液質(zhì)分析條件的優(yōu)化策略課件

液質(zhì)分析條件的優(yōu)化策略

（簡化板）第一頁，共四十三頁。液相色譜與液質(zhì)聯(lián)用儀使用的要點質(zhì)量校正的正確對于相關(guān)分析要有合適的支持軟件（Maxnet，TargeLyness）合適的液相色譜平臺合適的質(zhì)譜接口方式液相色譜分析中合適的色譜柱的選用質(zhì)譜檢測模式的選擇必要的后液流補充第二頁，共四十三頁。質(zhì)量校正的正確（Myoglobin校正）第三頁，共四十三頁。質(zhì)量校正的正確（Myoglobin校正）第四頁，共四十三頁。質(zhì)量校正的正確（CsI校正）的肽測定第五頁，共四十三頁。質(zhì)量校正的正確（CsI校正）的蛋白測定第六頁，共四十三頁。質(zhì)量校正的關(guān)鍵點針對不同的分析采用不同的校正，一般蛋白質(zhì)采用Myoglobin，對于小分子分析建議采用CsI校正。對于采用Myoglobin校正的建議采用高次方的校正曲線（3-4）；對CsI校正的建議采用低次方的校正曲線（1-2）第七頁，共四十三頁。合適的液相色譜平臺能提供一個連續(xù)、穩(wěn)定的液流環(huán)境真空脫氣設(shè)備系統(tǒng)的死體積盡可能小，減少管路的長度輸液泵的設(shè)計能適用于微徑柱的要求二極管陣列檢測器的池體積與質(zhì)譜儀匹配必要的液相色譜輔助配件必要的液相色譜與質(zhì)譜儀的軟件操作平臺第八頁，共四十三頁。合適的液相色譜柱柱內(nèi)徑：2.1mm柱長度：根據(jù)分析的目的選擇50mm或150mm柱填料選用新型的填料：Symmetry，Discovery，Vydac，Zorbax，Xterra，Intersil，Diamonsil等第九頁，共四十三頁。LC/MSFlowInjectionAnalysisofPeptidesandProteinsbyReversed-PhaseHPLC1.0%HOAcminAbundance10.0012.0014.0016.002.004.006.008.00500001000001500002000002500000.2%TFA2.004.006.008.0010.0012.0014.0016.0050000100000150000200000250000minAbundance第十頁，共四十三頁。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第十一頁，共四十三頁。Volatilebuffer–minimizeinstrumentdowntimeBufferconcentration:HighionsuppressiondecreasesESIsensitivityLowsystemadequatelybuffered?pHrangepermittedbystationaryphaseMethanoloracetonitrileStartwithacetonitrile01111ChangeRetentiontoImproveResolution–

SelectSolvents/ModifiersthatareMSCompatible第十二頁，共四十三頁。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第十三頁，共四十三頁。EffectofBufferonAnalyteResponsePhosphatebufferssuppresstheMSresponseofcaffeineatallpHsandalsotheMSresponseofOxazepamatpH8.Volatilebuffers(formate,acetate,ammonia)generallyprovidegoodresponses.

01121Mobilephase:A-10mMbufferpH6.0;B–methanolGradient:5%to75%in4min第十四頁，共四十三頁。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第十五頁，共四十三頁。EffectofMobilePhasepHon+ESIResponse01115第十六頁，共四十三頁。EffectofMobilePhasepHon-ESIResponse01116第十七頁，共四十三頁。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第十八頁，共四十三頁。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第十九頁，共四十三頁。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第二十頁，共四十三頁。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第二十一頁，共四十三頁。SignalSuppressionduetoAdditivesIonpairingwithanalyte,surfacetensioneffectSolutions:Post-columnadditionofasheathliquidofpropionicacid(10%)in2-propanol(TFAFix)UselowconcentrationsofTFAwithaceticacid(TFALight)ReplaceTFAESIsignalsuppressionbyTFA

01122AchievementofgaseousanalyteionizationatAPIinterfaceisthekeytoMSdetection第二十二頁，共四十三頁。離子化方式極性化合物多采用電噴霧（ESI），其中含氮的化合物一般在酸性條件下用ESI+（生物堿），含多羥基化合物采用中心條件下的ESI-（玉米赤酶醇）。非極性化合物多采用大氣壓化學(xué)電離源（APcI）（激素），原則上不建議采用添加其它化學(xué)試劑第二十三頁，共四十三頁。StepsforESIOptimizationIfanalyte’spKaisunknown,evaluate3pHregionsinpositiveandnegativeionmodes.Acids–NegativeIondetection,adjustpH2unitsabovepKaIncreasepHwithNH4OH,TEA,TMABases–PostiveIonDetection,adjustpH2unitsbelowpKa1

