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叶酸有关物质的液相色谱-质谱联用分离与鉴定

宁曼如, 徐柯卉, 杭太俊, 宋敏

宁曼如,徐柯卉,杭太俊,等. 叶酸有关物质的液相色谱-质谱联用分离与鉴定[J]. 中国药科大学学报,2025,56(1):56 − 64. DOI: 10.11665/j.issn.1000-5048.2024052201
引用本文: 宁曼如,徐柯卉,杭太俊,等. 叶酸有关物质的液相色谱-质谱联用分离与鉴定[J]. 中国药科大学学报,2025,56(1):56 − 64. DOI: 10.11665/j.issn.1000-5048.2024052201
NING Manru, XU Kehui, HANG Taijun, et al. Separation and identification of folic acid and its related substances by liquid chromatography-mass spectrometry[J]. J China Pharm Univ, 2025, 56(1): 56 − 64. DOI: 10.11665/j.issn.1000-5048.2024052201
Citation: NING Manru, XU Kehui, HANG Taijun, et al. Separation and identification of folic acid and its related substances by liquid chromatography-mass spectrometry[J]. J China Pharm Univ, 2025, 56(1): 56 − 64. DOI: 10.11665/j.issn.1000-5048.2024052201

叶酸有关物质的液相色谱-质谱联用分离与鉴定

详细信息
    通讯作者:

    宋敏: Tel:13584052217 E-mail:songmin@cpu.edu.cn

  • 中图分类号: R917

Separation and identification of folic acid and its related substances by liquid chromatography-mass spectrometry

  • 摘要:

    用ODS色谱柱和10 mmol/L乙酸铵缓冲溶液-甲醇流动相(pH 6.3)将叶酸有关物质进行线性梯度洗脱分离,电喷雾正离子化-四极杆-飞行时间质谱(ESI-Q-TOF/MS)高分辨测定各有关物质母离子及其子离子的准确质荷比和元素组成,通过质谱解析、有机反应机制分析、或对照品对照鉴定它们的结构。在所建立的LC-ESI-Q-TOF/MS条件下,叶酸及其有关物质分离良好,检测并鉴定叶酸及其强制降解试验样品中23个主要有关物质,其中2个为《欧洲药典》规定的已知杂质,4个与文献报道已知杂质一致,其余17个为新鉴定的未知杂质。鉴定结果可为叶酸质量控制提供参考依据。

    Abstract:

    The related substances in folic acid were separated on an ODS column by linear gradient elution using the mixture of 10 mmol/L ammonium acetate buffer solution and methanol (pH 6.3) as the mobile phase, and their accurate mass-charge ratio and elemental composition of parent and product ions were detected by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF/MS). The structures of the related substances were then determined by MS spectra elucidation, organic reaction mechanism analysis, or reference substances confirmation. Under the established LC-ESI-Q-TOF/MS conditions, folic acid and its related substances were adequately separated. Among the 23 main related substances found, 2 were known impurities listed in European Pharmacopoeia, 4 were consistent with the literature reports, and the remaining 17 were newly identified unknown related substances in this study. The results of separation and identification can provide some useful reference for the quality control of folic acid.

  • Figure  1.   Typical synthetic route for folic acid

    Figure  2.   Structures of folic acid known related substances

    Figure  3.   HPLC chromatograms of folic acid and its stressed test solutions

    API: Folic acid; 117: Related substances 117; a: Solvent blank; b: Test sample; c: Dry photolysis; d: Alkaline; e: Oxidation; f: Wet photolysis; g: Dry heat; h: Wet heat

    Figure  4.   MS/MS spectra of folic acid and its major related substances [M+H]+ ions

    Figure  5.   Chemical structures of folic acid and its related substances 123

    Figure  6.   MS/MS Fragmentation pathways of folic acid and its typical known related substances [M+H]+ ions

