXSingleMSOperationQ1FullScaSingleMSOperation(cont.)MultipleMasses/CenterWidthMode

MultiplesmallscanrangesofthesamewidthResolutiontuneandcalibratemassaxis(withPPG’s)orestablishexactmassofmolecularion/fragmentsQ3ScanusedonlytocalibrateQ3mass(canalsocalibrateQ3inMS/MSmode):CADgasison(=1)toslowionsdownQ1operatesinRF-onlymodeSingleMSOperation(cont.)MulSingleMSOperation(cont.)SIM-SelectedIonMonitoring(Width~0):UsedtooptimizeanalyzerforspecificionsSIMusedforquantitativeanalysesQ1SIMusedto“optimize”precursorionMaximizesignalinpreparationforMS/MSSingleMSOperation(cont.)SIMQ1MSScansQ1FullScan(Start–Stop)Q1alwaysusedassingleMSanalyzerUsedprimarilyforident.ofprecursorionQ3operatesinRF-onlymodeSIM-SelectedIonMonitoring(ormultipleions):UsedtooptimizeanalyzerforspecificionsforMS/MSSIMusedforquantitativeanalysesQ1MSScansQ1FullScan(StartQ3FullScan(Start–Stop)Q3alwaysusedassingleMSanalyzerUsedprimarilyforident.ofprecursorionorforIDAuseQ1operatesinRF-onlymodeQ3MSScansSIM-SelectedIonMonitoring(ormultipleions):UsedtooptimizeanalyzerforspecificionsforMS/MSSIMusedforquantitativeanalysesQ3FullScan(Start–Stop)Q3FragmentationWhenanIonfragmentsthefollowingformulasapply:Positivemode(Parent)+

