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)
Identifieur interne : 001759 ( Istex/Curation ); précédent : 001758; suivant : 001760A High-Performance-Liquid-Chromatography-Based Method for the Determination of Hydroxylated Testosterone Metabolites Formed In Vitro in Liver Microsomes from Gray Seal (Halichoerus grypus)
Auteurs : Hongxia Li [Canada] ; Robert J. Letcher [Canada]Source :
- Journal of Chromatographic Science [ 0021-9665 ] ; 2002-08.
Abstract
A reproducible and sensitive high-performance-liquidchromatography (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 6β-, 16β-, 16α-, and 7α-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 6β-, 7α-, 16α-, 16β-, and 2β-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 6β-, 7α-, 16α-, and 16β-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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DOI: 10.1093/chromsci/40.7.397
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Letcher</name><affiliation wicri:level="1"><mods:affiliation>Great Lakes Institute for Environmental Research, University of Windsor, Windsor, ON, N9B 3P4, Canada</mods:affiliation><country xml:lang="fr">Canada</country><wicri:regionArea>Great Lakes Institute for Environmental Research, University of Windsor, Windsor, ON, N9B 3P4</wicri:regionArea></affiliation><affiliation wicri:level="1"><mods:affiliation>E-mail: letcher@uwindsor.ca</mods:affiliation><country wicri:rule="url">Canada</country></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:CB723330190B15CF4F60C331F370D28CB4A159BE</idno><date when="2002" year="2002">2002</date><idno type="doi">10.1093/chromsci/40.7.397</idno><idno type="url">https://api-v5.istex.fr/document/CB723330190B15CF4F60C331F370D28CB4A159BE/fulltext/pdf</idno><idno type="wicri:Area/Istex/Corpus">001759</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001759</idno><idno type="wicri:Area/Istex/Curation">001759</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a">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)</title><author><name sortKey="Li, Hongxia" sort="Li, Hongxia" uniqKey="Li H" first="Hongxia" last="Li">Hongxia Li</name><affiliation wicri:level="1"><mods:affiliation>Great Lakes Institute for Environmental Research, University of Windsor, Windsor, ON, N9B 3P4, Canada</mods:affiliation><country xml:lang="fr">Canada</country><wicri:regionArea>Great Lakes Institute for Environmental Research, University of Windsor, Windsor, ON, N9B 3P4</wicri:regionArea></affiliation></author><author><name sortKey="Letcher, Robert J" sort="Letcher, Robert J" uniqKey="Letcher R" first="Robert J." last="Letcher">Robert J. 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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 6β-, 16β-, 16α-, and 7α-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 6β-, 7α-, 16α-, 16β-, and 2β-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 6β-, 7α-, 16α-, and 16β-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.</div></front></TEI></record>Pour manipuler ce document sous Unix (Dilib)
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