    Figure  7.   MS/MS fragmentation pathways of related substance 3 [M+H]+ ion

    Figure  8.   MS/MS fragmentation pathways of related substance 8 [M+H]+ ion

    Figure  9.   MS/MS fragmentation pathways of related substance 9 [M+H]+ ion

    Figure  10.   MS/MS fragmentation pathways of related substance 10 [M+H]+ ion

    Figure  11.   MS/MS fragmentation pathways of related substance 15 [M+H]+ ion

    Figure  12.   Acid-base isomerism of pterins

    Table  1   Mass balance of Folic Acid and its stressed test solutions

    Samplec/(mg/mL)AreaImpAreaTotalAreaTotal/cCalibration
    Test sample1.00388819835558327354519711.00
    Dry photolysis1.032103693334141675330830180.98
    Alkaline1.030461286136378124353185671.00
    Oxidation1.022470031435609756348432050.98
    Wet photolysis1.020225271135383000346892160.93
    Dry heat1.024390676034478456336703670.99
    Wet heat1.012299778635357191349379360.95
    下载: 导出CSV

    Table  2   Related substances identified in folic acid by LC-Q-TOF/MS

    No. [M+H]+(m/z) Ion formula Diff.(×10-6 Product ions(m/z UV λmax/nm Origins
    API 442.1480 [C19H20N7O6]+ −2.25 313.1045295.0941 281, 346 /
    1(Glu) 148.0603 [C5H10NO4]+ 0.91 130.0497102.054784.0447 281, 347 Dr
    2(EP-A) 267.1000 [C12H15N2O5]+ −9.21 130.0508120.0452 273 Dr/Pr
    3 324.1223 [C14H18N3O6]+ −9.18 195.0781177.0671 287 Dr
    4 193.0848 [C7H9N6O]+ −8.15 176.0580108.0562 267, 349 Dr
    5(CPT) 208.0483 [C7H6N5O3]+ −8.62 190.0372180.0530164.0524162.0424 286, 345 Dr
    6 343.1294 [C18H19N2O5]+ −1.61 299.1390281.1277253.1336214.0865169.0887 265 Dr
    7 295.0934 [C13H15N2O6]+ −3.19 295.0934176.0609148.0391130.049784.0449 266 Dr
    8 313.1036 [C13H17N2O7]+ −1.83 166.0498148.0391 279 Dr
    9 265.0675 [C9H9N6O4]+ 1.81 221.0780193.0828178.0721164.0560 275, 346 Dr
    10 196.0469 [C6H6N5O3]+ −1.97 168.0519140.056898.0346 279 Dr
    11 279.0918 [C10H11N6O4]+ −6.12 261.0753235.0968193.0848176.0580 274, 345 Dr
    12 152.0579 [C5H6N5O]+ −8.04 135.0311107.0364110.035582.0402 245, 273 Dr
    13 152.0580 [C5H6N5O]+ −8.70 135.0311107.0364110.035382.0402 284 Dr
    14 313.0663 [C12H13N2O8]+ 1.12 295.0559267.0611249.0503166.013084.0438 246, 278 Dr
    15 297.0744 [C12H13N2O7]+ −9.03 279.0640251.0683150.019684.0446 246, 274 Dr
    16(HPT) 194.0689 [C7H8N5O2]+ −8.54 176.0580134.0358106.040081.0480 274, 346 Dr
    17 137.0709 [C7H9N2O]+ 0.29 120.044194.0647 275 Dr
    18 456.1309 [C19H17N7O7]+ −7.34 438.1198327.0870309.0760281.0808 287, 376 Dr
    19(FPT) 192.0533 [C7H6N5O]+ −8.89 175.0255149.0455147.0311122.034594.0400 280, 346 Dr
    20 440.1317 [C19H18N7O6]+ −0.89 311.0885293.0782 281, 347 Dr
    21 443.1324 [C19H19N6O7]+ −3.23 296.0779 280 Dr
    22 442.1480 [C19H20N7O6]+ −2.36 313.1045295.0941 279, 346 Pr
    23(EP-D) 313.1072 [C14H13N6O3]+ −9.08 295.0965269.1169176.0581120.0453 278, 345 Dr
    Pr: Process related substance;Dr: Degradation related substance
    下载: 导出CSV
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出版历程
  • 收稿日期:  2024-05-21
  • 刊出日期:  2025-02-24

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