Studies on steroids : CCXXVII. Separation and determination of bile acid 7- and 12-sulphates in urine by high-performance liquid chromatography with fluorescence labelling

Abstract

A method for the characterization and determination of bile acid 7- and 12-sulphates in urine without prior deconjugation is described. The sulphate fraction was obtained from an urine specimen by passing it through a Sep-Pak C 18 cartridge, followed by group separation by ion-exchange chromatography on a lipophilic gel, piperidinohydroxypropyl Sephadex LH-20. Bile acid 7- and 12-sulphates were derivatized quantitatively into the fluorescence compounds through the hydroxyl group at C-3 by treatment with 1-anthroyl nitrile in the presence of quinuclidine in acetonitrile. Subsequent resolution into individual 7- and 12-sulphates was attained by high-performance liquid chromatography (HPLC) on a Cosmosil 5C 18 column using 0.3% potassium phosphate buffer (pH 4.0)-methanol (1:3) as a mobile phase. The 3-(1-anthroyl) derivatives of 7- and 12-sulphates were monitored by fluorescence detection. Taurochenodeoxycholate 7-sulphate in human urine was unequivocally identified on the basis of behaviour in HPLC using mobile phases of different pH values. The present method has proved to be applicable to the characterization and quantification of bile acid 7- and 12-sulphates in human urine.