Immobilized 3 alpha-hydroxysteroid dehydrogenase and dansyl hydrazine as a pre-labeling reagent for high-performance liquid chromatography with fluorescence detection of bile acids

A high-performance liquid chromatographic method with fluorescence detection is described for the determination of bile acids and their conjugates. After enzymatic conversion of bile acids to 3-oxo-bile acids using the immobilized 3 alpha-hydroxysteroid dehydrogenase reactor column, 3-oxo-bile acids were extracted with a Sep-Pak C18 cartridge, labelled with dansyl hydrazine and then separated by high-performance liquid chromatography on a reversed-phase column. The eluate is monitored by a fluorophotometer at 365 nm (excitation) and 520 nm (emission). Reactions proceed quickly under mild conditions to give fluorescent derivatives. Linearity of the fluorescence intensity (peak height) with the amounts of various bile acids and their conjugates was obtained above 0.5-1.0 pmol. The method is sensitive, reliable and useful for the simultaneous determination of bile acids in biological samples.