液质联用中质谱条件的优化策略

液质分析条件的优化策略

（简化板）液相色谱与液质联用仪使用的要点质量校正的正确对于相关分析要有合适的支持软件（Maxnet，TargeLyness）合适的液相色谱平台合适的质谱接口方式液相色谱分析中合适的色谱柱的选用质谱检测模式的选择必要的后液流补充质量校正的正确（Myoglobin校正）质量校正的正确（Myoglobin校正）质量校正的正确（CsI校正）的肽测定质量校正的正确（CsI校正）的蛋白测定质量校正的关键点针对不同的分析采用不同的校正，一般蛋白质采用Myoglobin，对于小分子分析建议采用CsI校正。对于采用Myoglobin校正的建议采用高次方的校正曲线（3-4）；对CsI校正的建议采用低次方的校正曲线（1-2）合适的液相色谱平台能提供一个连续、稳定的液流环境真空脱气设备系统的死体积尽可能小，减少管路的长度输液泵的设计能适用于微径柱的要求二极管阵列检测器的池体积与质谱仪匹配必要的液相色谱辅助配件必要的液相色谱与质谱仪的软件操作平台合适的液相色谱柱柱内径：2.1mm柱长度：根据分析的目的选择50mm或150mm柱填料选用新型的填料：Symmetry，Discovery，Vydac，Zorbax，Xterra，Intersil，Diamonsil等LC/MSFlowInjectionAnalysisofPeptidesandProteinsbyReversed-PhaseHPLC1.0%HOAcminAbundance10.0012.0014.0016.002.004.006.008.00500001000001500002000002500000.2%TFA2.004.006.008.0010.0012.0014.0016.0050000100000150000200000250000minAbundanceReverse-PhaseLC/MSSolventsACN,MeOH,H2O,IsopropanolNormal-PhaseLC/MSSolvents(forAPCI-MS)Hexane,MethyleneChloride,Acetone,EthanolCompatibleLC/MSBuffersandModifiers:Formicacid,aceticacid,ammoniumacetate,ammoniumformate,ammoniumhydroxide,trifluoroaceticacidTFAconcentrationshouldbe<0.1%v/vKeepvolatilebufferconcentrations<20mMtominimizeESIionsuppressionAvoidNon-volatileBuffersAlkali-metalphosphates,borates,etc.SuitableSolventsforLC/MSVolatilebuffer––minimizeinstrumentdowntimeBufferconcentration:HighionsuppressiondecreasesESIsensitivityLowsystemadequatelybuffered?pHrangepermittedbystationaryphaseMethanoloracetonitrileStartwithacetonitrile01111ChangeRetentiontoImproveResolution––SelectSolvents/ModifiersthatareMSCompatibleUsefulpHRangesforVolatileBuffersBuffersnormallyusedinLC/MS:01094BufferpKapHrangeFormate3.82.8–4.8Acetate4.83.8–5.89.28.2–10.2Triethylamine11.010–12Diethylamine10.59.5––11.5???Ammonia76––8BufferConcentrations/Additiveamounts:10to50mMformic,aceticacids0.01-1%v/vtrifluoroaceticacid<0.1%v/valkylaminetypebases<0.1%v/vEffectofBufferonAnalyteResponsePhosphatebufferssuppresstheMSresponseofcaffeineatallpHsandalsotheMSresponseofOxazepamatpH8.Volatilebuffers(formate,acetate,ammonia)generallyprovidegoodresponses.01121Mobilephase:A-10mMbufferpH6.0;B––methanolGradient:5%to75%in4minMobilePhasepHeffectonESIColumn:HyPURITY™C185m,50x2.1mmAqueousmobilephases:0.1%FormicacidpH3,Ammoniumformate20mMpH5,Ammoniumacetate20mMpH8.2,Ammoniumacetate20mMpH9,Ammoniumacetate20mMAqueous/methanol(50:50)Flowrate:0.2ml/minTemperature:25°CDetection:+ESI,450°°C,4.5kV,20V-ESI,450°C,3.5kV,20VScan:120––480uAnalytes:NortriptylinePropranololTetracyclineCaffeineParacetamolTryptophanSalicylicacidNicotinicacid01113EffectofMobilePhasepHon+ESIResponse01115EffectofMobilePhasepHon-ESIResponse01116SolventSystem50/50MeOH/H2O50/50ACN/H2O100%H2O100%MeOH100%ACN50/50MeOH/H2O1%Acetic50/50MeOH/H2O0.1%Formic50/50ACN/H2O1%Acetic50/50ACN/H2O0.1%Formic50/50MeOH/H2O5mMNH4OAc50/50MeOH/H2O10mMNH4OAc50/50MeOH/H2O0.1%TFA50/50MeOH/H2O0.05%TFA50/50MeOH/H2O0.02%TFA50/50ACN/H2O0.1%TFA50/50ACN/H2O0.05%TFA50/50ACN/H2O0.02%TFA50/50MeOH/H2O0.1%NH4OH50/50ACN/H2O0.1%NH4OH0100000200000300000400000500000600000IonSignal,Counts[M+H]+SolutionChemistryEffectsonPositiveIonESI-MSofLeu-EnkephalinLC/MSSensitivityvs.MobilePhaseModifierGluCDigestofBS5%Acetic0.001%TFA0.005%TFA0.01%TFAZorbax300SB-C3(2.1x150mm)HP1100MSDReversed-phaseHPLC/MSanalysisofaGluCdigestofBSAwasusedasamodeltotesttherecoveryandpeakshapeofpeptidesusingvaryingconcentrationsofTFAor5%aceticacidasamobile-phaseadditiveincombinationwiththeZorbax300SB-C3.DigestionofBSAwascarriedout37°Covernight,usingGluCina1:20ratiowithBSA(byweight).Thefinalmixturecontained1Mureaand25mMsodiumphosphate.Asignificantincreaseinsensitivityofpeptideswasobservedformostpeptidesanalyzedusing5%aceticacidratherthanTFA.ReducingTFAconcentrationto0.001%causedonlyaminorimprovementinsensitivity.Somepeptidesweremuchlessaffectedbyadditivechangethanothers.0for5min,0-40%B/55minthen40-100%B/20minF=0.2mL/min,A=5%AceticAcid,B=ACNMSD1TIC,MSStable,StericallyProtectedC3BondedPhaseinLCandLC/MSApplications,R.D.Ricker(1),B.E.Boyes(1),J.P.Nawrocki(2),andL.K.Pannell(2)(1)AgilentTechnologies,Inc.LCApplicatonsLab538FirstStateBlvd,Newport,DE19804-3552USA.(2)StructuralMassSpectrometryGroupNIDDK,NIH,Bethesda,MD,20892USA.EasternAnalyticalSymposium,Nov.,1999ProposedMechanismforTFASignalSuppressionandthe"TFA-Fix"-(M+H)+

