Quantitative isolation and gas–liquid chromatographic analysis of total dietary and fecal neutral steroids

Abstract

A method for isolation and quantification of fecal neutral steroids is described which allows studies to be made of sterol balance in man or in small laboratory animals without requiring the use of radioisotopes in vivo. The critical separations of cholesterol plant sterols and their conversion products depend upon preliminary separations into three subfractions by thin-layer chromatography. Individual components in the three subfractions thus obtained are then quantitatively measured by gas–liquid chromatography of the unsubstituted 3-ketosteroids and of the trimethylsilyl ethers of the sterols.Extractions of cholesterol-7-α-H3 added in vitro and of C14-labeled neutral steroids synthesized in vivo were quantitative and highly reproducible. Several lines of evidence validate the determination of individual fecal neutral steroids by GLC.Examples are given of the application of this technique: a sterol balance study of 27 days' duration is described in a patient whose diet included plant sterols as well as cholesterol. Representative results in man and in rats are compared to others obtained by previously described methods.The sensitivity of the method is such that 1-g fecal aliquots containing as little as 25 μg of mixed neutral steroids can be analyzed accurately, but the procedure lends itself well to preparative scale work for more definitive study of individual neutral steroids.

Keywords

• fecal neutral steroids
• quantitative recoveries
• thin-layer chromatography
• gas–liquid chromatography
• trimethylsilyl ethers
• hydrogen flame detection