中国药物警戒 ›› 2026, Vol. 23 ›› Issue (7): 790-796.
DOI: 10.19803/j.1672-8629.20260264

• 基础与临床研究 • 上一篇    下一篇

基于UPLC-MS/MS技术的大鼠肝肠循环多基质中37种胆汁酸检测方法研究

汪祺1,2, 杨颜榕1,3, 侯天宇1,4, 李妍怡1,5, 文海若6#, 宁霄7, *   

  1. 1中国食品药品检定研究院中药民族药检定所,北京 100050;
    2药品监管科学全国重点实验室,北京 102629;
    3沈阳药科大学功能食品与葡萄酒学院,辽宁 沈阳 110016;
    4沈阳药科大学中药学院,辽宁 沈阳 110016;
    5北京中医药大学中药学院,北京 102488;
    6中国食品药品检定研究院安全评价研究所,北京 100176;
    7中国食品药品检定研究院食品化妆品检定所,北京 100050
  • 收稿日期:2026-04-01 出版日期:2026-07-15 发布日期:2026-07-16
  • 通讯作者: #为共同通信作者。*宁霄,女,博士,主任药师,分析化学。E-mail: 506612164@qq.com
  • 作者简介:汪祺,女,博士,研究员,中药质量与安全性评价。
  • 基金资助:
    国家自然科学基金资助项目（82374033）; 药品监管科学全国重点实验室支持项目（2025SKLDRS0345)

A detection method for 37 types of bile acids in multiple matrices of hepatoenteric circulation of rats using UPLC-MS/MS technology

Wang Qi1,2, Yang Yanrong1,3, Hou Tianyu1,4, Li Yanyi1,5, Wen Hairuo6#, Ning Xiao7, *   

  1. 1Institute for Control of Chinese Traditional Medicine and Ethnic Medicine, National Institutes for Food and Drug Control, Beijing 100050, China;
    2State Key Laboratory of Drug Regulatory Science, Beijing 102629, China;
    3School of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang Liaoning 110016, China;
    4School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang Liaoning 110016, China;
    5School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China;
    6Institute of Safety Evaluation, National Institutes for Food and Drug Control, Beijing 100176, China;
    7Institute for Food and Cosmetics Testing, National Institutes for Food and Drug Control, Beijing 100050, China
  • Received:2026-04-01 Online:2026-07-15 Published:2026-07-16

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摘要： 目的 建立并验证基于超高效液相色谱-串联质谱（UPLC-MS/MS）的大鼠胆汁酸的高通量定量方法,用于测定大鼠6种基质中37种胆汁酸含量,为药物性肝损伤及肝肠微生态机制研究提供方法学参考。方法 采用多孔活性炭吸附法制备空白基质,结合内标校正复杂基质的内源性本底干扰;以预冷乙腈一步蛋白沉淀萃取法抑制肠道菌群介导的体外酶促降解;利用HSS T3色谱柱改善极性胆汁酸组分保留,实现鼠源特异性同分异构体的基线分离;针对游离型次级胆汁酸甾体母核碎裂困难的特点,优化采用低碰撞能（4~5 V）伪多反应监测模式,提升检测灵敏度与专属性。结果 37种胆汁酸在 0.09~3.60 μg·mL-1浓度范围内线性关系良好（r²≥0.995 2）,方法检出限 0.43~29.86 ng·mL-1,定量限1.44~99.53 ng·mL-1;6种基质中高、中、低浓度提取回收率为 80.17%~123.06%,经活性炭基质匹配校正后基质效应为 85.22%~114.75%,日内及日间精密度相对标准偏差均≤18.49%,准确度符合生物样品定量分析指导原则要求。结论 建立的多基质 UPLC-MS/MS 方法专属性强、灵敏度高、重现性好,通过前处理与色谱-质谱条件的协同优化,有效解决了胆汁酸靶向分析的关键技术难点,适用于大规模跨基质的药物警戒及毒理学代谢轮廓研究。

关键词: 多生物基质, 肝肠循环, 药物性肝损伤, 超高效液相色谱-串联质谱, 胆汁酸, 高通量靶向定量方法, 大鼠

Abstract: Objective To establish and validate a high-throughput quantitative method for determination of the contents of 37 types of bile acids in six types of matrices of rats based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in order to provide a reference for investigating drug-induced liver injury and hepatenteric microecological mechanisms. Methods A porous activated carbon adsorption method was employed to prepare blank matrices, which were used to correct endogenous background interference in complex matrices in combination with internal standards. A pre-cooled acetonitrile one-step protein precipitation extraction method was adopted to inhibit intestinal microbiota-mediated in vitro enzymatic degradation. The HSS T3 chromatographic column was used to improve the retention of components of polar bile acids to facilitate the baseline separation of murine-specific isomers. Given the difficult fragmentation of the steroid core of free-type secondary bile acids, a pseudo multi-reactor monitoring mode with low collision energy (4-5 V) was optimized to enhance the sensitivity and specificity of detection. Results The 37 types of bile acids showed good linear relationships at the concentrations ranging from 0.09 to 3.60 μg·mL-1 (r²≥0.995 2). The detection limit was 0.43-29.86 ng·mL-1 while the quantification limit was 1.44-99.53 ng·mL-1. In the six matrices, the recovery rates at high, medium, and low concentrations ranged from 80.17% to 123.06%. After matrix matching correction using activated carbon, the matrix effect was 85.22%-114.75%. The relative standard deviation of both intra-day and inter-day precisions was 18.49% or less, with the accuracy meeting the requirements in guidelines for quantitative analysis of biological samples. Conclusion The multi-matrix UPLC-MS/MS method established in this study is highly specific, sensitive and reproducible. Based on synergistic optimization of sample pretreatment and chromatography-mass spectrometry conditions, this method can effectively address the key bottlenecks in targeted analysis of bile acids and is applicable to large-scale studies on cross-matrix pharmacovigilance and toxicological metabolic profiling.

Key words: Multiple Biological Matrices, Enterohepatic Circulation (EHC), Drug-Induced Liver Injury, Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS), Bile Acids, High-Throughput Targeted Quantitative Methods, Rats

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