Development and Validation of a Sensitive UHPLC-MS/MS Method for the Simultaneous Determination of 19 Bile Acids in Liver and Feces: Application to Metabolic Dysfunction-Associated Steatotic Liver Disease Mice.

Recently, bile acids (BAs) have garnered significant attention due to their crucial roles in various metabolic diseases. However, the simultaneous quantification of BAs in biological samples is challenging due to the structural similarities, complex biological matrix, and tissue-specific distribution. We developed a sensitive UHPLC-MS/MS method for quantitation of 19 BAs in mouse liver (mainly conjugated BAs) and feces (mainly unconjugated BAs). We compared protein precipitation, liquid-liquid extraction, and solid-phase extraction (SPE) methods for sample preparation, and SPE was chosen due to the low background noise and high extraction efficiency for both conjugated and unconjugated BAs. The method was extensively validated by evaluating the linearity (R2 ≥ 0.991), extraction recovery (82%-112%), limits of detection (1-2 ng/mL) and low limits of quantification (2-5 ng/mL). Then, the method was applied to the detection of BAs in liver and feces of metabolic dysfunction-associated steatotic liver disease mice, and the correlations between BA levels and the mRNA expression levels of BA metabolic enzymes, transporters, and receptors were analyzed. This study provides a direct and reliable determination method for the in-depth investigation of the enterohepatic circulation of BAs.