Bile acids LVII. Analysis of bile acids by high pressure liquid chromatography and mass spectrometry.

Several high pressure liquid chromatographic methods for the separation of conjugated and free bile acids are presented. A mixture of synthetic conjugated bile acids has been separated by reverse-phase systems consisting of either a Waters Associated' "fatty-acid analysis" or a muBondapak/C18 column eluted with a mixture of 2-propanol/potassium phosphage buffer (pH 2.5 or 7.0). The major conjugated bile acids present in the gallbladder bile of obese subjects have been analyzed each in less than 30 min and quantitated with a U.V. detector set at 193 nm. Some of the 5 alpha- and 5 beta-isomers of conjugated bile salts could be resolved in straight-phase systems on Corasil II or muPorasil columns. Mass spectra of the conjugated bile acids obtained by electron impact were characteristics of the type of amino acid attached to the side chain, and the number of hydroxyl substituents on the nucleus. Most of the isomers could readily be differentiated by the relative intensities of the fragment ions.