Ion-pair high-performance liquid chromatography of bile salt conjugates: application to pig bile

The high-performance liquid chromatographic separation and quantitation of conjugated bile salts from pig bile is reported. Synthetic standards and bile samples were chromatographed on a C18 reversed phase column using acetonitrile/water/tetrabutyl ammonium phosphate as an isocratic mobile phase at a flow rate of 1 mL/min. Detection of the ion-pairs was at 214 nm. The method permits efficient separation of all conjugated pig biliary bile salts without prior modification or treatment of the samples. Analysis of 12 pig biles showed that 85% of the bile salts are conjugated to glycine. The three main conjugated bile salts were glyco-3 alpha,6 alpha,7 alpha-trihydroxy-5 beta-cholanoic acid (GHC), glyco-3 alpha,7 alpha-dihydroxy-5 beta-cholanoic acid (GCDC), and glyco-3 alpha,6 alpha-dihydroxy-5 beta-cholanoic acid (GHDC). Glyco-3 alpha-hydroxy-6-oxo-5 beta-cholanoic acid (G3 alpha 6oxo), tauro-3 alpha,7 alpha-dihydroxy-5 beta-cholanoic acid (TCDC), tauro-3 alpha,6 alpha,7 alpha-trihydroxy-5 beta-cholanoic acid (THC), and tauro-3 alpha,6 alpha-dihydroxy-5 beta-cholanoic acid (THDC) were found to contribute each for 4 to 5% to the total. An excellent correlation was found between the sum of conjugated bile salts quantitated by high-performance liquid chromatography (HPLC) and values obtained by conventional enzymatic assay. Simplicity, efficiency and relative rapidity of the method render it suitable for routine analyses.