Isolation of bile acid glucosides and N-acetylglucosaminides from human urine by ion-exchange chromatography and reversed-phase high-performance liquid chromatography

Abstract

A method for the isolation, separation and analysis of glucosides and N-acetylglucosaminides of non-amidated bile acids and of glycine- and taurine-conjugated bile acid glucosides from normal human urine is described. Total bile acids were extracted from 24-h collections of urine by repetitive use of Sep-Pak C 18 cartridges. After elution with 80% aqueous methanol, a group separation into non-amidated, glycine- and taurine-conjugated bile acids was performed by ion-exchange chromatography on Lipidex-DEAP. The glycosylated compounds were then separated from the corresponding non-glycosylated ones by high-performance liquid chromatography (HPLC) using a reversed-phase system with a linear methanol gradient. The glycosylated compounds isolated by HPLC were analysed by fast atom bombardment mass spectrometry and, after derivatization, by gas chromatography—mass spectrometry. Information about the sugar moieties of the bile acid glycosides was also obtained by treatment with different glycosidases.