A fast and reliable LC-MS/MS method for simultaneous quantitation of fluoxetine and mirtazapine in human plasma

A rapid and sensitive ultra-performance liquid chromatographic/tandem mass spectrometric assay (LC-MS/MS) was developed to simultaneously quantify fluoxetine and mirtazapine in human plasma using fluoxetine-D5 and olanzapine as internal standards (IS), respectively. The analytes and the internal standards (IS) were extracted from 400 μL aliquots of human plasma through liquid–liquid extraction. Chromatographic separation was achieved in a run time of 2.0 min on the X-terra RP8 (50 × 4.6 mm, 5 μm particle size) column. The isocratic mobile phase consisting of a mixture of acetonitrile and 10 mM ammonium acetate (90:10, v/v) at a flow-rate of 0.50 mL min−1 was found to be optimum. Quantitation of analytes was performed by electrospray ionization tandem mass spectrometry, operating in positive-ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursors to product ion transitions monitored for fluoxetine, mirtazapine, fluoxetine-D5 and olanzapine were at m/z 310.20 → 148.17, 266.35 → 195.31, 315.20 → 153.17 and 313.19 → 256.12, respectively. The method was validated over the concentration range of 0.050–50.037 ng mL−1 for fluoxetine and 0.100–100000 ng mL−1 for mirtazapine in human plasma. The method has shown high reproducibility with intra-batch and inter-batch precision (CV%) less than 10.16% across four quality control levels for both the analytes. The assay was linear over the concentration range of 0.050–50.037 ng mL−1 for fluoxetine (r2 = 0.9988) and 0.100–100000 ng mL−1 for mirtazapine (r2 = 0.9975). The method is suitable for measuring accurate concentration of the two analytes in bioequivalence study and therapeutic drug monitoring following combined administration.