Simultaneous Determination of Methadone, Fluoxetine, Venlafaxine and Their Metabolites in Rat Plasma by UPLC–MS/MS for Drug Interaction Study

Abstract

A sensitive and rapid ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed to determine methadone, EDDP, fluoxetine, norfluoxetine, venlafaxine and O-desmethylvenlafaxine in rat plasma using diazepam as the internal standard (IS). Sample preparation was accomplished through a simple one-step protein precipitation with 200 µL acetonitrile added to 100 µL rat plasma. The analytes were separated on an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm) and the mobile phase was acetonitrile and 0.1 % formic acid in water in gradient elution at a flow rate of 0.40 mL min⁻¹. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions. The linearity of this method was found to be within the concentration range of 0.5–250.0 ng mL⁻¹ for methadone, EDDP, fluoxetine and norfluoxetine and 0.4–200.0 ng mL⁻¹ for venlafaxine and O-desmethylvenlafaxine, respectively. The limit of quantification (LOQ) was 0.5 ng mL⁻¹ for methadone, EDDP, fluoxetine and norfluoxetine and 0.4 ng mL⁻¹ for venlafaxine and O-desmethylvenlafaxine, respectively. A time period of 3 min was needed for an analytical run. The method described above was successfully applied to the subsequent pharmacokinetic study.