High Performance Liquid Chromatographic Analysis of Individual Bile Acids: Free, Glycine- and Taurine-conjugated Bile Acids

Abstract

Abstract High performance liquid chromatographic analyses of individual bile acids (cholic, chenodeoxycholic, deoxycholic, and lithocholic acids), free and conjugated with glycine and taurine, are described. The analyses of the free and glycine-conjugated bile acids are based on the esterification of the carboxyl group of bile acids with O-(p-nitrobenzyl)-N,N′-diisopropylisourea(PNBDI). The bile acids in the biological samples were extracted by an Amberlite XAD-2 column, and separated by DEAE-Sepharose CL-6B into free, glycine and taurine-conjugated bile acids. After separation, the free and glycine-conjugated bile acids were directly esterified with PNBDI. Taurine-conjugated bile acids are unable to be esterified with PNBDI, the bile acids were hydrolyzed with NaOH to produce the free bile acids, and then esterified. The p-nitrobenzyl ester of bile acids has a characteristic ultraviolet absorption. Consequently the compounds were separated into the individual bile acids by high performance liquid chromatography, and detected by an UV-detector. An analysis of the individual bile acids in human bile is given for an example.