Separation and detection of uv-absorbing derivatives of fecal bile acid metabolites by high-performance liquid chromatography.

B Shaikh, N J Pontzer, J E Molina, M I Kelsey

Index: Anal. Biochem. 85 , 47, (1978)

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Abstract

Major fecal bile acid metabolites related to lithocholic acid were resolved by high-performance liquid chromatography (hplc). The uv-absorbing p-nitrobenzyl ester derivatives of lithocholic, isolithocholic, 3-keto-5β-cholanic, and 5β-cholanic acids were prepared using the reagent o,p-nitrobenzyl- N,N′-diisopropylisourea. Separation was achieved in less than 20 min on a microparticulate silica column using isocratic elution with 2% isopropanol in isooctane as the mobile phase. The p-chlorobenzoyl esters of methylated lithocholic and isolithocholic acids were also prepared but required purification by thin-layer chromatography before separation by hplc. These derivatives were eluted from a Porasil T column using 5% diisopropyl ether in isooctane as the mobile phase. Lithocholic and isolithocholic acids produced by microbial metabolism of [ 14COOH]taurolithocholic acid were separated and identified by preparing p-nitrobenzyl derivatives and monitoring the column effluent for both uv and radioactivity. This technique is a rapid and sensitive method for isolating bile acid metabolites.