Development and Validation of a Rapid LC‐MS/MS Method for Quantification of 20 Bile Acids in Serum and Application to Murine Hepatotoxicity Models

Bile acids function both as facilitators of biliary lipid transport and as signaling mediators that maintain metabolic homeostasis. Perturbations in bile acid metabolism accompany diverse forms of liver injury, yet conventional clinical assays lack the sensitivity and specificity required for comprehensive bile acid profiling. To address this, we aimed to develop and validate a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of 20 bile acids using minimal serum volume and shortened analysis time. The developed method requires only 20 μL of serum and completes analysis within 13 min. Following protein precipitation, analytes were separated on a C18 column with gradient elution. The method demonstrated excellent sensitivity with limits of detection of 1-2 ng/mL and a uniform limit of quantification of 5 ng/mL for all analytes. Validation results showed satisfactory performance: accuracy within ± 14.8%, intra-assay and interassay precision below 16.5%, extraction recovery of 72.6%-108.4%, and acceptable matrix effects. Application to murine hepatotoxicity models induced by α-amanitin (α-AMA), ethanol, and acetaminophen (APAP) revealed distinct, compound-specific bile acid alterations. This work establishes a rapid, miniaturized, and validated LC-MS/MS approach, demonstrating its utility in revealing toxin-specific bile acid signatures, which provides a valuable tool for preclinical hepatotoxicity research and potential translational applications.