Quantitative profiling of bile acids in rat bile using ultrahigh-performance liquid chromatography–orbitrap mass spectrometry: Alteration of the bile acid composition with aging

• Development of an analytical method for profiling 41 BAs in rat bile.
• Optimization of the sample preparation method for accurate and simple analysis.
• Quantification of 19 BAs and detection of 22 unknown BAs.
• Application of the method to the age-related rat bile.

Bile acids (BAs) play important roles in physiological functions, including the homeostasis of cholesterol and lipids and as ligands for G protein-coupled receptors (GPCRs). With the increasing importance of BAs, analytical methods for their quantification and screening have been developed. However, due to the diverse range and variety of BAs with different activation potency, a simple, effective, and sensitive method is required to screen BAs for accurate quantification and identification. This paper presents an application of ultrahigh-performance liquid chromatography-orbitrap mass spectrometry (UHPLC-LTQ–Orbitrap MS) for profiling BAs in bile. Using this method, along with the accurate quantification of 19 targeted BAs, 22 unknown BAs were detected and characterized by their fragmentation patterns. The method is beneficial for screening most of the BAs (quantitatively and qualitatively) in rat bile with simple preparation in a single run. The sample dilution ranges of each BA were optimized depending on the concentration of BAs in the bile to obtain good peak separation and accurate data. The method validation was performed successfully using charcoal-treated bile and the intra and inter-day coefficients of variation were less than 20% for all BAs while the recovery were above 88.5% except for the lithocholic acid. The method was applied to profile the age-dependent changes in the contents of rat BAs. Through statistical analysis, we found that as the rats aged, unconjugated BAs and glycine-conjugated BAs decreased or were unaffected, while taurine-conjugated BAs were increased in general. Among the unknown BAs, 5 of the taurine-conjugated BAs increased, while a glycine-conjugated BA decreased, in agreement with the trends of the targeted BAs.