1、PropranololWith TFA-FixDigestion of BSA was carried out 37C overnight,using GluC in a 1:20 ratio with BSA(by weight).1mm column or lower,flow rates of 200-400 uL/minParacetamolsource conditions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。50/50 MeOH/H2O 0.Weak Ion Pairing with
2、Acid-Anion导入电压（cone voltage）Flow rate:0.Remove salts which may cause ion suppression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液相色谱与液质联用仪使用的要点 质量校正的正确质量校正的正确 对于相关分析要有合适的支持软件（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液相色谱平台 合适的质谱接口方式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质谱检测模式的选择 必要的后液流补充必要的后液
3、流补充质量校正的正确（质量校正的正确（Myoglobin校正）校正）质量校正的正确（质量校正的正确（Myoglobin校正）校正）质量校正的正确（质量校正的正确（CsI校正）的肽测定校正）的肽测定质量校正的正确（质量校正的正确（CsI校正）的蛋白测定校正）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合适的液相色谱平台 能提供一个连续、稳定的液流环境能提供一个连续、稳定的液流环境 真
4、空脱气设备真空脱气设备 系统的死体积尽可能小，减少管路的长度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辅助配件 必要的液相色谱与质谱仪的软件操作平台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
5、/MS Flow Injection Analysis of Peptides and Proteins by Reversed-Phase HPLC1.0%HOAcminAbundance10.0012.0014.0016.002.004.006.008.00500001000001500002000002500000.2%TFA2.004.006.008.0010.0012.0014.0016.0050000100000150000200000250000minAbundance Reverse-Phase LC/MS Solvents ACN,MeOH,H2O,Isopropanol N
6、ormal-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 m
7、inimize ESI ion suppression Avoid Non-volatile Buffers Alkali-metal phosphates,borates,etc.Suitable Solvents for LC/MS Volatile buffer minimize instrument downtime Buffer concentration:High ion suppression decreases ESI sensitivity Low system adequately buffered?pH range permitted by stationary phas
8、e Methanol or acetonitrile Start with acetonitrile 01111Change Retention to Improve Resolution Select Solvents/Modifiers that are MS CompatibleUseful pH Ranges for Volatile BuffersBuffers normally used in LC/MS:01094BufferpKapH rangeFormate3.82.8 4.8Acetate4.83.8 5.89.28.2 10.2Triethylamine11.010 12
9、 Diethylamine10.59.5 11.5?Ammonia76 8Buffer Concentrations/Additive amounts:10 to 50 mM formic,acetic acids 0.01-1%v/v trifluoroacetic acid 0.1%v/v alkylamine type bases 159.Flow rate:0.Mobile Phase pH effect on ESIColumn:HyPURITY C18 5m,50 x2.1mmAqueous mobile phases:0.1%Formic acidpH 3,Ammonium fo
10、rmate 20 mMpH 5,Ammonium acetate 20 mMpH 8.2,Ammonium acetate 20 mMpH 9,Ammonium acetate 20 mMAqueous/methanol(50:50)Flow rate:0.2 ml/minTemperature:25CDetection:+ESI,450C,4.5kV,20V-ESI,450C,3.5kV,20VScan:120 480uAnalytes:NortriptylinePropranololTetracyclineCaffeineParacetamolTryptophanSalicylic aci
11、dNicotinic acid01113Effect of Mobile Phase pH on+ESI Response01115Variation of peak area with pH in positive ESI0.E+002.E+064.E+066.E+068.E+061.E+071.E+071.E+07NortriptylinePropranololTetracyclineCaffeineTryptophanParacetamolNicotinic acidSalicylic acidCompoundPeak area0.1%formicpH 3pH 5pH 8pH 9 Eff
12、ect of Mobile Phase pH on-ESI Response01116Variation of peak area with pH in negative ESI0.E+005.E+041.E+052.E+052.E+053.E+053.E+054.E+054.E+055.E+055.E+05NortriptylinePropranololTetracyclineCaffeineTryptophanParacetamolNicotinicacidSalicylicacidCompoundArea0.1%formicpH 3pH 5pH 8pH 9 Solvent System5
13、0/50 MeOH/H2O50/50 ACN/H2O100%H2O100%MeOH100%ACN50/50 MeOH/H2O 1%Acetic50/50 MeOH/H2O 0.1%Formic50/50 ACN/H2O 1%Acetic50/50 ACN/H2O 0.1%Formic50/50 MeOH/H2O 5mM NH4OAc50/50 MeOH/H2O 10mM NH4OAc50/50 MeOH/H2O 0.1%TFA50/50 MeOH/H2O 0.05%TFA50/50 MeOH/H2O 0.02%TFA50/50 ACN/H2O 0.1%TFA50/50 ACN/H2O 0.05
