Analysis of Unsulfated and Sulfated Bile Acids without Hydrolysis by High-Performance Liquid Chromatography

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A method for simultaneous determination of unconjugated and conjugated bile acids in human bile has been described. Bile acids were extracted with a Sep-pack C<SUB>18</SUB> cartridge and separated into the unconjugated, glycine-, taurine-conjugated and sulfated groups by ion-exchange chromatography on piperidinohydroxypropyl Sephadex LH-20. Resolution of each unsulfated group into ursodeoxycholate, cholate, chenodeoxycholate, deoxycholate and lithocholate was attained into two stages by high-performance liquid chromatography (HPLC) on a Radial-Pak A column. First, 0.3% ammonium phosphate (pH7.7) /acetonitrile (19: 8, v/v) was used for the separation of the latter threee as a mobile phase. Ursodeoxycholate and cholate were efficiently resolved in 0.3% ammonium phosphate (pH7.7) /acetonitrile (23: 8, v/v). The UV (205nm) detector showed a linear response to unconjugated, glycine- and taurine-conjugated bile acids with a quantitation limit of 500, 50 and 150ng, respectively. The present method was found to be applicable to determination of bile acids in human bile with satisfactory accuracy and pre- cision. Separation of monosulfated bile acids by HPLC was also carried out. Effects of pH and salt concentration of a mobile phase on the k' values of sulfated bile acids were investigated with the reversed-phase columns. The 3-monosulfates were efficiently separated on ODS SC-02 using three aqueous ammonium carbonate/acetonitrile systems.
Japan Society of Clinical Chemistryの論文

Analysis of Unsulfated and Sulfated Bile Acids without Hydrolysis by High-Performance Liquid Chromatography

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