Simultaneous assay for individual sulphated 3 alpha- and beta-hydroxysteroids in serum using high-performance liquid chromatography combined with 3 alpha- and beta-hydroxysteroid dehydrogenases immobilized on one column.

A high-performance liquid chromatographic method for the simultaneous determination of individual sulphated 3 alpha- and beta-hydroxysteroids in serum using 3 alpha- and beta-hydroxysteroid dehydrogenases (3 alpha-HSD and beta-HSD, respectively) immobilized on one column and a fluorimeter to detect the resulting NAD+ to NADH transformation is described. Individual sulphated 3 alpha- and beta-hydroxysteroids in serum are extracted with ethanol, solvolysed with sulphuric acid in ethyl acetate and then separated by high-performance liquid chromatography. The hydroxysteroids thus separated are subsequently mixed with NAD+ and then passed through the column in which the following catalytic reaction occurs: (formula, see text) The detection limits are as low as 0.5-1.0 microgram/dl for sulphated 3 alpha- or beta-hydroxysteroids in serum. The present assay method is highly specific, reliable and reproducible and is thus applicable to a clinical study on the metabolism of sulphated 3 alpha- and beta-hydroxysteroids in patients with adrenal or gonadal diseases.