液质分析条件的优化策略课件

液质分析条件的优化策略

（简化板）第一页，共四十三页。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第二页，共四十三页。质量校正的正确（Myoglobin校正）第三页，共四十三页。质量校正的正确（Myoglobin校正）第四页，共四十三页。质量校正的正确（CsI校正）的肽测定第五页，共四十三页。质量校正的正确（CsI校正）的蛋白测定第六页，共四十三页。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）第七页，共四十三页。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第八页，共四十三页。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等第九页，共四十三页。LC/MSFlowInjectionAnalysisofPeptidesandProteinsbyReversed-PhaseHPLC1.0%HOAcminAbundance10.0012.0014.0016.002.004.006.008.00500001000001500002000002500000.2%TFA2.004.006.008.0010.0012.0014.0016.0050000100000150000200000250000minAbundance第十页，共四十三页。Reverse-PhaseLC/MSSolventsACN,MeOH,H2O,IsopropanolNormal-PhaseLC/MSSolvents(forAPCI-MS)Hexane,MethyleneChloride,Acetone,Ethanol

CompatibleLC/MSBuffersandModifiers:Formicacid,aceticacid,ammoniumacetate,ammoniumformate,ammoniumhydroxide,trifluoroaceticacidTFAconcentrationshouldbe<0.1%v/vKeepvolatilebufferconcentrations<20mMtominimizeESIionsuppressionAvoidNon-volatileBuffersAlkali-metalphosphates,borates,etc.SuitableSolventsforLC/MS第十一页，共四十三页。Volatilebuffer–minimizeinstrumentdowntimeBufferconcentration:HighionsuppressiondecreasesESIsensitivityLowsystemadequatelybuffered?pHrangepermittedbystationaryphaseMethanoloracetonitrileStartwithacetonitrile01111ChangeRetentiontoImproveResolution–

SelectSolvents/ModifiersthatareMSCompatible第十二页，共四十三页。UsefulpHRangesforVolatileBuffersBuffersnormallyusedinLC/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/v第十三页，共四十三页。EffectofBufferonAnalyteResponsePhosphatebufferssuppresstheMSresponseofcaffeineatallpHsandalsotheMSresponseofOxazepamatpH8.Volatilebuffers(formate,acetate,ammonia)generallyprovidegoodresponses.

01121Mobilephase:A-10mMbufferpH6.0;B–methanolGradient:5%to75%in4min第十四页，共四十三页。MobilePhasepHeffectonESIColumn: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:NortriptylinePropranololTetracyclineCaffeineParacetamolTryptophanSalicylicacidNicotinicacid01113第十五页，共四十三页。EffectofMobilePhasepHon+ESIResponse01115第十六页，共四十三页。EffectofMobilePhasepHon-ESIResponse01116第十七页，共四十三页。SolventSystem50/50MeOH/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-Enkephalin第十八页，共四十三页。LC/MSSensitivityvs.MobilePhaseModifier

GluCDigestofBS5%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.,1999第十九页，共四十三页。ProposedMechanismforTFASignalSuppressionandthe"TFA-Fix"-(M+H)+

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

AcidCompetition(TFAmorevolatile)(M+H)+

+CF3COO- [(M+H)+•CF3COO-]"±0”

StrongIonPairingwithTFA-Anion第二十页，共四十三页。HPLCConditionsColumn: 2.1x250mmVydacC-18FlowRate: 200µl/minSolventA: Water+0.1%TFA SolventB:CH3CN+0.1%TFAGradient: 0-60%Bin60minTemp:50°C

200000600000AbundanceWithout"TFA-Fix"18.0022.0026.0030.0034.0038.00200000600000minAbundanceWith"TFA-Fix"1:2post-columnadditionof75%propionicacidinIPATrypticDigestMapbyES-LC/MS

1nmolChickenLysozyme第二十一页，共四十三页。SignalSuppressionduetoAdditivesIonpairingwithanalyte,surfacetensioneffectSolutions:Post-columnadditionofasheathliquidofpropionicacid(10%)in2-propanol(TFAFix)UselowconcentrationsofTFAwithaceticacid(TFALight)ReplaceTFAESIsignalsuppressionbyTFA

01122AchievementofgaseousanalyteionizationatAPIinterfaceisthekeytoMSdetection第二十二页，共四十三页。离子化方式极性化合物多采用电喷雾（ESI），其中含氮的化合物一般在酸性条件下用ESI+（生物碱），含多羟基化合物采用中心条件下的ESI-（玉米赤酶醇）。非极性化合物多采用大气压化学电离源（APcI）（激素），原则上不建议采用添加其它化学试剂第二十三页，共四十三页。StepsforESIOptimizationIfanalyte’spKaisunknown,evaluate3pHregionsinpositiveandnegativeionmodes.Acids–NegativeIondetection,adjustpH2unitsabovepKaIncreasepHwithNH4OH,TEA,TMABases–PostiveIonDetection,adjustpH2unitsbelowpKa1

DecreasepHuseformicacid,aceticacid,TFARemovesaltswhichmaycauseionsuppressionAdjustsourcetemperatureandsourcevoltagestomaximizesignalInnegativeionmode,uselowersprayvoltagetominimizedischarge1

