A high-performance-liquid-chromatography-based method for the determination of hydroxylated testosterone metabolites formed in vitro in liver microsomes from gray seal (Halichoerus grypus).

A reproducible and sensitive high-performance-liquid-chromatography (HPLC)-based method with UV-vis detection is developed and optimized for the determination of hydroxytestosterone compounds formed via the cytochrome P450 enzyme-mediated metabolism of testosterone. The method is used to characterize and quantitate hydroxytestosterone metabolites formed in vitro via testosterone incubation with hepatic microsomes from the liver of gray seals (Halichoerus grypus). The HPLC method employs a Zorbax Eclipse XDB-C18 column (5 microm, 250- x 4.6-mm i.d.) and a combination of step gradient and solvent systems of mixtures of acetonitrile, methanol, and water. Metabolites are detected at 254 nm. The eluted peaks of 10 testosterone metabolite standards are well-resolved and a flat baseline is maintained over the elution period of the entire chromatogram. The instrumental detection limits (signal-to-noise ratio = 3) of 6beta-, 16beta-, 16alpha-, and 7alpha-hydroxytestostone and androstenedione are 14, 3, 3, 14, and 3 pmol (20 microL injection), respectively. Eleven hydroxytestosterone metabolites are detected after in vitro testosterone incubation with hepatic microsomes of gray seals. Six are identified as 6beta-, 7alpha-, 16alpha-, 16beta-, and 2beta-hydroxytestosterone and androstenedione. In order of abundance, the formation rates are 2100, 39.6, 12.8, 26.2, and 132 pmol/mg protein/min for 6beta-, 7alpha-, 16alpha-, and 16beta-hydroxytestosterone and androstenedione, respectively. The within-day precision (relative standard deviation) is less than 3% for testosterone metabolites. Five relatively substantial peaks are detected but not identified.