Rapid and sensitive on-line precolumn purification and high-performance liquid chromatographic assay for bile acids...

A simple and rapid technique for the simultaneous isolation and analysis of fifteen kinds of bile acid was developed using reversed-phase high-performance liquid chromatography with an automatic dual-precolumn switching system. The serum samples were directly injected onto a first precolumn (hydroxyapatite), which was flushed with 1 mM phosphate buffer. Serum proteins were strongly retained on the hydroxyapatite column, but bile acids were unretained. The bile acids were absorbed on a second precolumn (Serumout-25) and eluted onto the analytical column with solvent B (acetonitrile-methanol-30 mM ammonium acetate, 20:20:60, v/v/v). For the separation of each bile acid, the gradient elution technique was used (solvent A was acetonitrile-methanol-30 mM ammonium acetate, 30:30:40). After separation of the bile acids, NADH was produced by use of immobilized 3 alpha-hydroxysteroid dehydrogenase column and then determined fluorimetrically (gamma em = 460 nm, gamma ex = 350 nm). The recoveries of bile acids in serum generally approached 100%.