High performance liquid chromatographic separation of stereoisomeric bile acids as their UV-sensitive esters

High performance liquid chromatographic separation of a series of mono-, di- and trihydroxylated 5 beta-cholanic acids, which differ only in position and configuration of hydroxyl groups at positions C-3, C-7 and/or C-12, is reported. The C-24 free acids were derivatized to four different classes of UV-sensitive esters, i.e., p-bromophenacyl (BP), m-methoxyphenacyl (MP), 4-nitrophthalimidemethyl (NPM) and 9-anthrylmethyl (AM) esters, and chromatographed on two variants of C18 reversed-phase columns (Nova-Pak C18 and Zorbax ODS) with methanol-water systems as mobile phase. Separation efficiency and elution order of some isomeric pairs were influenced by both the structure of the C-24 ester groups and the nature of the columns used. Excellent chromatographic properties were found for those derivatives, particularly for the NPM esters.