Published:Journal of Chromatographic Science,
ISSN 0021-9665Volume
40, Number 7, August 2002, pp. 397-402
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)
Hongxia Li and Robert J. Letcher
Great Lakes Institute for Environmental Research, University of Windsor, Windsor,
ON, N9B 3P4, Canada
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 µm,
250- ¥ 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 6b-, 16b-,
16a-, and 7a-hydroxytestostone
and androstenedione are 14, 3, 3, 14, and 3 pmol (20 µL injection), respectively.
Eleven hydroxytestosterone metabolites are detected after in vitro testosterone
incubation with hepatic microsomes of gray seals. Six are identified as 6b-,
7a-, 16a-, 16b-,
and 2b-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 6b-, 7a-,
16a-, and 16b-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.
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