Validation of a liquid chromatography–mass spectrometry method to assess the metabolism of dextromethorphan in rat everted gut sacs

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 μM (27.1 ng/mL–0.677 μg/mL) for dextromethorphan and 0.005–0.5 μM for dextrorphan and 3-methoxymorphinan (1.28 ng/mL–0.128 μg/mL) and 3-hydroxymorphinan (1.22 ng/mL–0.122 μg/mL). The limits of quantification (LOQ) were 0.0025 μM (12.5 fmoles, 3.4 pg, 5 μL injected) for dextromethorphan; 0.0025 μM for dextrorphan, 3-methoxymorphinan (24.9 fmoles, 6.4 pg injected), and 3-hydroxymorphinan (25.1 fmoles, 6.1 pg injected) with 10 μL 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.