Separation and Determination of Bile Acids in Human Bile by High-Performance Liquid Chromatography

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Abstract
A method for simultaneous determination of major bile acids in human bile is described. The unconjugated, glycine- and taurine-conjugated bile acids are extracted with Sep-pak C18 and separated into groups by ion-exchange chromatography on a lipo-philic gel. Subsequently, resolution of each group into ursodeoxycholate, cholate, chenodeoxycholate, deoxycholate and lithocholate is attained into 2 stages by high-performance liquid chromatography on a Radial-PAK A column. First, 0.3% ammonium phosphate (pH 7.7)/acetonitrile (19:8, vol/vol) is used for separation of the latter 3 as a mobile phase. Ursodeoxycholate and cholate are efficiently separated in 0.3% ammonium phosphate (pH 7.7)/acetonitrile (23:8, vol/vol). The present method is applicable to quantitation of bile acids in human bile with satisfactory accuracy and precision.

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