液质联用中质谱条件优化策略

1、液质联用中质谱条件优化策略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

2、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 Reverse

3、d-Phase HPLC1.0% HOAcminAbundance10.0012.0014.0016.002.004.006.008.00500001000001500002000002500000.2% TFA2.004.006.008.0010.0012.0014.0016.0050000100000150000200000250000minAbundanceReverse-Phase LC/MS SolventsACN, MeOH, H2O, IsopropanolNormal-Phase LC/MS Solvents (for APCI-MS)Hexane, Methylene Chl

4、oride, 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 Buffer

5、sAlkali-metal phosphates, borates, etc.Suitable Solvents for LC/MSVolatile 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 0111

6、1Change 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 Diethylamine10.59.5 11.5?Ammonia76 8Buffer Concentra

7、tions/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/vEffect 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 (for

8、mate, acetate, ammonia) generally provide good responses. 01121Mobile phase: A - 10mM buffer pH 6.0; B methanolGradient: 5% to 75% in 4 minMobile 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

9、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 acid01113Effect of

10、 Mobile Phase pH on +ESI Response01115Effect of Mobile Phase pH on -ESI Response01116Solvent 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 NH4OAc

11、50/50 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

12、 Leu-EnkephalinLC/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 concentrati

13、ons 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 pep

14、tides 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

15、, 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

16、 Mass Spectrometry Group NIDDK, NIH, Bethesda, MD, 20892 USA. Eastern Analytical Symposium, Nov., 1999Proposed 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

17、)(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 B: CH3CN + 0.1% TFAGradient: 0-60% B in 60 minTemp: 50 C 2000006000001000000AbundanceWithout TFA-Fix18.0022.0026.0030.0034.0038.0020

18、00006000001000000minAbundanceWith TFA-Fix1:2 post-column addition of 75% propionic acid in IPATryptic Digest Map by ES-LC/MS1 nmol Chicken LysozymeSignal Suppression due to AdditivesIon pairing with analyte, surface tension effectSolutions:Post-column addition of a sheath liquid of propionic acid (1

19、0%) 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

20、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 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 Dec

21、rease 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 observed01564Maximizing

22、 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 c

23、olumn 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)02383Solvents Compatible With ESIMethanolAcetonitrilePropanolI

24、sopropanolButanol2-methoxy ethanolAceticFormicTFAAmm. AcetateAmm. FormateHFBATEAAmm. HydroxideTetrabutyl amm. hydroxideHexafluorobutanolSamples can be dissolved in any HPLC compatible solventModifiers Between 0.05 - 1% and 5 - 50 mM02382Electrospray SummaryAnalyte type:preformed ions (acids and base

25、s)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, Oligonuc

26、leotides01328Steps 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 wi

27、th NH4OH, TEA, TMAAdjust corona discharge voltageAdjust nebulization temperatureConsider possible thermal decomposition of analyte01565APCI 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):35-8

28、0Auxiliary 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”02386APCI-TipsEnsure that the APCI probe is hot enough so that the spray s

29、hield 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 periodically02385APCI SummaryAnalyte type:low to mid polarityhigh pro