A++BNeutralNegativemode(Parent)-

A-+BNeutralTwocommontypesmodesoffragmentation:CAD=CollisionallyActivatedDissociationincollisioncell,gasmoleculescollideandcauseweakbondstobreak.CID=CollisionallyInducedDissociationincurtaingasregion,OR/DP&EP/Q0,non-selectiveFragmentationWhenanIonfragMS/MS-ProductIonScanProductionscan-commonMS/MSmodeAfteridentification,theprecursorionissentintothecollisioncellandfragmentedbyCIDQ1isfixed,Q3sweepsagivenmassrangeUsedforstructuralinformationgatheringandidentificationofproductionsFirststeptodevelopingquantitativemethodMS/MS-ProductIonScanProduMS/MS-ProductIonScan(cont.)ProductIonScanm1+m2+m2+m2+Productionspectrumofaparticularcompoundm1+setm2+scanMS/MS-ProductIonScan(contm1+fixedm3+scannedMS/MSProductIonScanAfteridentification,theprecursorionissentintothecollisioncellandfragmentedbyCIDQ1isfixed,Q3sweepsagivenmassrangeUsedforstructuralelucidationFirststeptodevelopingquantitativemethodm1+fixedm3+scannedMS/MSProEphedrine,MW=165(M+H)+166ExampleofProductIonSpectrumEphedrine,MW=165(M+H)+ExampMS/MS-PrecursorIonScanPrecursorionscanQ1sweepsagivenmassrange,Q3isfixedUsedtodeterminethe“origin”ofparticularproduction(s)createdinthecollisioncellFrequentlyusedfordrugmetaboliteidentification(commonproductionobservedinthemetabolites)MS/MS-PrecursorIonScanPrecMS/MS-PrecursorIonScan(cont.)PrecursorIonScanm1+m2+m1+m1+Asetofcompoundswithacommonproductionm1+scanm2+setMS/MS-PrecursorIonScan(com3+fixedm1+scannedQ1sweepsagivenmassrange,Q3isfixedUsedtodeterminethe“origin”ofparticularproduction(s)createdinthecollisioncellFrequentlyusedfordrugmetaboliteidentification(commonproductionobservedinthemetabolites)MS/MSPrecursorIonScanm3+fixedm1+scannedQ1sweeps600900120015001800m/z,amu587.5745.6895.6802.4633.4520.31105.01704.4600900120015001800m/z,amu3.0e66.0e69.0e61.2e7Intensity,cps608.3520.3802.4600900120015001800m/z,amu3.0e46.0e49.0e4Intensity,cpsPrecursorsof86(Ile/Leu)Q1Scan745.6ExampleofPrecursorIonSpectrumAnalysisofpeptidesthathaveLucineorisolucine600900120015001800m/z,amu587.MS/MSConstantNeutralLossNeutrallossscanQ1&Q3bothscanagivenmassrangebutwithaconstantdifferencebetweenrangesscannedSpectrumindicateswhichionsloseaneutralspeciesequaltoQ1-Q3differenceComplementtoPrecursorIonScanNeutral“gain”indicatesamultiplychargedprecursorionwasfragmentedMS/MSConstantNeutralLossNeuMS/MSConstantNeutralLoss(cont.)ConstantNeutralLossScanm1+m2+m2+m1+Asetofcompoundswithacommonneutralfragmentm1+scanm2+scanm-m-mMS/MSConstantNeutralLoss(cm1+scannedm3+scannedΔmQ1&Q3bothscanagivenmassrangebutwithaconstantdifferencebetweenrangesscannedSpectrumindicateswhichionsloseaneutralspeciesequaltoQ1-Q3differenceComplementtoPrecursorIonScanNeutral“gain”indicatesamultiplychargedprecursorionwasfragmentedMS/MSConstantNeutralLossScanm1+scannedm3+scannedΔmQ1&24681012141618Time,min010002000300040005000600070008000Intensity,cps1-Hydroxy-BuspironeGlucuronide2-BuspironeGlucuronide21ConstantNLScan-PhaseIIMetabolitesRet.Time6.6minNL176MS/MSBuspironeGlucuronideEPIof578NNNNNOOGluc-OBuspironeGlucuronideMS3of578402NNNNNOO138265222HO24681012141618Time,min0100020MS/MS-MultiplereactionMonitoring(MRM)IfQ1andQ3width=0,thenMRMManyprecursortoproductionpairscanbemonitored(A-B,A’-B’,A”-B”,etc.)MRManalysisisthebestwaytomaximizesignalintensityofproductionsMRMusedprimarilyforquantitationstudiesMS/MS-MultiplereactionMoniMS/MS-MultipleReactionMonitoring(MRM)(cont.)PrecursorionsetProductionsetFragmentation(CAD)MS/MS-MultipleReactionMoniPrecursorionfixedProductionfixedFragmentation(CAD)Manyprecursortoproductionpairscanbemonitored(A-B,A’-B’,etc.)MRMisthebestwaytomaximizesignalintensityofproductionsMRMusedprimarilyforquantitationMS/MSMultipleReactionMonitoring(MRM)PrecursorionProductionFragHighThroughputMRM:LC/MS/MSBioAnalysisofMultipleDrugs

atenolol,pindolol,acebutololamoxicillin,dicloxicillin,ampicillinHighThroughputMRM:atenololSelectivity:LC/MSvs.LC/MS/MS

Cruderacehorseurineextractdexamethazone(21hrpostdose)2nginsampleinjectedbothcases1.02.03.04.001020304050607080901001.02.03.04.05.06.00102030405060708090100IonselectionToDetectorPrecursorionselectionProductionselectionFragmentation(CAD)ToDetectorSelectivity:LC/MSvs.LC/MS/MWhyLC/MS/MSforQuantitation?SelectbyMWSelectbyStructureIncreasedSensitivity&SpecificityRetentionTime

SeparateonHPLCRetentionTimeIntensitye.g.howmuchcocaineisinurine?WhyLC/MS/MSforQuantitationAPI-3000Analyzer-Q3OperationInallMS/MSmodes,Q3isamassfilterIonsareseparatedbasedonmass/chargeratioQ2is“source”ofproductionsenteringQ3InsingleMSscans,Q3isRF-onlymodeQ3transmitsallionstowarddetectorregionQ3actsas“ionguide”forproductionsAPI-3000Analyzer-Q3OperatioGasControllersNebulizer(Gas1)operatingpre