Quantification of multiple bile acids in uninephrectomized rats using ultra-performance liquid chromatography-tandem mass spectrometry

Abstract

In order to study the roles of individual BAs and due to limited blood sample volumes available from experimental animals, improved methods for the simultaneous quantification of multiple BAs are needed. We developed and validated an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the quantification of 24 BAs, including 11 unconjugated, 6 glycine-conjugated and 7 taurine-conjugated BAs, in 50 μL of rat serum or plasma. The UPLC-MS/MS method, operated in negative and positive ion mode, allows quantification of BAs using multiple-reaction monitoring (MRM), with specific fragmentation of BAs. The method showed acceptable intra- and inter-day accuracy, precision, extraction recovery and high sensitivity, with a lower limit of quantification (LLOQ) in the pM range for several taurine-conjugated BAs. We applied the established method to investigate potential time-dependent changes of BAs in plasma from sham-operated and uninephrectomized male Sprague-Dawley rats. The levels of several primary and secondary BAs were transiently elevated one week after uninephrectomy, followed by normalization thereafter. In contrast, several conjugated BAs were slightly increased after the second week post-surgery. The established UPLC-MS/MS method, employing specific fragmentation of free and conjugated BAs by MRM, allows the simultaneous quantification of multiple BAs in 50 μL serum or plasma samples, and can be used to assess BA profiles in patho-physiological situations.