Simultaneous determination of bile acids in rat bile and serum by high-performance liquid chromatography

A method for the simultaneous determination of bile acids in rat bile and serum by high-performance liquid chromatography with a post-column enzymic reaction and fluorescence detection has been developed. Without prior fractionation and alkaline hydrolysis, 26 unconjugated, glycine- and taurine-conjugated bile acids were determined. They were separated on a reversed-phase column using a linear gradient solvent system of 200 mM dibasic ammonium phosphate buffer (pH 7.9)-acetonitrile-methanol (73:19:8, v/v/v) and 20 mM dibasic ammonium phosphate buffer (pH 7.9)-acetonitrile-methanol (2:1:2, v/v/v). The limits of detection were 1-5 pmol, and calibration curves were linear for concentrations between 10 and 4000 pmol. This rapid and reliable method is effective for measuring bile acid levels in the bile and serum not only of rats but also of patients with hepatobiliary and other diseases.