Development of an analytical method for simultaneous determination of 38 bile acids and its application in studying the mechanism of WuZhi Pian against rifampicin-induced liver injury

Bile acids (BAs) are crucial signaling molecules involved in liver metabolism regulation. The development of quantitative detection methods for BAs is essential for understanding the molecular mechanisms of liver-related diseases. In this study, we optimized an analytical method to quantify 38 BAs in serum using ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-QQQ-MS/MS). We evaluated and optimized various sample preparation methods, chromatographic columns, mobile phase compositions, and mass spectrometry conditions. After method validation, BA profiles were analyzed in mice with rifampicin (RIP)-induced liver injury, with or without WuZhi Pian treatment. According to the results, the optimal serum preparation involved a 1% ammonia-methanol solution at a 1:3 sample-to-solvent ratio, vortex-mixing for 5 min, and incubating for 60 min at 4°C. All 38 BAs achieved optimal separation on an ACQUITY UPLC® HSS T3 column (2.1 × 100 mm, 1.8 μm) with a 20-min run time. The best peak shapes and responses were obtained using mobile phases consisted of 4.3 mM ammonia with 0.1% acetic acid in water and acetonitrile. This method demonstrated high specificity, linearity (> 0.9991), and a lower limit of quantification (LLOQ) ranging from 0.0054 to 2.4414 nM across different BAs, with good repeatability (RSD% < 15.35%) and reproducibility (RSD% < 19.68%). Average recoveries for all BAs ranged from 70% to 122%. Using this method, 13 BAs were found to be altered in RIP-induced liver injury, of which nine BAs were modulated by WuZhi Pian treatment. Therefore, the optimized UPLC-QQQ-MS/MS method is a powerful tool for monitoring BA metabolism-related diseases.