Separation and Characterization of Ursodeoxycholate 7-N-Acetylglu-Cosaminides in Human Urine by High-Performance Liquid Chromatography with Fluorescence Detection

Abstract

The separation and characterization of unconjugated, glycine- and taurine-conjugated ursodeoxycholate 7-N-acetyl-glucosaminides in human urine without prior deconjugation have been carried out by high-performance liquid chromatography (HPLC) on a reversed phase column. The bile acid fraction was obtained from a urine specimen by the combined use of a Sep-Pak C₁₈ cartridge for solid phase extraction and lipophilic gel, piperidinohydroxypropyl Sephadex LH-20 (PHP-LH-20), for ion-exchange chromatography. Bile acid N-acetyl-glucosaminides were derivatized quantitatively into the corresponding fluorescent esters through the inherent primary hydroxyl group by treatment with 9-anthroyl cyanide. The derivatives were separated into unconjugated, glycine- and taurine-conjugated fractions on PHP-LH-20. Subsequent resolution into individual N-acetylglucosaminides was attained by HPLC with fluorescence detection on a Cosmosil 5C₁₈ column using 0.3% potassium phosphate buffer-methanol as a mobile phase. Unconjugated, glycine- and taurine-conjugated urso-deoxycholate 7-N-acetylglucosaminides in human urine from a patient with primary biliary cirrhosis were unambiguously identified on the basis of their chromatographic behavior using mobile phases of different pHs and organic modifiers. It has proved that ursodeoxycholate 7-N-acetylglucosaminides in urine were dominantly conjugated with glycine, while any 3-N-acetylglucosaminides of ursodeoxycholates as well as other common bile acids were not present.