An automated column-switching technique coupled to isocratic high-performance liquid chromatography (HPLC) with fluorescence detection was developed for simultaneous determination of dextromethorphan and its three major metabolites, dextrorphan, hydroxymorphinan, and methoxymorphinan. After cleavage of conjugates by incubation with glucuronidasearylsulfatase at 37 degrees C for 15 h, plasma samples were injected directly into the HPLC system. Dextromethorphan and metabolites were retained on a cleanup column (10 x 4.6 mm internal diameter [ID]) filled with cyanopropyl (CN) material (Hypersil CPS, 10-microns article size) while interfering proteins and lipids were washed to waste. After column switching, the drugs were eluted from the cleanup column and separated on Spherisorb CN material (5-microns particle size, column size 250 x 4.6 mm ID). Fluorescence detection was carried out with an excitation wavelength of 220 nm and an emission wavelength of 305 nm. Sample cleanup and HPLC separation were completed within 20 min. Regression analyses found linearity (r > 0.99) between drug concentration and detector response over a wide range-5-220 ng/ml for dextromethorphan, 5-550 ng/ml for dextrorphan, 5-500 ng/ml for hydroxymorphinan, and 5-200 ng/ml for methoxymorphinan. The limit of quantification was approximately 5 ng/ml, and the recovery was > 90% for all compounds. At concentrations of 20-500 ng/ml, the intra- and interassay coefficients of variation ranged from 3.5 to 14.6% and from 7.0 to 14.0%, respectively. The method is suitable for in vivo phenotyping of CYP2D6 activity, which catalyzes the O-demethylation of dextromethorphan to dextrorphan, and is also applicable to pharmacokinetic studies in man.
This pilot study was conducted to evaluate the potential of the new antidepressant moclobemide to inhibit the cytochrome enzyme P4502D6 (CYP2D6) using the cough suppressant dextromethorphan as a substrate in four extensive metabolizers (EM) of debrisoquine. The subjects received seven oral doses of 20 mg dextromethorphan at 4-h intervals over 2 days (1 and 2) and subsequently moclobemide (300 mg b.i.d.) for 9 days. On days 10 and 11, they received seven doses of 20 mg dextromethorphan in addition to moclobemide. During monotreatment and combined treatment, blood was collected on days 2 and 11, respectively, for determination of dextromethorphan and its demethylated metabolites using automated high-performance liquid chromatography with column switching. Concurrent administration of moclobemide markedly reduced the O-demethylation of dextromethorphan, whereas the N-demethylation of dextrorphan to hydroxymorphinan was not affected. The findings indicate that moclobemide can affect the pharmacokinetics of drugs that are mainly metabolized by CYP2D6.
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