Journal of Pharmaceutical and Biomedical Analysis 90 (2014) 64–71 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis jou rn al hom epage: www.elsevier.com/locate/jpba Simultaneous determination of Olanzapine and Fluoxetine in human plasma by LC–MS/MS: Its pharmacokinetic application S.L. Bonde a , R.P. Bhadane a , Avinash Gaikwad b,∗ , S.R. Gavali b , D.U. Katale b , A.S. Narendiran b a Nowrosjee Wadia College, University of Pune, India b Bioanalytical Department of VerGo Clinicals, Goa, India a r t i c l e i n f o Article history: Received 12 April 2013 Received in revised form 21 October 2013 Accepted 23 October 2013 Keywords: Olanzapine Fluoxetine LC–MS/MS Human plasma Bioequivalence a b s t r a c t 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 mm i.d., 5 m) analytical column with isocratic elution using methanol: 2 mM 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 Flu- oxetine. 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 coef- ficient 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. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Treatment-resistant major depressive disorder remains a major therapeutic challenge in clinical practice. Between 10% and 30% of depressed patients fail to respond to antidepressant treatment. Augmentation strategies have proved effective for many suffering from this treatment-resistant depression [1]. The FDA-approved SYMBYAX combines Fluoxetine and Olanzapine produced signifi- cant improvement over monotherapy with either drug. In addition, the Olanzapine–Fluoxetine combination also appears to be ben- eficial for other difficult-to-treat depressive disorders, including psychotic depression and bipolar depression [2]. The adult dosage regimen is 25 and 6 mg for Fluoxetine and Olanzapine, respectively. Severe depression and suicidal ideation are generally manifested in patients with a pre-existing history of depression [3]. Therefore, patient’s therapy should be closely monitored through a validated method. ∗ Corresponding author. Tel.: +91 9765550740. E-mail addresses: avinashbg171@gmail.com, avinash.gaikwad@vergopharma.com (A. Gaikwad). Literature survey reveals several methods for the determina- tion of Olanzapine in biological fluids using UV detection [4–6], amperometric detection [7], electrochemical detection [8–12] and liquid chromatography–tandem mass spectroscopy (LC–MS/MS) [13–16]. Similarly, a number of methods for the determination of Fluoxetine alone or Fluoxetine with its metabolites in bio- logical fluids using Fluorescence detection [17–23] and liquid chromatography–tandem mass spectroscopy (LC–MS/MS) [24–27]. More recently, Gopinath et al. [28] reported a method for the simultaneous determination of Olanzapine and Fluoxetine in plasma samples by liquid chromatography–tandem mass spec- troscopy (LC–MS/MS). The aim of the present article was to develop and validate a simple, specific and sensitive LC–MS/MS method, using a solid phase extraction procedure to quantify Olanzapine and Fluoxe- tine. The present work employs single step solid phase extraction technique using Oasis HLB Cartridges for sample preparation and liquid chromatography with electrospray ionization–tandem mass spectrometry for simultaneous quantitation of Fluoxetine and Olanzapine in human plasma. Use of deuterated internal standard helped in getting improved precision and accuracy for the quan- tification of the analytes and the shorter runtime of the method is 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.10.033