Development and validation of a sensitive UPLC-MS/MS platform for comprehensive bile acid profiling in multiple biological matrices: Application to a 17α-ethinylestradiol-induced cholestatic rat model.

Bile acids are crucial indicators for the diagnosis of liver diseases, including cholestasis, which necessitates robust and versatile analytical methods for comprehensive analysis. This study presents a validated UPLC-MS/MS method for the quantitative determination of 23 bile acids in rat plasma, ileal tissues, and feces, encompassing free primary and secondary bile acids as well as taurine-conjugated and glycine-conjugated bile acids. All bile acids were qualitatively and quantitatively assessed within 24 min. The method demonstrated detection limits of 0.01-0.1 ng/mL, and quantification limits of 0.1-1 ng/mL. Performance evaluation demonstrated excellent specificity and good method robustness. Both intra-day and inter-day precision were below 12.4%, with accuracy ranging from 86.8% to 113%. Extraction recoveries were 90.7-108% with RSD of 1.11-11.9%, while matrix effects were 93.5-109% with RSD of 0.30-11.6%, indicating high extraction efficiency and negligible matrix interference. The analytes were stable under the tested conditions, with stability values from 86.1% to 113%. In an estrogen-induced cholestatic rat model, the UPLC-MS/MS method detected increased conjugated bile acids in plasma and a disturbed primary-to-secondary bile acid ratio in ileal and fecal samples, indicating impaired enterohepatic circulation. The method was fully validated in accordance with the Guideline on Bioanalytical Method Validation and demonstrated reliable, stable, and consistent quantification of bile acids across complex biological matrices. This work supports mechanistic studies of cholestasis and facilitates the identification of potential clinical biomarkers.