Studies on steroids : CLXXXVII. Determination of serum bile acids by high-performance liquid chromatography with fluorescence labeling

Abstract

A method for the simultaneous determination of bile acids in serum by high-performance liquid chromatography (HPLC) with fluorescence labeling is described. The bile acid fraction was obtained from a serum specimen by passing it through a BondElut cartridge. Bile acids were derivatized quantitatively into the fluorescent compounds through the hydroxyl group at C-3 by treatment with 1-anthroyl nitrile in the presence of quinuclidine in acetonitrile. These derivatives were separated into the free, glycine- and taurine-conjugate fractions by ion-exchange chromatography on a lipophilic gel, piperidinohydroxypropyl Sephadex LH-20. Subsequent resolution of each fraction into cholate, ursodeoxycholate, chenodeoxycholate, deoxycholate and lithocholate was attained by HPLC on a Cosmosil 5C 18 column using 0.3% potassium phosphate buffer (pH 6.0)—methanol (1:5) and 0.1% potassium phosphate buffer (pH 6.0)—methanol (1:8) as mobile phases. The anthroyl bile acids were monitored by fluorescence detection (excitation wavelength 370 nm; emission wavelength 470 nm), the limit of detection being 20 fmol. The proposed method proved to be applicable to the quantitation of bile acids in serum with satisfactory reliability and sensitivity.