RESUMEN
A rapid, sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method was developed for the simultaneous assay of dextromethorphan and its metabolites in tissue culture medium and its intestinal metabolism studied with the rat everted gut sac model. The method was validated in the concentration range of 0.1-2.5 microM (27.1 ng/mL-0.677 microg/mL) for dextromethorphan and 0.005-0.5 microM for dextrorphan and 3-methoxymorphinan (1.28 ng/mL-0.128 microg/mL) and 3-hydroxymorphinan (1.22 ng/mL-0.122 microg/mL). The limits of quantification (LOQ) were 0.0025 microM (12.5 fmoles, 3.4 pg, 5 microL injected) for dextromethorphan; 0.0025 microM for dextrorphan, 3-methoxymorphinan (24.9 fmoles, 6.4 pg injected), and 3-hydroxymorphinan (25.1 fmoles, 6.1 pg injected) with 10 microL injected. The detection of dextrorphan and 3-methoxymorphinan showed that both the P450 isoforms CYP3A and 2D were active in the intestinal mucosa and metabolised dextromethorphan during its passage across the mucosa.
Asunto(s)
Cromatografía Liquida/métodos , Dextrometorfano/análogos & derivados , Dextrometorfano/análisis , Dextrometorfano/metabolismo , Intestino Delgado/metabolismo , Espectrometría de Masas/métodos , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A , Dextrorfano/análisis , Dextrorfano/metabolismo , Técnicas In Vitro , Mucosa Intestinal/enzimología , Masculino , Oxidorreductasas N-Desmetilantes/metabolismo , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
A rapid, sensitive and specific method was developed for the simultaneous assay of testosterone, androstenedione and 6beta-hydroxytestosterone (6beta-OHT) in the TC199 tissue culture medium used in intestinal drug metabolism studies with the rat everted gut sac model. An electrospray LC-MS method was validated in the concentration range of 0.025-9.5 microM (7.2 ng-2.7 microg/mL) for testosterone and androstenedione and 0.01-4 microM (3 ng-1.2 microg/mL) for 6beta-hydroxytestosterone. The limits of quantification (LOQ) with an injection volume of 10 microL were 0.0005 microM (4.9 fmol, 1.4 pg injected), 0.004 microM (0.04 pmol, 11.4 pg injected) and 0.03 microM (0.3 pmol, 91 pg injected), respectively. The method also detected the other testosterone metabolites, the 16alpha-, 16beta-, 2beta- and 2alpha-hydroxytestosterones and was then used to study the metabolism of testosterone during its absorption by rat intestine in vitro, using everted gut sacs.