Incomplexmolecules,manyexceptionstotheserulesareobserved01564第二十四页，共四十三页。MaximizingHighFlowESISensitivitySelectappropriatechromatographygradeHPLCsolventsAvoidexoticsolventmixes(MeOH,MeCN,Water,0.1%formicworkbestfor98%ofLC/MSapplications)Avoidaddingexcessivemodifiers(egamm.acetate@10mMnot50mM)Choosetherightcolumnchemistry(C-8vs.C-18);changecolumnchemistrybeforechangingsolventmixorcomposition2.1mmcolumnorlower,flowratesof200-400uL/minPeakwidthsforquantificationnotgreaterthan8-10secsDissolvesampleinstartmobilephasesolvent(weakestsolventpossible)02383第二十五页，共四十三页。SolventsCompatibleWithESIMethanolAcetonitrilePropanolIsopropanolButanol2-methoxyethanolAceticFormicTFAAmm.AcetateAmm.FormateHFBATEAAmm.HydroxideTetrabutylamm.hydroxideHexafluorobutanolSamplescanbedissolvedinanyHPLCcompatiblesolventModifiersBetween0.05-1%and5-50mM02382第二十六页，共四十三页。ElectrospraySummaryAnalytetype:preformedions(acidsandbases)polarneutralsmultiplychargedionsofbiopolymers<100daupto200000daTypicalflowrates:lownL-1.0ml/minPromoteionization:correctpHfavorableHPLCsolventcompositionPost-columnadditionofreagentsSoftionizationtechniqueTypicalapplications:Drugs,Sugars,Peptides,Proteins,Oligonucleotides01328第二十七页，共四十三页。StepsforAPCIOptimizationIfanalyte’spKaisunknown,evaluate3pHregionsinpositiveandnegativeionmodes.Acids–NegativeIondetection,adjustpH2unitsbelowpKaDecreasepHuseformicacid,aceticacid,TFABases–PositiveIondetection,adjustpH2unitsabovepKaIncreasepHwithNH4OH,TEA,TMAAdjustcoronadischargevoltageAdjustnebulizationtemperatureConsiderpossiblethermaldecompositionofanalyte01565第二十八页，共四十三页。APCI–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”02386第二十九页，共四十三页。APCI-TipsEnsurethattheAPCIprobeishotenoughsothatthesprayshieldisnotdrippingwetVaporizerTemp:400-450Cisagoodstart(400-1000uL/min)ReducetemperatureofheatedcapillaryifneededChecksheathgascarefullyBeginwithauxgasat0MemoryeffectduetocompoundsburningontoprobepartsifinjectedinlargeconcentrationsBakeoutsourceperiodically02385第三十页，共四十三页。APCISummaryAnalytetype:lowtomidpolarityhighprotonaffinityhighgasphaseacidity<1200daTypicalflowrates:0.2-2.0ml/minToleratesbuffersbetterthanESIProducessinglychargedionsonlySoftionizationtechniqueTypicalapplications:Drugs,Pesticides,Steroids,Azodyes01332第三十一页，共四十三页。ObtainingaReferenceSpectrumUnliketheEIprocess,spectrageneratedbyAPILC/MSvarydependingonmobilephasesourceconditionsionizationmodein-sourceCIDconditions第三十二页，共四十三页。SpectralVariations-mobilephasem/z751001251501752002250.02mMNaOAc+0.1%HOAc195.1217.0196.1138.1m/z7510012515017520022501000020000300004000050000040000800001200000.1%HOAc195.1138.1196.1第三十三页，共四十三页。SpectralVariations-CIDconditionsm/z100150200250300010000200003000040000Fragmentor=120Volts124.1186.0279.0156.0108.092.1213.0204.0301.0m/z10015020025030004000080000120000Fragmentor=75Volts279.0186.0第三十四页，共四十三页。SpectralVariations-ionpolaritym/z100150200250300API-ESNegative283.0284.992.0156.0128.1195.9183.0m/z100150200250300020406080100020406080100API-ESPositive156.0285.0108.092.1286.9第三十五页，共四十三页。质谱参数的控制电离电压（capillaryvoltage）导入电压（conevoltage）Probe位置雾化气流与干燥气流聚焦棱镜电压（Len）根据需求条件一级、三级的分辨率，原则上scan、daughter条件下，选用单位质量分辨条件，在mrm条件下，选用2-3单位质量分辨率。合适的碰撞能量和碰撞室的聚焦电压。合适稳定的碰撞室气压。检测器的倍增电压，有选择性的调节。第三十六页，共四十三页。多级质谱提高灵敏度的方法通过合适的电离方式进行，尤其是对于能否将[M+Na]+改变为[M+H]+通过合适的方法提高电离效率，如后补液来调整流动相的PH值和有机溶剂比例关系等。研究scan、daughter谱图，寻找特征具有结构特征的碎片离子，一般而言碎片越稳定、丰度越大而得到的灵敏度越高。第三十七页，共四十三页。MRMAnalysisof192>159.9(Carbendazim)SIRAnalysisof192(Carbendazim)SIR:MRMComparisonfor