A sensitive LC-MS/MS assay for the determination of dextromethorphan and metabolites in human urine--application for drug interaction studies assessing potential CYP3A and CYP2D6 inhibition

The commonly used antitussive dextromethorphan can be used to simultaneously assess potential cytochrome P450 3A (CYP3A) and CYP2D6 inhibition during drug development. The metabolism of dextromethorphan to dextrorphan and subsequently to 3-hydroxymorphinan are via the 2D6 pathway, while the metabolism of dextromethorphan to 3-methoxymorphinan is via the 3A pathway. A sensitive and specific LC-MS/MS assay has been developed to determine the human urine concentrations of dextromethorphan and three metabolites (dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan) in support of drug interaction studies. Urine samples (0.5 ml), after enzymatic hydrolysis of the conjugates and containing 3-ethylmorphine as an internal standard, were extracted with chloroform under basic conditions. Following concentration and reconstitution, the samples were analyzed by LC-MS/MS. The assay was linear over the range of 5.00-500 ng/ml for dextromethorphan and 3-methoxymorphinan; and 200-3000 ng/ml for dextrorphan and 3-hydroxymorphinan using a Perkin-Elmer Sciex triple quadrupole mass spectrometer (API 300). The intra- and inter-day relative standard deviation (RSD) across three validation runs over the entire concentration range for all analytes was less than 15%. Accuracy determined at three or four concentrations (9.00, 200, and 400 ng/ml for dextromethorphan and 3-methoxymorphinan; 250, 400, 1300 and 2500 ng/ml for dextrorphan and 3-hydroxymorphinan) ranged between 96.3 and 113.8%. The stability of analytes in urine was demonstrated for 9 months at -20 degrees C, 24 h under ambient conditions and for up to three freeze/thaw cycles. The method described herein is suitable for the rapid and efficient measurement of dextromethorphan and different metabolites to estimate potential CYP3A inhibition by drug candidates and for screening of extensive and poor metabolizers of CYP2D6 in the heterogeneous population. The method has subsequently been validated on a Sciex API 3000 with lower limit of quantitation; 1.00 ng/ml for dextromethorphan and 3-methoxymorphinan; 60.0 ng/ml for dextrorphan and 100 ng/ml for 3-hydroxymorphinan.