High-performance liquid chromatography assay for simultaneous determination of dextromethorphan and its main metabolites in urine and in microsomal preparations

An HPLC method has been developed and validated for the determination of de xtromethorphan, dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan in u rine samples. Deconjugated compounds were extracted on silica cartridges us ing dichloromethane/hexane (95:05, v/v) as an eluent. Chromatographic separ ation was accomplished on a Phenyl analytical column serially connected wit h a Nitrile analytical column. The mobile phase consisted of a mixture of a n aqueous solution, containing 1.5% acetic acid and 0.1% triethylamine, and acetonitrile (75:25, v/v). Compounds were monitored using a fluorescence d etector. Calibration curves were linear over the range investigated (0.2-8. 0 muM) with correlation coefficients >0.999. The method was reproducible an d precise. Coefficients of variation and deviations from nominal values wer e both below 10%, For all the analytes, recoveries exceeded 77% and the lim its of detection were 0.01 muM The validated assay proved to be suitable fo r the determination of DEM metabolic indexes reported to reflect the enzyma tic activity of the cytochrome P450s, CYP2D6 and CYP3A, both in vivo, when applied to urine samples from patients. and in vitro, when applied to sampl es from the incubation of liver microsomes with dextromethorphan. (C) 2001 Elsevier Science B.V. All rights reserved.