Rapid and sensitive on-line precolumn purification and high-performance liquid chromatographic assay for bile acids in serum.
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%.
Yoshida
S
S
Kyoto Pharmaceutical University, Japan.
Murai
H
H
Hirose
S
S
D001647:Bile Acids and Salts / Q000097:blood* / Q000302:isolation & purification; D001798:Blood Proteins / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D017886:Durapatite; D006505:Hepatitis / Q000097:blood; D006801:Humans; D006882:Hydroxyapatites / Q000032:analysis; D007202:Indicators and Reagents; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D001647:Bile Acids and Salts; D001798:Blood Proteins; D006882:Hydroxyapatites; D007202:Indicators and Reagents; D017886:Durapatite