Method for the separation of the unconjugates and conjugates of chenodeoxycholic acid and deoxycholic acid by two-dimensional reversed-phase thin-layer chromatography with methyl β-cyclodextrin

Abstract

A simple and efficient method for the separation of individual unconjugated bile acids and their glycine- and taurine-amidated, 3-sulfated, 3-glucosylated and 3-glucuronidated conjugates is described. The method involves the use of a two-dimensional (2D) reversed-phase (RP) high-performance thin-layer chromatographic (HPTLC) technique with methyl β-cyclodextrin (Me-β-CD). Five major unconjugated bile acids, cholic acid, chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid and lithocholic acid, and their conjugates were examined as the solutes. A high degree of separation of individual bile acids in each homologous series was achieved on a RP–HPTLC plate by developing with aqueous methanol in the first dimension and the same solvent system containing Me-β-CD in the second dimension. In particular, all of the six `difficult-to-separate' pairs, unconjugated CDCA and DCA and their conjugated forms with glycine, taurine, sulfuric acid, d-glucose and d-glucuronic acid, were effectively resolved by adding Me-β-CD in the aqueous mobile phases with the formers having larger mobilities than the latter. The application of this 2D inclusion RP–HPLC method to the separation of glycine-conjugated bile acids in human bile is also described. The present method would be useful for separating and characterizing these bile acids present in biological materials.