Thin-layer separation and quantification of bile acids

A method has been developed for quantification of total free and conjugated bile acids separated on silica gel HR coated thin-layer chromatography plates. Aliquots of bile acid solutions are applied to channeled plates which are developed with either ethyl acetates: isooctane:glacial acetic acid 10:10:2 v/v for free bile acid separation, or chloroform:methanol:glacial acetic acid:water 130:50:4:8 V/V for conjugated bile acid separation. Bile acids are determined directly in serial areas of silica gel by treating gel areas suspended in tris buffer with resazurin reagent. The method is quantitative and as little as 0.1 microgram of bile acid is readily determined. Application of the method to determinations of bile acids in crude fecal extracts is described.