+CH3COO- [(M+H)+•CH3COO-]"±0” WeakIonPairingwithAcid-AnionggCF3COO-+RCOOH CF3COOH+RCOO-

AcidCompetition(TFAmorevolatile)(M+H)+

+CF3COO- [(M+H)+•CF3COO-]"±0”

StrongIonPairingwithTFA-AnionHPLCConditionsColumn: 2.1x250mmVydacC-18FlowRate: 200µl/minSolventA: Water+0.1%TFA SolventB:CH3CN+0.1%TFAGradient: 0-60%Bin60minTemp:50°C

2000006000001000000AbundanceWithout"TFA-Fix"18.0022.0026.0030.0034.0038.002000006000001000000minAbundanceWith"TFA-Fix"1:2post-columnadditionof75%propionicacidinIPATrypticDigestMapbyES-LC/MS1nmolChickenLysozymeSignalSuppressionduetoAdditivesIonpairingwithanalyte,surfacetensioneffectSolutions:Post-columnadditionofasheathliquidofpropionicacid(10%)in2-propanol(TFAFix)UselowconcentrationsofTFAwithaceticacid(TFALight)ReplaceTFAESIsignalsuppressionbyTFA01122AchievementofgaseousanalyteionizationatAPIinterfaceisthekeytoMSdetection离子化化方式式极性化化合物物多采采用电电喷雾雾（ESI），，其中中含氮氮的化化合物物一般般在酸酸性条条件下下用ESI+（生物物碱）），含含多羟羟基化化合物物采用用中心心条件件下的的ESI-（玉米米赤酶酶醇））。非极性性化合合物多多采用用大气气压化化学电电离源源（APcI））（激激素）），原原则上上不建建议采采用添添加其其它化化学试试剂StepsforESIOptimizationIfanalyte’’spKaisunknown,evaluate3pHregionsinpositiveandnegativeionmodes.Acids––NegativeIondetection,adjustpH2unitsabovepKaIncreasepHwithNH4OH,TEA,TMABases––PostiveIonDetection,adjustpH2unitsbelowpKa1DecreasepHuseformicacid,aceticacid,TFARemovesaltswhichmaycauseionsuppressionAdjustsourcetemperatureandsourcevoltagestomaximizesignalInnegativeionmode,uselowersprayvoltagetominimizedischarge1Incomplexmolecules,manyexceptionstotheserulesareobserved01564MaximizingHighFlowESISensitivitySelectappropriatechromatographygradeHPLCsolventsAvoidexoticsolventmixes(MeOH,MeCN,Water,0.1%formicworkbestfor98%ofLC/MSapplications)Avoidaddingexcessivemodifiers(egamm.acetate@10mMnot50mM)Choosetherightcolumnchemistry(C-8vs.C-18);changecolumnchemistrybeforechangingsolventmixorcomposition2.1mmcolumnorlower,flowratesof200-400uL/minPeakwidthsforquantificationnotgreaterthan8-10secsDissolvesampleinstartmobilephasesolvent(weakestsolventpossible)02383SolventsCompatibleWithESIMethanolAcetonitrilePropanolIsopropanolButanol2-methoxyethanolAceticFormicTFAAmm.AcetateAmm.FormateHFBATEAAmm.HydroxideTetrabutylamm.hydroxideHexafluorobutanolSamplescanbedissolvedinanyHPLCcompatiblesolventModifiersBetween0.05-1%and5-50mM02382ElectrospraySummaryAnalytetype:preformedions(acidsandbases)polarneutralsmultiplychargedionsofbiopolymers<100daupto200000daTypicalflowrates:lownL-1.0ml/minPromoteionization:correctpHfavorableHPLCsolventcompositionPost-columnadditionofreagentsSoftionizationtechniqueTypicalapplications:Drugs,Sugars,Peptides,Proteins,Oligonucleotides01328