14、%TFA50/50 ACN/H2O 0.02%TFA50/50 MeOH/H2O 0.1%NH4OH50/50 ACN/H2O 0.1%NH4OH0100000200000300000400000500000600000Ion Signal,Counts M+H+Solution Chemistry Effects on Positive Ion ESI-MS of Leu-Enkephalin3x106min0102030405060LC/MS Sensitivity vs.Mobile Phase ModifierGluC Digest of BS5%Acetic0.001%TFA0.00
15、5%TFA0.01%TFAZorbax 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.Dige
16、stion 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 concentra
17、tion 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 minF=0.2mL/min,A=5%Acetic Acid,B=ACN MSD1 TIC,MSStable,Sterically Protected C3 Bonded Phase in LC and LC/MS Applications,R.D.Ric
18、ker(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.,1999Proposed Mechanism for TFA Signal Suppressio
19、n and the TFA-Fix-(M+H)+CH3COO-(M+H)+CH3COO-0”Weak Ion Pairing with Acid-Anion g gCF3COO-+RCOOH CF3COOH +RCOO-Acid Competition(TFA more volatile)(M+H)+CF3COO-(M+H)+CF3COO-0”Strong Ion Pairing with TFA-AnionHPLC ConditionsColumn:2.1 x 250 mm Vydac C-18Flow Rate:200 l/minSolvent A:Water+0.1%TFASolvent
20、 B:CH3CN+0.1%TFAGradient:0-60%B in 60 minTemp:50 C 2000006000001000000AbundanceWithout TFA-Fix18.0022.0026.0030.0034.0038.002000006000001000000minAbundanceWith TFA-Fix1:2 post-column addition of 75%propionic acid in IPATryptic Digest Map by ES-LC/MS1 nmol Chicken LysozymeSignal Suppression due to Ad
21、ditives Ion pairing with analyte,surface tension effect Solutions:Post-column addition of a sheath liquid of propionic acid(10%)in 2-propanol(TFA Fix)Use low concentrations of TFA with acetic acid(TFA Light)Replace TFAESI signal suppression by TFA 01122Achievement of gaseous analyte ionization at AP
22、I interface is the key to MS detection离子化方式离子化方式 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Steps for ESI Optimization If analytes pKa is unknown,evaluate 3 pH regions in positive and negative ion modes.Acids Negative Ion detect
23、ion,adjust pH 2 units above pKa Increase pH with NH4OH,TEA,TMA Bases Postive Ion Detection,adjust pH 2 units below pKa 1 Decrease pH use formic acid,acetic acid,TFA Remove salts which may cause ion suppression Adjust source temperature and source voltages to maximize signal In negative ion mode,use
24、lower spray voltage to minimize discharge1 In complex molecules,many exceptions to these rules are observed01564Maximizing High Flow ESI Sensitivity Select appropriate chromatography grade HPLC solvents Avoid exotic solvent mixes(MeOH,MeCN,Water,0.1%formic work best for 98%of LC/MS applications)Avoi
25、d adding excessive modifiers(eg amm.acetate 10mM not 50mM)Choose the right column chemistry(C-8 vs.C-18);change column chemistry before changing solvent mix or composition 2.1mm column or lower,flow rates of 200-400 uL/min Peak widths for quantification not greater than 8-10 secs Dissolve sample in
26、start mobile phase solvent(weakest solvent possible)02383Solvents Compatible With ESIMethanolAcetonitrilePropanolIsopropanolButanol2-methoxy ethanolAceticFormicTFAAmm.AcetateAmm.FormateHFBATEAAmm.HydroxideTetrabutyl amm.hydroxideHexafluorobutanolSamples can be dissolved in any HPLC compatible solven
27、tModifiers Between 0.05-1%and 5-50 mM02382Electrospray Summary Analyte type:*preformed ions(acids and bases)*polar neutrals*multiply charged ions of biopolymers*100 da up to 200 000 da Typical flow rates:low nL-1.0 ml/min Promote ionization:*correct pH*favorable HPLC solvent composition*Post-column
