液质联用中质谱条件的优化策略课件

路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索07 七月 2024液质联用中质谱条件的液质联用中质谱条件的优化策略优化策略PPT42页页路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索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流补充路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索质量校正的正确（质量校正的正确（Myoglobin校正）校正）路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索质量校正的正确（质量校正的正确（Myoglobin校正）校正）路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索质量校正的正确（质量校正的正确（CsI校正）的肽测定校正）的肽测定路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索质量校正的正确（质量校正的正确（CsI校正）的蛋白测定校正）的蛋白测定路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索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）路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索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路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索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等路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索LC/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路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Reverse-Phase LC/MS SolventsACN,MeOH,H2O,IsopropanolNormal-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 acidTFA concentration should be 0.1%v/vKeep volatile buffer concentrations 20 mM to minimize ESI ion suppressionAvoid Non-volatile BuffersAlkali-metal phosphates,borates,etc.Suitable Solvents for LC/MS路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Volatile buffer minimize instrument downtimeBuffer concentration:High ion suppression decreases ESI sensitivity Low system adequately buffered?pH range permitted by stationary phaseMethanol or acetonitrileStart with acetonitrile 01111Change Retention to Improve Resolution Select Solvents/Modifiers that are MS Compatible路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Useful 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 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 0.1%v/v路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Effect of Buffer on Analyte ResponsePhosphate 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.01121Mobile phase:A-10mM buffer pH 6.0;B methanolGradient:5%to 75%in 4 min路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Mobile Phase pH effect on ESIColumn:HyPURITY C18 5m,50 x2.1mmAqueous mobile phases:0.1%Formic acidpH 3,Ammonium formate 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 acidNicotinic acid01113路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Effect of Mobile Phase pH on+ESI Response01115路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Effect of Mobile Phase pH on-ESI Response01116路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Solvent System50/50 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%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-Enkephalin路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索LC/MS Sensitivity vs.Mobile Phase ModifierGluC Digest of BS5%Acetic0.001%TFA0.005%TFA0.01%TFAZorbax 300SB-C3(2.1 x 150 mm)HP1100 MSDReversed-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 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.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路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Proposed Mechanism for TFA Signal Suppression 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-Anion路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索HPLC ConditionsColumn:2.1 x 250 mm Vydac C-18Flow Rate:200 l/minSolvent A:Water+0.1%TFASolvent 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 Lysozyme路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Signal Suppression due to AdditivesIon pairing with analyte,surface tension effectSolutions: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 API interface is the key to MS detection路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索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路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Steps for ESI OptimizationIf analytes pKa is unknown,evaluate 3 pH regions in positive and negative ion modes.Acids Negative Ion detection,adjust pH 2 units above pKaIncrease pH with NH4OH,TEA,TMABases Postive Ion Detection,adjust pH 2 units below pKa 1 Decrease pH use formic acid,acetic acid,TFARemove salts which may cause ion suppressionAdjust source temperature and source voltages to maximize signalIn negative ion mode,use lower spray voltage to minimize discharge1 In complex molecules,many exceptions to these rules are observed01564路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Maximizing High Flow ESI SensitivitySelect appropriate chromatography grade HPLC solvents Avoid exotic solvent mixes(MeOH,MeCN,Water,0.1%formic work best for 98%of LC/MS applications)Avoid 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 composition2.1mm column or lower,flow rates of 200-400 uL/minPeak widths for quantification not greater than 8-10 secsDissolve sample in start mobile phase solvent(weakest solvent possible)02383路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Solvents Compatible With ESIMethanolAcetonitrilePropanolIsopropanolButanol2-methoxy ethanolAceticFormicTFAAmm.AcetateAmm.FormateHFBATEAAmm.HydroxideTetrabutyl amm.hydroxideHexafluorobutanolSamples can be dissolved in any HPLC compatible solventModifiers Between 0.05-1%and 5-50 mM02382路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Electrospray SummaryAnalyte type:preformed ions(acids and bases)polar neutralsmultiply charged ions of biopolymers100 da up to 200 000 daTypical flow rates:low nL-1.0 ml/minPromote ionization:correct pHfavorable HPLC solvent compositionPost-column addition of reagentsSoft ionization techniqueTypical applications:Drugs,Sugars,Peptides,Proteins,Oligonucleotides01328路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Steps for APCI OptimizationIf 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 use formic acid,acetic acid,TFABases Positive Ion detection,adjust pH 2 units above pKaIncrease pH with NH4OH,TEA,TMAAdjust corona discharge voltageAdjust nebulization temperatureConsider possible thermal decomposition of analyte01565路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索APCI Typical Operating ConditionsFlow Rates:50L/min.-2mL/min.Vaporizer Temp(C):400-550(600 max)Discharge Current(A):5(20A max.)Sheath Gas Flow Rate(arb):35-80Auxiliary Gas:0Capillary Temp(C):250-350CTube Lens Offset(V):30-60VHigher flow rate means more solvent for plasma productionPosition of corona discharge needle is critical for sensitivity“Works better at higher flow rates”02386路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索APCI-TipsEnsure that the APCI probe is hot enough so that the spray shield is not dripping wetVaporizer Temp:400-450C is a good start(400-1000uL/min)Reduce temperature of heated capillary if neededCheck sheath gas carefullyBegin with aux gas at 0Memory effect due to compounds burning onto probe parts if injected in large concentrationsBake out source periodically02385路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索APCI SummaryAnalyte type:low to mid polarityhigh proton affinity high gas phase acidity159.9(Carbendazim)SIR Analysis of 192(Carbendazim)SIR:MRM Comparison for Carbendazim(0.05pg/m mL std)路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索液相色谱/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。路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Guidelines for Choosing Ionization MethodYesIs compound polar?Is compound thermally labile?Is compound basic,acidic or neutral?APCI or APPINoYesESINeutralBasicAcidicESI(positive)ESI(negative)APCINo