Targeted profiling of circulating and hepatic bile acids in human, mouse, and rat using a UPLC-MRM-MS-validated method

Abstract

Bile acids (BAs) are a group of chemically related steroids recognized as regulatory molecules whose profiles can change in different physio-pathological situations. We have developed a sensitive, fast, and reproducible ultraperformance liquid chromatography/multiple reaction monitoring/mass spectrometry method to determine the tissue and sera BA profiles in different species (human, rat, and mouse) by quantifying 31 major and minor BA species in a single 21-min run. The method has been validated according to FDA guidelines, and it generally provides good results in terms of intra- and interday precision (less than 8.6% and 16.0%, respectively), accuracy (relative error measurement between -11.9% and 8.6%), and linearity (R(2) > 0.996 and dynamic ranges between two and four orders of magnitude), with limits of quantification between 2.5 and 20 nM. The new analytical approach was applied to determine BA concentrations in human, rat, and mouse serum and in liver tissue. Our comparative study confirmed and extended previous reports, showing marked interspecies differences in circulating and hepatic BA composition. The targeted analysis revealed the presence of unexpected minoritary BAs, such as tauro-alpha-Muricholic acid in human serum, thus allowing us to obtain a thorough profiling of human samples. Its great sensitivity, low sample requirements (25 µl of serum, 5 mg of tissue), and comprehensive capacity to profile a considerable number of BAs make the present method a good choice to study BA metabolism in physiological and pathological situations, particularly in toxicological studies.

Fig. 1.

Chromatographic separation of the BAs standard mix solution. A: Nonconjugated BAs. Coeluting BAs (i.e., N5/N6) get separated and individually quantified by BA-specific MRM transition (Table 1). B: Glycine-conjugated BAs. C: Taurine-conjugated BAs. D: Deuterium-labeled internal standards. All the BAs were separated and detected in a single analytical run. Green: DHCA; blue: tri-hydroxylated BAs; red: di-hydroxylated BAs; dark blue: mono-hydroxylated BAs.

Fig. 2.

An unsupervised hierarchical clustering analysis based on the quantitative analysis of the 31 BAs. Each row shows the data for a specific BA, and each column shows the BAs profiles for the different species. The clustering analysis efficiently distinguishes between serum and liver samples and, more importantly, between the studied species.