Quantitative analysis of bile acids in serum and bile, using gas -liquid chromatography

Abstract

The results of a gas—liquid chromatographical analysis of individual bile acids in serum and bile are presented. The analysis of bile acids in serum involved enzymatic hydrolysis prior to protein precipitation, purification according to Roovers et al. [2], diethyl ether extraction, preparation of trifluoroacetate methylester derivatives and gas—liquid chromatographical analysis with 1% OV-210 on Gaschrom Q as stationary phase. Protein precipitation and special purification steps were omitted in the routine analysis of bile acids in bile. With 7-ketodeoxycholic acid as internal standard adequate separation of the individual bile acids-cholic acid (chenodeoxycholic acid, deoxycholic acid and lithocholic acid) was obtained. There was a linear correlation between the dose injected and the peak height response. Recovery studies with unconjugated and conjugated bile acids, added to serum, water and bile, revealed good reproducibility. The only significant loss, mainly of cholic and lithocholic acid, occurred during the hydrolysis step. Special attention was paid to purification and diethyl ether extraction to minimize loss of bile acids during these steps. Recovery of conjugated bile acids, after correction for loss in preparation of trifluoroacetate methylester derivatives and in the gas-chromatographic procedure, was 80–95% in serum and 83–100% in bile. Duplicate analyses in serum and bile samples proved that reproducibility was good. Numerous estimates were made in serum and bile from fasting normal persons in order to establish valid and reliable normal values.