Simultaneous determination of Olanzapine and Fluoxetine in human plasma by LC-MS/MS: its pharmacokinetic application.

A simple and rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous quantitation of Olanzapine and Fluoxetine in human plasma using Olanzapine-d3 and Fluoxetine-d5 HCl as internal standard (IS), respectively. After solid phase extraction of the plasma samples on Waters Oasis HLB Catridges, Olanzapine, Fluoxetine and IS were chromatographed on Thermo Hypersil Gold C18 (50 mm × 4.6 mmi.d., 5 μm) analytical column with isocratic elution using methanol: 2mM Ammonium acetate buffer (90:10). Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique and operating in multiple reaction monitoring (MRM) and positive ion mode with transitions at 313/256 for Olanzapine and 310/148 for Fluoxetine. The total chromatographic run time was 2.0 min and calibration curves were linear over the concentration range of 0.10-20.00 ng/mL for Olanzapine and 0.50-50.00 ng/mL for Fluoxetine. The method was validated for selectivity, sensitivity, recovery, linearity, accuracy and precision and stability studies. The recoveries obtained for the Olanzapine and its IS was ≥87% and Fluoxetine and its IS was ≥91%. Recoveries obtained were consistent and reproducible. Inter-batch and intra-batch coefficient of variation across three validation runs (LLOQ, LQC, MQC1, MQC and HQC) was less than 3.6 for Olanzapine and less than 5.2% for Fluoxetine. The method was successfully applied to a pharmacokinetic study of fixed dose combination of Olanzapine/Fluoxetine in healthy male volunteers.