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采用柱后抑制器技术降低流动相中三氟乙酸对质谱信号抑制作用的研究

张婷婷 刘绿叶 张艳海 金燕

张婷婷, 刘绿叶, 张艳海, 金燕. 采用柱后抑制器技术降低流动相中三氟乙酸对质谱信号抑制作用的研究[J]. 分析测试技术与仪器, 2017, 23(1): 18-23. doi: 10.16495/j.1006-3757.2017.01.004
引用本文: 张婷婷, 刘绿叶, 张艳海, 金燕. 采用柱后抑制器技术降低流动相中三氟乙酸对质谱信号抑制作用的研究[J]. 分析测试技术与仪器, 2017, 23(1): 18-23. doi: 10.16495/j.1006-3757.2017.01.004
ZHANG Ting-ting, LIU Lv-ye, ZHANG Yan-hai, JIN Yan. Enhancement of Sensitivity for Bioanalysis by Liquid Chromatography-Electrospray Mass Spectrometry with Trifluoroacetic Acid in Mobile Phase Using a Suppressor[J]. Analysis and Testing Technology and Instruments, 2017, 23(1): 18-23. doi: 10.16495/j.1006-3757.2017.01.004
Citation: ZHANG Ting-ting, LIU Lv-ye, ZHANG Yan-hai, JIN Yan. Enhancement of Sensitivity for Bioanalysis by Liquid Chromatography-Electrospray Mass Spectrometry with Trifluoroacetic Acid in Mobile Phase Using a Suppressor[J]. Analysis and Testing Technology and Instruments, 2017, 23(1): 18-23. doi: 10.16495/j.1006-3757.2017.01.004

采用柱后抑制器技术降低流动相中三氟乙酸对质谱信号抑制作用的研究

doi: 10.16495/j.1006-3757.2017.01.004
详细信息
    作者简介:

    张婷婷 (1982-), 硕士, 主要研究方向为高效液相色谱系统在生物制药领域的应用, Tel:13818636877, E-mail:tingting.zhang@thermofisher.com

  • 中图分类号: O657.3

Enhancement of Sensitivity for Bioanalysis by Liquid Chromatography-Electrospray Mass Spectrometry with Trifluoroacetic Acid in Mobile Phase Using a Suppressor

  • 摘要: 研究柱后抑制器(CSRS)技术有效降低流动相中三氟乙酸(TFA)对质谱信号的抑制.采用双三元液相系统,左泵以反相模式分离细胞色素C酶解肽段(流动相含0.1 %TFA,流速0.25 mL/min),柱后选择CSRS作为抑制器.同时右泵提供碳酸氢铵(浓度为0.05 mol/L,流速1.00 mL/min)作为再生液.碳酸氢铵和三氟乙酸在抑制器CSRS中通过阴离子交换膜进行离子交换,降低流动相中TFA离子抑制效应,提高肽段在质谱上的响应(S/N提高1~16倍).采用柱后抑制器技术对硫酸依替米星主成分及杂质进行定性分析(0.2 mol/L TFA),流动相经过抑制器后由强酸性变成中性,实现样品在LC和MS之间无缝连接分析.
  • 图  1  柱后抑制器CSRS的抑制原理图

    Figure  1.  Schematic diagram of CSRS

    图  2  柱后抑制器技术的HPLC-MS/MS系统结构图

    Figure  2.  Configuration of HPLC-MS/MS with CSRS

    图  3  柱后抑制器CSRS中再生液流速对多肽的MS灵敏度影响

    Figure  3.  Effect of flow rate of NH4HCO3 on MS/MS sensitivity

    图  4  硫酸依替米星直接进TSQ/经过CSRS抑制后进TSQ谱图

    Figure  4.  Mass chromatograms of etimicin sulfate with and without CSRS

    (a) Without CSRS, (b) With CSRS

    表  1  细胞色素C酶解多肽片断的信息

    Table  1.   Information of Cytochrome C digest

    多肽片断 多肽序列 (质量/电荷)/(m/z) 理论分子量
    s-1 KYIPGTK 403.8(+2) 805.5
    s-2 YIPGTK 339.7(+2) 677.4
    s-3 IFVQK 317.7 (+2) 633.4
    s-4 KTGQAPGFSYTDANK 793.0(+2) 1 583.8
    s-5 TGQAPGFSYTDANK 728.9 (+2) 1 455.7
    s-6 TGPNLHGLFGR 584.75(+2) 1 167.6
    s-7 GEREDLIAYLKK 717.74(+2) 1 433.8
    s-8 MIFAGIK 779.5 (+1) 778.4
    s-9 EDLIAYLK 964.08(+1) 963.5
    s-10 IFVQKCAQCHTVEK 817.31(+2) 1 632.8
    s-11 GITWGEETLMEYLENPKK 713.5(+3) 2 137.0
    s-12 GITWGEETLMEYLENPK 1006.0(+2) 2 008.9
    下载: 导出CSV

    表  2  柱后抑制器技术对多肽质谱灵敏度影响

    Table  2.   Effect of CSRS on MS/MS sensitivity

    多肽片断 (质量/电荷比值)
    /(m/z)
    保留时间
    / min
    无抑制器
    (S/N)
    有抑制器
    CSRS with 0.05 mol/L NH4HCO3(S/N)
    提高倍数
    S-1 403.81 8.33 630 2 776 4.41
    S-2 339.72 8.39 1 786 6 076 3.40
    S-3 317.72 10.08 1 001 11 321 11.31
    S-4 793.01 10.42 363 519 1.43
    S-5 728.93 10.56 559 1 132 2.02
    S-6 584.75 14.74 3 141 51 827 16.50
    S-7 717.74 14.91 896 1 294 1.44
    S-8 779.57 15.10 965 11 521 11.94
    S-10 817.31 15.39 415 253 0.61
    S-12 1 006.0 19.59 1 071 322 0.30
    下载: 导出CSV

    表  3  再生液NH4HCO3浓度对多肽质谱灵敏度影响

    Table  3.   Effect of NH4HCO3 on MS/MS sensitivity

    多肽片断 再生液0.1 mol/L NH4HCO3 再生液0.05 mol/L NH4HCO3 再生液0.025 mol/L NH4HCO3
    信噪比S/N 提高比例* 信噪比S/N 提高比例* 信噪比S/N 提高比例*
    S-1 2483 3.94 2776 4.41 1708 2.71
    S-2 3673 2.06 6076 3.40 3367 1.89
    S-3 4728 4.72 11321 11.31 7148 7.14
    S-4 376 1.04 519 1.43 163 0.45
    S-5 957 1.71 1132 2.02 725 1.30
    S-6 22726 7.24 51827 16.50 31495 10.03
    S-7 1126 1.26 1294 1.44 1867 2.08
    S-8 7700 7.98 11521 11.94 6701 6.94
    *:提高比例为该再生液浓度条件下, 质谱峰的信噪比 (S/N) 与无再生液条件下质谱峰的信噪比 (S/N) 的比值.
    下载: 导出CSV

    表  4  硫酸依替米星主成分及杂质进行定性分析

    Table  4.   Summary of results of LCMSMS

    名称 保留时间/min 正离子模式 分子量
    杂质1 8.89 350.20 [M+H]+ 349
    杂质2 10.92 450.03 [M+H]+ 449
    杂质3 12.50 464.10 [M+H]+ 463
    硫酸依替米星 13.47 478.10 [M+H]+ 477
    杂质4 15.86 492.17 [M+H]+ 491
    下载: 导出CSV
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出版历程
  • 收稿日期:  2017-01-06
  • 修回日期:  2017-02-20
  • 刊出日期:  2017-03-01

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