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,*,液质分析条件的优化策略（简化板）,1,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,2,质量校正的正确（Myoglobin校正）,3,质量校正的正确（Myoglobin校正）,4,质量校正的正确（CsI校正）的肽测定,5,质量校正的正确（CsI校正）的蛋白测定,6,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）,7,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,8,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,9,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,10,Reverse-Phase LC/MS Solvents,ACN,MeOH,H,2,O,Isopropanol,Normal-Phase LC/MS Solvents(for APCI-MS),Hexane,Methylene Chloride,Acetone,Ethanol,Compatible LC/MS Buffers and Modifiers:,Formic acid,acetic acid,ammonium acetate,ammonium formate,ammonium hydroxide,trifluoroacetic acid,TFA concentration should be 0.1%v/v,Keep volatile buffer concentrations 20 mM to minimize ESI ion suppression,Avoid Non-volatile Buffers,Alkali-metal phosphates,borates,etc.,Suitable Solvents for LC/MS,11,Volatile buffer minimize instrument downtime,Buffer concentration:,High,ion suppression decreases ESI sensitivity,Low system adequately buffered?,pH range permitted by stationary phase,Methanol or acetonitrile,Start with acetonitrile,01111,Change Retention to Improve Resolution Select Solvents/Modifiers that are MS Compatible,12,Useful pH Ranges for Volatile Buffers,Buffers normally used in LC/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,Buffer Concentrations/Additive amounts:,10 to 50 mM,formic,acetic acids 0.01-1%v/v,trifluoroacetic acid 0.1%v/v,alkylamine type bases 0.1%v/v,13,Effect of Buffer on Analyte Response,Phosphate buffers suppress the MS response of caffeine at all pHs and also the MS response of Oxazepam at pH 8.Volatile buffers(formate,acetate,ammonia)generally provide good responses.,01121,Mobile phase:,A-10mM buffer pH 6.0;,B methanol,Gradient:5%to 75%in 4 min,14,Mobile Phase pH effect on ESI,Column:HyPURITY,C18 5,m,50 x2.1mm,Aqueous mobile phases:,0.1%Formic acid,pH 3,Ammonium formate 20 mM,pH 5,Ammonium acetate 20 mM,pH 8.2,Ammonium acetate 20 mM,pH 9,Ammonium acetate 20 mM,Aqueous/methanol(50:50),Flow rate:0.2 ml/min,Temperature:25C,Detection:+ESI,450C,4.5kV,20V,-ESI,450C,3.5kV,20V,Scan:120 480u,Analytes:,Nortriptyline,Propranolol,Tetracycline,Caffeine,Paracetamol,Tryptophan,Salicylic acid,Nicotinic acid,01113,15,Effect of Mobile Phase pH on+ESI Response,01115,16,Effect of Mobile Phase pH on-ESI Response,01116,17,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,+,Solution Chemistry Effects on Positive Ion ESI-MS of Leu-Enkephalin,18,LC/MS Sensitivity vs.Mobile Phase Modifier,GluC Digest of BS,5%Acetic,0.001%TFA,0.005%TFA,0.01%TFA,Zorbax 300SB-C3,(,2.1 x 150,mm,),HP1100 MSD,Reversed-phase HPLC/MS analysis of a,GluC,digest of BSA was used as a model to test the recovery and,peakshape,of peptides using varying concentrations of TFA or 5%acetic acid as a mobile-phase additive in combination with the,Zorbax,300SB-C3.,Digestion of BSA was carried out 37C overnight,using,GluC,in a 1:20 ratio with BSA(by weight).The final mixture contained 1M urea and 25mM sodium phosphate.,A significant increase in sensitivity of peptides was observed for most peptides analyzed using 5%acetic acid rather than TFA.,Reducing TFA concentration to 0.001%caused only a minor improvement in sensitivity.Some peptides were much less affected by additive change than others.,0 for 5min,0-40%B/55 min then 40-100%B/20 min,F=0.2mL/min,A=5%Acetic Acid,B=ACN,MSD1 TIC,MS,Stable,Sterically Protected C3 Bonded Phase in LC and LC/MS Applications,R.D.Ricker(1),B.E.Boyes(1),J.P.Nawrocki(2),and L.K.Pannell(2)(1)Agilent Technologies,Inc.LC Applicatons Lab 538 First State Blvd,Newport,DE 19804-3552 USA.(2)Structural Mass Spectrometry Group NIDDK,NIH,Bethesda,MD,20892 USA.Eastern Analytical Symposium,Nov.,1999,19,Proposed Mechanism 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),+