USE OF AN IMMOBILIZED ENZYME REACTOR FOR THE ANALYSIS OF RESIDUES OF 17-ALPHA-METHYLTESTOSTERONE IN TROUT BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

We have recently observed in trout that 48 h after ingestion of a single dose of [C-14]-17-alpha-methyltestosterone ([C-14]-17-MT), 25% of the radioactivity was still in the carcass, corresponding to metabolites of 17-MT. These compounds have no appreciable chromophore, fluorophore or electrophore, therefore the usual detection systems were not satisfactory for their analysis. Consequently a method was developed for high-performance liquid chromatographic separation and detection of two of the major tissue metabolites of 17-MT: 5-alpha-androstane-17-alpha-methyl-3-alpha, 17-beta-diol and 5-beta-androstane-17-alpha-methyl-3-alpha, 17-beta-diol. A column of immobilized 3-alpha-hydroxysteroid dehydrogenase was prepared and used for detection. The NADH produced from 3-hydroxysteroids by this immobilized enzyme reactor was monitored fluorimetrically. The detection limit of this method, as obtained from the calibration curve, was at the picomole level; the limit of quantification in muscle was 1-mu-g/kg, at a signal-to-noise ratio of 4.