Chinese Journal of Pharmacovigilance ›› 2026, Vol. 23 ›› Issue (7): 790-796.
DOI: 10.19803/j.1672-8629.20260264

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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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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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