Sensitive assay system for bile acids and steroids having hydroxyl groups utilizing high-performance liquid chromatography with peroxyoxalate chemiluminescence detection.

3 alpha- or 3 beta-hydroxysteroids, such as bile acids (free and glycine and taurine conjugates), 3 beta-hydoxy-5-cholenic acid, pregnanediol, 5-pregnene-3 beta, 20 beta-diol and 5-pregnene-3-beta,20 alpha-diol, were converted to 3-oxosteroids by enzymatic reaction using immobilized hydroxysteroid dehydrogenase, derivatized with dansylhydrazine to the corresponding dansyl hydrazones and purified by gel permeation chromatography. The dansyl hydrazones were chromatographed on a C18 column with a tetrahydrofuran-containing eluent and detected at the level of a few femtomoles by a peroxyoxalate chemiluminescence post-column reaction using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate as a chemilumigenic reagent. The dansyl hydrazones of chenodeoxycholic acid and deoxycholic acid (free and glycine and taurine conjugates) in particular, which coeluted under the chromatographic conditions above, were separated using an eluent including acetonitrile and 2,6-di-O-methyl-beta-cyclodextrin and detected in the same way.