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 0.100-100 000 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 0.100-100 000 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.