Separation of Bile Acid N-Acetylglucosaminides by High-Performance Liquid Chromatography with Precolumn Fluorescence Labeling

Abstract

A sensitive and reliable method for the separation and characterization of bile acid N-acetylglucosaminides has been developed by precolumn labeling followed by high-performance liquid chromatography (HPLC) with fluorescence detection. The 3-N-acetylglucosaminides of unconjugated, glycine- and taurine-conjugated bile acids were derivatized into fluorescent esters through a primary hydroxyl group on the sugar moiety by treatment with 9-anthroyl cyanide in the presence of quinuclidine in acetonitrile. Subsequent separation into each 3-N-acetylglucosaminide was achieved by reversed-phase chromatography on a Cosmosil 5C18 column using a 0.3% potassium phosphate buffer (pH7.0)/methanol (1:4, v/v) as a mobile phase. The derivatized 3-N-acetylglucosaminides were monitored by fluorescence detection (excitation wavelength 362nm; emission wavelength 470nm), the limit of detection being 100fmol. The chromatographic separation of 3- and 7-N-acetylglucosaminides of ursodeoxycholates is also described.