Determination of bile acids and their conjugates in serum by HPLC using immobilized 3α‐hydroxysteroid dehydrogenase for detection

A method is described for the separation of unconjugated bile acids and their glycine and taurine conjugates in a single step with adequate sensitivity for analysis of serum samples. Separation was carried out over a period of 80 min using a linear gradient with increasing concentrations of methanol in aqueous ammonium dihydrogen phosphate buffer, on an ODS Spherisorb column. Spectrofluorometric detection of NADH, formed as the column eluate passed through a column of immobilized 3 alpha-hydroxysteroid dehydrogenase, enabled amounts less than 40 pmol to be quantified. The reaction was carried out at neutral pH so that the lifetime of the enzyme column was increased, compared to other methods where a pH nearer 9.0 is commonly used. Flow rates were optimized to give comparable peak area for both primary and secondary bile acids.