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.