质谱条件的优化策略(简化板)

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
Intensity (peak area)
Mobile phase: A - 10mM buffer pH 6.0; B – methanol
3.E+08
Buffer type
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.
01111
Change Retention to Improve Resolution – Select Solvents/ Modifiers that are MS Compatible • 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
Peak area
8.E +06
Compound
01116
Effect of Mobile Phase pH on -ESI Response
Variation of peak area with pH in negative ESI
5.E+05 5.E+05 4.E+05 4.E+05 3.E+05 3.E+05 2.E+05 2.E+05 1.E+05 5.E+04 0.E+00 Nortriptyline Tetracycline Tryptophan Paracetamol Propranolol Nicotinic acid Caffeine Salicylic acid
01113
Mobile Phase pH effect on ESI
Column : HyPURITY™ C18 5µm, 50x2.1mm Analytes: Aqueous mobile phases: Nortriptyline 0.1% Formic acid pH 3, Ammonium formate 20 mM Propranolol pH 5, Ammonium acetate 20 mM Tetracycline pH 8.2, Ammonium acetate 20 mM Caffeine pH 9, Ammonium acetate 20 mM Paracetamol Aqueous/methanol (50:50) Tryptophan Flow rate: 0.2 ml/min Salicylic Temperature: 25°C acid Detection: +ESI, 450°C, 4.5kV, 20V -ESI, 450°C, 3.5kV, 20V Nicotinic acid Scan: 120 – 480u
质量校正的正确（ 校正） 质量校正的正确（Myoglobin校正） 校正
质量校正的正确（ 校正） 质量校正的正确（Myoglobin校正） 校正
质量校正的正确（ 校正 校正） 质量校正的正确（CsI校正）的肽测定
质量校正的正确（ 校正 校正） 质量校正的正确（CsI校正）的蛋白测定
质量校正的关键点
250000 Abundance 200000 150000 100000 50000 min
1.0% HOAc
Байду номын сангаас
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
250000 Abundance 200000 150000 100000 50000
0.2% TFA
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
min
Suitable Solvents for LC/MS
• Reverse-Phase LC/MS Solvents – ACN, MeOH, H2O, 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.
01094
Useful pH Ranges for Volatile Buffers
Buffers normally used in LC/MS :
Buffer
Formate Acetate ??? Ammonia Diethylamine Triethylamine
pKa
3.8 4.8 7 9.2 10.5 11.0
01115
Effect of Mobile Phase pH on +ESI Response
Variation of pe ak ar ea with pH in positive ESI
1.E +07 1.E +07 1.E +07 0.1%f ormic pH 3 pH 5 6.E +06 4.E +06 2.E +06 0.E +00 Tetracycline Tryptophan Nicotinic acid Paracetamol Nortriptyline Propranolol Salic ylic acid Caffeine pH 8 pH 9
pH range
2.8 – 4.8 3.8 – 5.8 6–8 8.2 – 10.2 9.5 – 11.5 10 – 12
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
0.1%formic pH 3 pH 5 pH 8 pH 9
Area
Compound
Solution Chemistry Effects on Positive Ion ESI-MS of Leu-Enkephalin
50/50 ACN/H2O 0.1% NH4OH 50/50 MeOH/H2O 0.1% NH4OH 50/50 ACN/H2O 0.02% TFA 50/50 ACN/H2O 0.05% TFA 50/50 ACN/H2O 0.1% TFA 50/50 MeOH/H2O 0.02% TFA 50/50 MeOH/H2O 0.05% TFA 50/50 MeOH/H2O 0.1% TFA 50/50 MeOH/H2O 10mM NH4OAc 50/50 MeOH/H2O 5mM NH4OAc 50/50 ACN/H2O 0.1% Formic 50/50 ACN/H2O 1% Acetic 50/50 MeOH/H2O 0.1% Formic 50/50 MeOH/H2O 1% Acetic 100% ACN 100% MeOH 100% H2O 50/50 ACN/H2O 50/50 MeOH/H2O
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 37°C 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.
01121
Effect of Buffer on Analyte Response
3.E+08 2.E+08 Procainamide Caffeine Propranolol Nortriptyline Oxazepam
Gradient: 5% to 75% in 4 min
2.E+08 1.E+08 5.E+07 0.E+00 Formate pH 2.5 Acetate pH 6.0 Ammonia pH 8.0 Phosphate pH 2.5 Phosphate pH 6.0 Phosphate pH 8.0
• 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、 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， 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
0 100000 200000 300000 400000 500000 600000
Solvent System
Ion Signal, Counts [M+H]+
LC/MS Sensitivity vs. Mobile Phase Modifier GluC Digest of BS
MSD1 TIC, MS
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， 对于相关分析要有合适的支持软件（ ， 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