28、addition of reagents Soft ionization technique Typical applications:Drugs,Sugars,Peptides,Proteins,Oligonucleotides01328Steps for APCI Optimization If analytes pKa is unknown,evaluate 3 pH regions in positive and negative ion modes.Acids Negative Ion detection,adjust pH 2 units below pKa Decrease pH
29、 use formic acid,acetic acid,TFA Bases Positive Ion detection,adjust pH 2 units above pKa Increase pH with NH4OH,TEA,TMA Adjust corona discharge voltage Adjust nebulization temperature Consider possible thermal decomposition of analyte01565APCI Typical Operating Conditions Flow Rates:50L/min.-2mL/mi
30、n.Vaporizer Temp(C):400-550(600 max)Discharge Current(A):5(20A max.)Sheath Gas Flow Rate(arb):35-80 Auxiliary Gas:0 Capillary Temp(C):250-350C Tube Lens Offset(V):30-60VHigher flow rate means more solvent for plasma productionPosition of corona discharge needle is critical for sensitivity“Works bett
31、er at higher flow rates”02386APCI-Tips Ensure that the APCI probe is hot enough so that the spray shield is not dripping wet Vaporizer Temp:400-450C is a good start(400-1000uL/min)Reduce temperature of heated capillary if needed Check sheath gas carefully Begin with aux gas at 0 Memory effect due to
32、 compounds burning onto probe parts if injected in large concentrations Bake out source periodically02385APCI Summary Analyte type:*low to mid polarity*high proton affinity*high gas phase acidity*1200 da Typical flow rates:0.2-2.0 ml/min Tolerates buffers better than ESI Produces singly charged ions
33、 only Soft ionization technique Typical applications:Drugs,Pesticides,Steroids,Azo dyes01332Obtaining a Reference Spectrum Unlike the EI process,spectra generated by API LC/MS vary depending on mobile phase source conditions ionization mode in-source CID conditionsSpectral Variations-mobile phasem/z
34、751001251501752002250.02mM NaOAc+0.1%HOAc 195.1 217.0 196.1 138.1m/z7510012515017520022501000020000300004000050000040000800001200000.1%HOAc 195.1 138.1 196.1Spectral Variations-CID conditionsm/z100150200250300010000200003000040000Fragmentor=120 Volts 124.1 186.0 279.0 156.0 108.0 92.1 213.0 204.0 30
35、1.0m/z10015020025030004000080000120000Fragmentor=75 Volts 279.0 186.0Spectral Variations-ion polaritym/z100150200250300API-ES Negative 283.0 284.9 92.0 156.0 128.1 195.9 183.0m/z100150200250300020406080100020406080100API-ES Positive 156.0 285.0 108.0 92.1 286.9质谱参数的控制 电离电压（capillary voltage）导入电压（con
36、e voltage）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。多级质谱提高灵敏度的方法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
37、的灵敏度越高。Sheath Gas Flow Rate(arb):35-80Solvent B:CH3CN+0.Detection:+ESI,450C,4.在样品基质对于电离有明显抑制效应时，考虑采用空白样品提取溶液作为标准系列的基质溶剂。Increase pH with NH4OH,TEA,TMAtrifluoroacetic acid 159.9(Carbendazim)SIR Analysis of 192(Carbendazim)SIR:MRM Comparison for Carbendazim(0.05pg/L std)液相色谱/质谱联用关键点 分清分析的目的是分离定性为主，还是高
38、灵敏度定量为主。定性优先考虑色谱分离的好坏，兼顾考虑质谱要求；定量优先考虑质谱结果的可靠性，而无需考虑色谱行为的科学性。色谱行为服从于质谱行为要求，尤其是在电离要求上。对于以高灵敏度定量为主的分析，样品要求统一于一般液相色谱的残留分析要求。在有些条件下，可采用柱后补液的方法来协调分析物电离条件、液相色谱流动相的分离条件与质谱仪的接口条件等的关系。在样品基质对于电离有明显抑制效应时，考虑采用空白样品提取溶液作为标准系列的基质溶剂。Guidelines for Choosing Ionization MethodYesIs compound polar?Is compound thermally
39、labile?Is compound basic,acidic or neutral?APCI or APPINoYesESINeutralBasicAcidicESI(positive)ESI(negative)APCINo残留分析的一般工作策略 确定目标化合物，研究目标化合物结构特性，预期可能的电离模式。确定目标化合物的检测浓度要求，确定液质联用的可行性。查阅有关技术文献，确定样品处理的技术路线和色谱分离条件、质谱参数条件等 按设计的色谱流动相条件进行目标化合物质谱直接进样分析，获得的scan、daughters图。对所获得的scan、daughters图可能的谱图结构表现、碎片离子的裂解规律进行解释。残留分析的一般工作策略 对目标化合物采用适当的方式进行电量优化，如直接进样、载流恒比进样、载流定量管进样等，得到最优化的质谱参数。研究目标化合物的色谱保留行为，原则上采用50mm柱，保留时间在1-3分钟较为理想。研究目标化合物的样品提取条件，注意样品进样条件必须满足质谱分析要求，尤其是在含有可能抑制电离化合物，如盐、离子对试剂等。组合所有的分离要素，建立分析方法，实施有关质量控制技术规范要求。
