液质联用中质谱条件的优化策略（PPT42页)

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%TFA2.004.006.008.0010.0012.0014.0016.0050000100000150000200000250000minAbundance•Reverse-PhaseLC/MSSolvents–ACN,MeOH,H2O,Isopropanol•Normal-PhaseLC/MSSolvents(forAPCI-MS)–Hexane,MethyleneChloride,Acetone,Ethanol•CompatibleLC/MSBuffersandModifiers:–Formicacid,aceticacid,ammoniumacetate,ammoniumformate,ammoniumhydroxide,trifluoroaceticacid–TFAconcentrationshouldbe0.1%v/v–Keepvolatilebufferconcentrations20mMtominimizeESIionsuppression•AvoidNon-volatileBuffers–Alkali-metalphosphates,borates,etc.SuitableSolventsforLC/MS•Volatilebuffer–minimizeinstrumentdowntime•Bufferconcentration:–HighionsuppressiondecreasesESIsensitivity–Lowsystemadequatelybuffered?•pHrangepermittedbystationaryphase•Methanoloracetonitrile–Startwithacetonitrile01111ChangeRetentiontoImproveResolution–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:•10to50mM•formic,aceticacids0.01-1%v/v•trifluoroaceticacid0.1%v/v•alkylaminetypebases0.1%v/vEffectofBufferonAnalyteResponsePhosphatebufferssuppresstheMSresponseofcaffeineatallpHsandalsotheMSresponseofOxazepamatpH8.Volatilebuffers(formate,acetate,ammonia)generallyprovidegoodresponses.01121Mobilephase:A-10mMbufferpH6.0;B–methanolGradient:5%to75%in4min0.E+005.E+071.E+082.E+082.E+083.E+083.E+08FormatepH2.5PhosphatepH2.5AcetatepH6.0PhosphatepH6.0AmmoniapH8.0PhosphatepH8.0BuffertypeIntensity(peakarea)ProcainamideCaffeinePropranololNortriptylineOxazepamMobilePhasepHeffectonESIColumn:HyPURITY™C185m,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+ESIResponse01115VariationofpeakareawithpHinpositiveESI0.E+002.E+064.E+066.E+068.E+061.E+071.E+071.E+07NortriptylinePropranololTetracyclineCaffeineTryptophanParacetamolNicotinicacidSalicylicacidCompoundPeakarea0.1%formicpH3pH5pH8pH9EffectofMobilePhasepHon-ESIResponse01116VariationofpeakareawithpHinnegativeESI0.E+005.E+041.E+052.E+052.E+053.E+053.E+054.E+054.E+055.E+055.E+05NortriptylinePropranololTetracyclineCaffeineTryptophanParacetamolNicotinicacidSalicylicacidCompoundArea0.1%formicpH3pH5pH8pH9SolventSystem50/50MeOH/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-Enkephalin3x106min0102030405060LC/MSSensitivityvs.MobilePhaseModifierGluCDigestofBS5%Acetic0.001%TFA0.005%TFA0.01%TFAZorbax300SB-C3(2.1x150mm)HP1100MSD•Reversed-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.,1999ProposedMechanismforTFASignalSuppressionandtheTFA-Fix-(M+H)++CH3COO-[(M+H)+•CH3COO-]±0”WeakIonPairingwithAcid-AnionggCF3COO-+RCOOHCF3COOH+RCOO-AcidCompetition(TFAmorevolati