DecreasepHuseformicacid,aceticacid,TFARemovesaltswhichmaycauseionsuppressionAdjustsourcetemperatureandsourcevoltagestomaximizesignalInnegativeionmode,uselowersprayvoltagetominimizedischarge1

Incomplexmolecules,manyexceptionstotheserulesareobserved01564第二十四頁，共四十三頁。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第二十五頁，共四十三頁。SolventsCompatibleWithESIMethanolAcetonitrilePropanolIsopropanolButanol2-methoxyethanolAceticFormicTFAAmm.AcetateAmm.FormateHFBATEAAmm.HydroxideTetrabutylamm.hydroxideHexafluorobutanolSamplescanbedissolvedinanyHPLCcompatiblesolventModifiersBetween0.05-1%and5-50mM02382第二十六頁，共四十三頁。ElectrospraySummaryAnalytetype:preformedions(acidsandbases)polarneutralsmultiplychargedionsofbiopolymers<100daupto200000daTypicalflowrates:lownL-1.0ml/minPromoteionization:correctpHfavorableHPLCsolventcompositionPost-columnadditionofreagentsSoftionizationtechniqueTypicalapplications:Drugs,Sugars,Peptides,Proteins,Oligonucleotides01328第二十七頁，共四十三頁。StepsforAPCIOptimizationIfanalyte’spKaisunknown,evaluate3pHregionsinpositiveandnegativeionmodes.Acids–NegativeIondetection,adjustpH2unitsbelowpKaDecreasepHuseformicacid,aceticacid,TFABases–PositiveIondetection,adjustpH2unitsabovepKaIncreasepHwithNH4OH,TEA,TMAAdjustcoronadischargevoltageAdjustnebulizationtemperatureConsiderpossiblethermaldecompositionofanalyte01565第二十八頁，共四十三頁。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第二十九頁，共四十三頁。APCI-TipsEnsurethattheAPCIprobeishotenoughsothatthesprayshieldisnotdrippingwetVaporizerTemp:400-450Cisagoodstart(400-1000uL/min)ReducetemperatureofheatedcapillaryifneededChecksheathgascarefullyBeginwithauxgasat0MemoryeffectduetocompoundsburningontoprobepartsifinjectedinlargeconcentrationsBakeoutsourceperiodically02385第三十頁，共四十三頁。APCISummaryAnalytetype:lowtomidpolarityhighprotonaffinityhighgasphaseacidity<1200daTypicalflowrates:0.2-2.0ml/minToleratesbuffersbetterthanESIProducessinglychargedionsonlySoftionizationtechniqueTypicalapplications:Drugs,Pesticides,Steroids,Azodyes01332第三十一頁，共四十三頁。ObtainingaReferenceSpectrumUnliketheEIprocess,spectrageneratedbyAPILC/MSvarydependingonmobilephasesourceconditionsionizationmodein-sourceCIDconditions第三十二頁，共四十三頁。SpectralVariations-mobilephasem/z751001251501752002250.02mMNaOAc+0.1%HOAc195.1217.0196.1138.1m/z7510012515017520022501000020000300004000050000040000800001200000.1%HOAc195.1138.1196.1第三十三頁，共四十三頁。SpectralVariations-CIDconditionsm/z100150200250300010000200003000040000Fragmentor=120Volts124.1186.0279.0156.0108.092.1213.0204.0301.0m/z10015020025030004000080000120000Fragmentor=75Volts279.0186.0第三十四頁，共四十三頁。SpectralVariations-ionpolaritym/z100150200250300API-ESNegative283.0284.992.0156.0128.1195.9183.0m/z100150200250300020406080100020406080100API-ESPositive156.0285.0108.092.1286.9第三十五頁，共四十三頁。質(zhì)譜參數(shù)的控制電離電壓（capillaryvoltage）導(dǎo)入電壓（conevoltage）Probe位置霧化氣流與干燥氣流聚焦棱鏡電壓（Len）根據(jù)需求條件一級、三級的分辨率，原則上scan、daughter條件下，選用單位質(zhì)量分辨條件，在mrm條件下，選用2-3單位質(zhì)量分辨率。合適的碰撞能量和碰撞室的聚焦電壓。合適穩(wěn)定的碰撞室氣壓。檢測器的倍增電壓，有選擇性的調(diào)節(jié)。第三十六頁，共四十三頁。多級質(zhì)譜提高靈敏度的方法通過合適的電離方式進行，尤其是對于能否將[M+Na]+改變?yōu)閇M+H]+通過合適的方法提高電離效率，如后補液來調(diào)整流動相的PH值和有機溶劑比例關(guān)系等。研究scan、daughter譜圖，尋找特征具有結(jié)構(gòu)特征的碎片離子，一般而言碎片越穩(wěn)定、豐度越大而得到的靈敏度越高。第三十七頁，共四十三頁。MRMAnalysisof192>159.9(Carbendazim)SIRAnalysisof192(Carbendazim)SIR:MRMComparisonfor