Reversed-phase high-performance liquid chromatographic separation and quantification of individual human bile acids

The reversed-phase high-performance liquid chromatographic separation and quantification of individual bile acids is described. Taurine- and glycine-conjugated bile acids were separated and detected directly by an ultraviolet absorbance detector operating at 200 nm. Simultaneous quantitation of at least 100 ng of each conjugated bile acid is possible. Carboxylic (free and glycine-conjugated) bile acids were esterified with p-bromophenacyl-bromide. The reaction, using N,N-diisopropylethylamine as catalyst, yields quantitatively the strongly absorbing p-bromophenacyl esters whch can be determined by absorbance measurement at 254 nm. Simultaneous quantitation of less than 20 ng of each bile acid is possible. The present method is applied to the quantitation of individual bile acids in ten human gallbladder bile samples.