Separation and determination of bile acids by high-performance liquid chromatography using immobilized 3 alpha-hydroxysteroid dehydrogenase and an electrochemical detector.

A high-performance liquid chromatographic method using an immobilized 3 alpha-hydroxysteroid dehydrogenase column and an electrochemical detector was developed for the determination of individual bile acids in serum and bile. Bile acids in the eluate from a Radial-Pak A column reacted with NAD in the enzyme column to generate NADH, which was monitored by a voltammetric detector after mixing with phenazine methosulphate solution. Each bile acid was measurable at the 20 pmole level at the highest sensitivity of the detector. The mean recoveries and reproducibilities of bile acids were 86.7-104.6% [coefficient of variation (C.V.) = 0.3-8.7%] within-assay and 83.8-103.4% (C.V. = 1.6-7.0%] between-assay.