液质条件的优化策略课件

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,2021/2/8,#,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/MS Flow Injection Analysis of Peptides and Proteins by Reversed-Phase HPLC,1.0% HOAc,min,Abundance,10.00,12.00,14.00,16.00,2.00,4.00,6.00,8.00,50000,100000,150000,200000,250000,0.2% TFA,2.00,4.00,6.00,8.00,10.00,12.00,14.00,16.00,50000,100000,150000,200000,250000,min,Abundance,Reverse-PhaseLC/MSSolvents,ACN,MeOH,H,2,O,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,Startwithacetonitrile,01111,ChangeRetentiontoImproveResolution,SelectSolvents/ModifiersthatareMSCompatible,UsefulpHRangesforVolatileBuffers,BuffersnormallyusedinLC/MS:,01094,Buffer,pK,a,pH range,Formate,3.8,2.8 4.8,Acetate,4.8,3.8 5.8,9.2,8.2 10.2,Triethylamine,11.0,10 12,Diethylamine,10.5,9.5,11.5,?,Ammonia,7,6,8,BufferConcentrations/Additiveamounts:,10to50mM,formic,aceticacids0.01-1%v/v,trifluoroaceticacid0.1%v/v,alkylaminetypebases0.1%v/v,EffectofBufferonAnalyteResponse,Phosphate buffers suppress the MS response of caffeine at all pHs and also the MS response of Oxazepam at pH8.Volatilebuffers(formate, acetate,ammonia)generally provide goodresponses.,01121,Mobile phase:,A -10mMbufferpH 6.0;,B methanol,Gradient: 5%to75%in 4min,Mobile PhasepHeffect on ESI,Column :HyPURITY,C185,m,50x2.1mm,Aqueousmobile phases:,0.1%Formicacid,pH 3, Ammonium formate 20 mM,pH 5, Ammonium acetate 20 mM,pH 8.2,Ammoniumacetate20mM,pH 9, Ammonium acetate 20 mM,Aqueous/methanol(50:50),Flowrate: 0.2 ml/min,Temperature:25C,Detection:+ESI,450,C,4.5kV, 20V,-ESI, 450C,3.5kV,20V,Scan: 120 480u,Analytes:,Nortriptyline,Propranolol,Tetracycline,Caffeine,Paracetamol,Tryptophan,Salicylic acid,Nicotinic acid,01113,Effect of MobilePhase pH on+ESI Response,01115,Effect of MobilePhase pH on-ESI Response,01116,Solvent System,50/50 MeOH/H2O,50/50 ACN/H2O,100% H2O,100% MeOH,100% ACN,50/50 MeOH/H2O 1% Acetic,50/50 MeOH/H2O 0.1% Formic,50/50 ACN/H2O 1% Acetic,50/50 ACN/H2O 0.1% Formic,50/50 MeOH/H2O 5mM NH4OAc,50/50 MeOH/H2O 10mM NH4OAc,50/50 MeOH/H2O 0.1% TFA,50/50 MeOH/H2O 0.05% TFA,50/50 MeOH/H2O 0.02% TFA,50/50 ACN/H2O 0.1% TFA,50/50 ACN/H2O 0.05% TFA,50/50 ACN/H2O 0.02% TFA,50/50 MeOH/H2O 0.1% NH4OH,50/50 ACN/H2O 0.1% NH4OH,0,100000,200000,300000,400000,500000,600000,Ion Signal, Counts M+H,+,SolutionChemistry Effects on Positive Ion ESI-MS ofLeu-Enkephalin,LC/MS Sensitivity vs. MobilePhase ModifierGluCDigestof BS,5% Acetic,0.001% TFA,0.005% TFA,0.01% TFA,Zorbax 300SB-C3,(,2.1x 150,mm,),HP1100 MSD,Reversed-phase HPLC/MS analysisof aGluC digestofBSAwasusedasa modelto testtherecoveryandpeakshape of peptides usingvaryingconcentrationsof TFA or 5%aceticacidasa mobile-phase additivein combination withtheZorbax 300SB-C3.,Digestion ofBSAwascarriedout37C overnight,using GluCin a1:20 ratiowithBSA(byweight). The finalmixturecontained 1Murea and 25mM sodium phosphate.,A significant increase in sensitivity ofpeptides was observed for mostpeptidesanalyzed using5% acetic acid rather than TFA.,ReducingTFAconcentration to 0.001%causedonlya minorimprovementinsensitivity.Some peptides weremuchless affected by additive change than others.,0 for 5min,0-40% B/55 minthen40-100% B/20 min,F=0.2mL/ min,A= 5%AceticAcid, B= ACN,MSD1TIC, MS,Stable,Sterically ProtectedC3Bonded PhaseinLC and LC/MSApplications, R.D.Ricker (1),B.E.Boyes (1),J.P. Nawrocki (2),andL.K.Pannell(2)(1)AgilentTechnologies, Inc. LC Applicatons Lab 538 FirstState Blvd,Newport,DE19804-3552 USA.