Capillary gas-liquid chromatography of glycine-conjugated bile acids without prior hydrolysis.

Abstract

A method is described for the gas-liquid chromatographic (GLC) analysis of intact glycine conjugates of the major bile acids present in human plasma. It is, therefore, now possible to analyze glycine-conjugated and unconjugated bile acids together on a single GLC column without the necessity for a hydrolytic step. A large number of derivatives of bile acid glycine conjugates were examined, but only acetate- and silyl ether-derivatives of carboxylic acid methyl esters were found initially to be suitable. It was not possible to make acetates consistently, and trimethylsilyl ethers did not allow resolution of the glycine conjugates of cholic and chenodeoxycholic acids. Dimethylethylsilyl ether methyl ester derivatives were subsequently found to give the best results. Chromatographic conditions for successful analysis of these derivatives were examined and it was found to be necessary to use wall-coated capillary columns of thin film thickness (0.12 micron) and very high carrier gas flow rates (ca. 20 ml/min hydrogen). Using acetonitrile and Bond Elut extraction, fractionation on Sep-Pak SIL cartridges, and derivatization as dimethylethylsilyl ether methyl esters, the capillary gas-liquid chromatography of intact glycine-conjugated bile acids from human plasma was demonstrated for the first time.