StepsforAPCIOptimizationIfanalyte’spKaisunknown,evaluate3pHregionsinpositiveandnegativeionmodes.Acids––NegativeIondetection,adjustpH2unitsbelowpKaDecreasepHuseformicacid,aceticacid,TFABases––PositiveIondetection,adjustpH2unitsabovepKaIncreasepHwithNH4OH,TEA,TMAAdjustcoronadischargevoltageAdjustnebulizationtemperatureConsiderpossiblethermaldecompositionofanalyte01565APCI–TypicalOperatingConditionsFlowRates:50µL/min.-2mL/min.VaporizerTemp(°C):400-550(600max)DischargeCurrent(µA):5(20µAmax.)SheathGasFlowRate(arb):35-80AuxiliaryGas:0CapillaryTemp(°C):250-350CTubeLensOffset(V):30-60VHigherflowratemeansmoresolventforplasmaproductionPositionofcoronadischargeneedleiscriticalforsensitivity“Worksbetterathigherflowrates”02386APCI-TipsEnsurethattheAPCIprobeishotenoughsothatthesprayshieldisnotdrippingwetVaporizerTemp:400-450Cisagoodstart(400-1000uL/min)ReducetemperatureofheatedcapillaryifneededChecksheathgascarefullyBeginwithauxgasat0MemoryeffectduetocompoundsburningontoprobepartsifinjectedinlargeconcentrationsBakeoutsourceperiodically02385APCISummaryAnalytetype:lowtomidpolarityhighprotonaffinityhighgasphaseacidity<1200daTypicalflowrates:0.2-2.0ml/minToleratesbuffersbetterthanESIProducessinglychargedionsonlySoftionizationtechniqueTypicalapplications:Drugs,Pesticides,Steroids,Azodyes01332ObtainingaReferenceSpectrumUnliketheEIprocess,spectrageneratedbyAPILC/MSvarydependingonmobilephasesourceconditionsionizationmodein-sourceCIDconditionsSpectralVariations-mobilephasem/z751001251501752002250.02mMNaOAc+0.1%HOAc195.1217.0196.1138.1m/z7510012515017520022501000020000300004000050000040000800001200000.1%HOAc195.1138.1196.1SpectralVariations-CIDconditionsm/z100150200250300010000200003000040000Fragmentor=120Volts124.1186.0279.0156.0108.092.1213.0204.0301.0m/z10015020025030004000080000120000Fragmentor=75Volts279.0186.0SpectralVariations-ionpolaritym/z100150200250300API-ESNegative283.0284.992.0156.0128.1195.9183.0m/z100150200250300020406080100020406080100API-ESPositive156.0285.0108.092.1286.9质谱参数数的控制制电离电压压（capillaryvoltage）导入电压压（conevoltage）Probe位位置雾化气流流与干燥燥气流聚焦棱镜镜电压（（Len）根据需求求条件一一级、三三级的分分辨率，，原则上上scan、daughter条件件下，选选用单位位质量分分辨条件件，在mrm条条件下，，选用2-3单单位质量量分辨率率。合适的碰碰撞能量量和碰撞撞室的聚聚焦电压压。合适稳定定的碰撞撞室气压压。检测器的的倍增电电压，有有选择性性的调节节。多级质谱谱提高灵灵敏度的的方法通过合适的的电离方式式进行，尤尤其是对于于能否将[M+Na]+改变为[M+H]+通过合适的的方法提高高电离效率率，如后补补液来调整整流动相的的PH值和和有机溶剂剂比例关系系等。研究scan、daughter谱图图，寻找特特征具有结结构特征的的碎片离子子，一般而而言碎片越越稳定、丰丰度越大而而得到的灵灵敏度越高高。MRMAnalysisof192>159.9(Carbendazim)SIRAnalysisof192(Carbendazim)SIR:MRMComparisonforCarbendazim(0.05pg/mLstd)液相色谱/质谱联用用关键点分清分析的的目的是分分离定性为为主，还是是高灵敏度度定量为主主。定性优优先考虑色色谱分离的的好坏，兼兼顾考虑质质谱要求；