Simultaneous determination of keto and non-keto bile acids in human serum by gas chromatography with selected ion monitoring

Abstract

A reliable method for the simultaneous determination of keto and non-keto bile acids in human serum was developed. Carbonyl substituents of bile acid ethyl esters were converted into methyloxime and hydroxyl substituents into dimethylethylsilyl ethers and the products were analysed directly by capillary gas chromatography with selected ion monitoring using [ 2H 4]chenodeoxycholic and [ 2H 4]3α-hydroxy-7-oxo-5β-cholanoic acids as internal standards. The bile acid peaks on the selected ion chromatogram were separated without interference from endogenous substances present in serum. Recoveries of individual keto bile acids added to serum range from 74.4 to 94.7% with a mean of 87.1%. Eight kinds of keto bile acids not previously found in sera of normal subjects, namely 3-oxo-, 3-oxo-7α-hydroxy-, 3-oxo-12α-hydroxy, 3α-hydroxy-7-oxo, 3α-hydroxy-12-oxo-, 3-oxo-7α, 12α-dihydroxy-, 3α,7α-dihydroxy-12-oxo- and 3α,12α-dihydroxy-7-oxo-5β-cholanoic acids were identified and quantified. The total concentration of keto bile acids was found to be 0.16±0.08 nmol/ml and constituted 2.9±1.5% of that of the usual non-keto bile acids in peripheral venous serum.