Isomer-specific separation and analysis of 42 bile acids in intestinal contents using ultra-high performance liquid chromatography-tandem mass spectrometry.

Bile acids (BAs) are recognized as established disease biomarkers, yet quantitative studies have largely focused on serum or plasma, with insufficient attention paid to intestinal contents that hold significant pathophysiological importance. Accurate quantification of intestinal BAs is key to elucidating disease mechanisms. However, this analysis remains highly challenging due to the complex matrix of intestinal contents and the presence of numerous bile acid isomers. This study focuses on developing and validating a rapid, accurate, and sensitive UPLC-MS/MS method for the simultaneous quantification of 42 BAs (covering 11 isomer groups) in intestinal contents, and its application to depression research. Samples were pretreated using lyophilization followed by composite solvent extraction. Chromatographic separation used a CORTECSTM Premier T3 column with gradient elution. Among the 11 isomer groups, baseline separation was achieved for most of them. The method validation results showed excellent linearity with r2 ≥ 0.996; recoveries ranged 86.73% - 111.03%; stability tests under various conditions including room temperature storage for 24 h, 4 °C for 7 days, -80 °C for 90 days, and three freeze-thaw cycles yielded RSD ≤ 14.41%. Intra-day and inter-day precision RSD ≤ 16.10%. The lower limits of quantification ranged from 0.1 to 2.0 ng/mL. Applying to intestinal contents from a CUMS-induced depression SD rat model, the method revealed significant differences in 14 BAs compared with normal rats, among which Iso-HDCA was upregulated as much as 6-fold, suggesting that BAs detection should be given attention in exploring the mechanisms of depression.