(2)Structural MassSpectrometryGroup NIDDK, NIH, Bethesda,MD,20892 USA.EasternAnalytical Symposium, Nov.,1999,ProposedMechanism for TFA Signal Suppression and the TFA-Fix,-,(M+H),+,+ CH,3,COO,-, (M+H),+,CH,3,COO,-, 0”,Weak Ion Pairing with Acid-Anion,g g,CF,3,COO,-,+ RCOOH,CF,3,COOH,+ RCOO,-,Acid Competition (TFA more volatile),(M+H),+,+ CF,3,COO,-, (M+H),+,CF,3,COO,-, 0”,Strong Ion Pairing with TFA-Anion,HPLC Conditions,Column: 2.1 x 250 mm Vydac C-18,Flow Rate: 200 l/min,Solvent A: Water + 0.1% TFA,Solvent B: CH,3,CN + 0.1% TFA,Gradient: 0-60% B in 60 min,Temp: 50 C,200000,600000,1000000,Abundance,Without TFA-Fix,18.00,22.00,26.00,30.00,34.00,38.00,200000,600000,1000000,min,Abundance,With TFA-Fix,1:2post-columnadditionof75%propionic acid in IPA,TrypticDigest Map by ES-LC/MS1nmol Chicken Lysozyme,Signal Suppression due to Additives,Ionpairingwithanalyte, surface tension effect,Solutions:,Post-columnadditionofa sheathliquidof propionicacid (10%)in 2-propanol (TFA Fix),Uselowconcentrations of TFA with acetic acid (TFALight),ReplaceTFA,ESIsignal suppression by TFA,01122,Achievementof gaseous analyte ionization atAPIinterface is the key toMSdetection,离子化方,式,式,极性化合,物,物多采用,电,电喷雾（ESI）,，,，其中含,氮,氮的化合,物,物一般在,酸,酸性条件,下,下用ESI,+,（生物碱,）,），含多,羟,羟基化合,物,物采用中,心,心条件下,的,的ESI,-,（玉米赤,酶,酶醇）。,非极性化,合,合物多采,用,用大气压,化,化学电离,源,源（APcI）（,激,激素），,原,原则上不,建,建议采用,添,添加其它,化,化学试剂,Steps for ESI Optimization,If analytespK,a,is unknown,evaluate3 pH regions inpositive and negative ion modes.,Acids NegativeIondetection,adjust pH 2units abovepK,a,IncreasepHwithNH,4,OH,TEA,TMA,Bases PostiveIonDetection,adjust pH 2units belowpK,a,1,DecreasepHuseformic acid, aceticacid, TFA,Remove saltswhich may causeionsuppression,Adjust source temperature and sourcevoltages tomaximize signal,In negativeionmode, use lowerspray voltage tominimize discharge,1,In complex molecules, many exceptions tothese rulesareobserved,01564,Maximizing HighFlowESISensitivity,Select appropriate chromatography gradeHPLCsolvents,Avoid exoticsolventmixes (MeOH, MeCN,Water, 0.1%formic workbestfor98%ofLC/MS applications),Avoid addingexcessive modifiers(egamm.acetate 10mMnot50mM),Choose the rightcolumnchemistry (C-8 vs. C-18); changecolumnchemistry beforechanging solvent mix orcomposition,2.1mm columnorlower, flowrates of200-400uL/min,Peakwidthsforquantification not greater than8-10secs,Dissolvesamplein startmobilephase solvent (weakest solvent possible),02383,SolventsCompatibleWithESI,Methanol,Acetonitrile,Propanol,Isopropanol,Butanol,2-methoxy ethanol,Acetic,Formic,TFA,Amm.Acetate,Amm.Formate,HFBA,TEA,Amm.Hydroxide,Tetrabutyl