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An LC/MS/MS method for the simultaneous determination of cilostazol, a quinolinone derivative, and three active metabolites, OPC-13015, OPC-13213, and OPC-13217, in human plasma was developed and validated. Cilostazol, its metabolites, and the internal standard, OPC-3930 were extracted from human plasma by liquid-liquid partitioning followed by solid-phase extraction (SPE) on a Sep-Pak silica column. The eluent from the SPE column was then evaporated and the residue reconstituted in a mixture of methanol/ammonium acetate buffer (pH 6.5) (2:8 v/v). The analytes in the reconstituted solution were resolved using reversed-phase chromatography on a Supelcosil LC-18-DB HPLC column by an 17.5-min gradient elution. Cilostazol, its metabolites, and the internal standard were detected by tandem mass spectrometry with a Turbo Ionspray interface in the positive ion mode. The method was validated over a linear range of 5.0-1200.0 ng/ml for all the analytes. This method was demonstrated to be specific for the analytes of interest with no interference from endogenous substances in human plasma or from several potential concomitant medications. For cilostazol and its metabolites, the accuracy (relative recovery) of this method was between 92.1 and 106.4%, and the precision (%CV) was between 4.6 and 6.5%. During the validation, standard curve correlation coefficients equalled or exceeded 0.999 for cilostazol and its metabolites. These data demonstrate the reliability and precision of the method. The method was successfully cross-validated with an established HPLC method.

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.