High-performance liquid chromatographic method for the determination of moclobemide and its two major metabolites in human plasma

Abstract

A selective, sensitive, and simple high-performance liquid chromatographic (HPLC) method was developed for the determination of moclobemide and its two major metabolites, Ro 12-5637 and Ro 12-8095, in human plasma. Sample preparation (0.5 ml of plasma) involved solid-phase extraction (SPE) using Speedisk (R) H2O-Philic DVB columns. Separations were performed on a Waters XTerra (TM) RP18 column (5 mu m, 150 mm x 4.6 mm). The mobile phase consisted of 10 mM KH2PO4 with 1% triethylamine (pH 3.9) and acetonitrile (93:17, v/v), and a flow-rate was 1.2 ml/min. The total run time was 13 min. UV detection was performed at 240 nm. Mean absolute recoveries were >= 90% and the limit of quantification (LOQ) for all analytes was 0.02 mg/l. Calibration curves were linear (r > 0.995) over a wide range of the analyte concentrations in plasma; thus, the method is suitable for different clinical studies when large variations in the drug/metabolites concentrations are observed. During a 5-day assay validation procedure the accuracy and precision were tested and proven (relative errors (RE) lt = 13%; intra-day coefficient of variation (CV) lt = 7%; inter-day CV lt = 13%). Many drugs frequently used in the target patient population were evaluated for potential interference in order method selectivity to be ensured. The assay has been used in a clinical pharmacokinetic study to assess steady-state pharmacokinetics of moclobenide and two metabolites in depressive patients on mono- and combined therapy.