amm.hydroxide,Hexafluorobutanol,Samplescanbe dissolvedinanyHPLCcompatiblesolvent,Modifiers Between 0.05 -1%and5 -50 mM,02382,ElectrospraySummary,Analytetype:,preformed ions (acids and bases),polar neutrals,multiplychargedions ofbiopolymers,100daup to 200 000 da,Typicalflowrates:lownL -1.0ml/min,Promoteionization:,correctpH,favorable HPLC solvent composition,Post-columnadditionofreagents,Softionizationtechnique,Typicalapplications: Drugs, Sugars, Peptides,Proteins, Oligonucleotides,01328,Steps for APCI Optimization,If analytespK,a,is unknown,evaluate3 pH regions inpositive and negativeionmodes.,Acids NegativeIondetection,adjust pH 2units belowpK,a,DecreasepHuseformic acid, aceticacid, TFA,Bases PositiveIondetection,adjust pH 2units abovepK,a,IncreasepHwithNH4OH,TEA,TMA,Adjust corona dischargevoltage,Adjust nebulizationtemperature,Considerpossible thermal decompositionof analyte,01565,APCI, Typical OperatingConditions,FlowRates:50,L/min. -2mL/min.,Vaporizer Temp (,C):400-550 (600 max),Discharge Current (,A):5 (20,A max.),Sheath Gas FlowRate(arb):35-80,Auxiliary Gas:0,Capillary Temp (,C):250-350C,TubeLens Offset(V):30-60V,Higher flowratemeans moresolventforplasma production,Positionofcorona dischargeneedleis criticalforsensitivity,“ Worksbetter athigher flowrates”,02386,APCI-Tips,Ensure thattheAPCIprobe is hot enoughsothatthespray shield isnotdripping wet,Vaporizer Temp:400-450Cisa good start(400-1000uL/min),Reduce temperature of heatedcapillary if needed,Check sheathgascarefully,Begin with aux gas at 0,Memory effect due tocompounds burning ontoprobe partsif injectedin largeconcentrations,Bakeoutsourceperiodically,02385,APCISummary,Analytetype:,lowto mid polarity,highprotonaffinity,highgasphase acidity,159.9,(Carbendazim),SIR Analysis of192,(Carbendazim),SIR :MRMComparisonforCarbendazim,(0.05pg/,m,L std),液相色谱/,质,质谱联用关,键,键点,分清分析的,目,目的是分离,定,定性为主，,还,还是高灵敏,度,度定量为主,。,。定性优先,考,考虑色谱分,离,离的好坏，,兼,兼顾考虑质,谱,谱要求；定,量,量优先考虑,质,质谱结果的,可,可靠性，而,无,无需考虑色,谱,谱行为的科,学,学性。,色谱行为服,从,从于质谱行,为,为要求，尤,其,其是在电离,要,要求上。,对于以高灵,敏,敏度定量为,主,主的分析，,样,样品要求统,一,一于一般液,相,相色谱的残,留,留分析要求,。,。,在有些条件,下,下，可采用,柱,柱后补液的,方,方法来协调,分,分析物电离,条,条件、液相,色,色谱流动相,的,的分离条件,与,与质谱仪的,接,接口条件等,的,的关系。,在样品基质,对,对于电离有,明,明显抑制效,应,应时，考虑,采,采用空白样,品,品提取溶液,作,作为标准系,列,列的基质溶,剂,剂。,GuidelinesforChoosing IonizationMethod,Yes,Is compound polar?,Is compound thermally labile?,Is compound basic, acidicor neutral?,APCIor APPI,No,Yes,ESI,Neutral,Basic,Acidic,ESI (positive),ESI (negative),APCI,No,残留分析的,一,一般工作策,略,略,确定目标化,合,合物，研究,目,目标化合物,结,结构特性，,预,预期可能的,电,电离模式。,确定目标化,合,合物的检测,浓,浓度要求，,确,确定液质联,用,用的可行性,。,。,查阅有关技,术,术文献，确,定,定样品处理,的,的技术路线,和,和色谱分离,条,条件、质谱,参,参数条件等,按设计的色,谱,谱流动相条,件,件进行目标,化,化合物质谱,直,直接进样分,析,析，获得的scan、daughters图,。,。,对所获得的scan、daughters图,可,可能的谱图,结,结构表现、,碎,碎片离子的,裂,裂解规律进,行,行解释。,残留分析的,一,一般工作策,略,略,对目标化合,物,物采用适当,的,的方式进行,电,电量优化，,如,如直接进样,、,、载流恒比,进,进样、载流,定,定量管进样,等,等，得到最,优,优化的质谱,参,参数。,研究目标化,合,合物的色谱,保,保留行为，,原,原则上采用50mm柱,，,，保留时间,在,在1-3分,钟,钟较为理想,。,。,研究目标化,合,合物的样品,提,提取条件，,注,注意样品进,样,样条件必须,满,满足质谱分,析,析要求，尤,其,其是在含有,可,可能抑制电,离,离化合物，,如,如盐、离子,对,对试剂等。,组合所有的,分,分离要素，,建,建立分析方,法,法，实施有,关,关质量控制,技,技术规范要,求,求。,