Quantification of Fluoxetine in Human Plasma by the Development of Liquid Chromatography-tandem Mass Spectrometry Method

Aim: The aim of this research paper is the development and validation of an easy, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of fluoxetine in human K3EDTA plasma and application of this method on bioequivalence studies of fluoxetine. Methods: As Amitriptyline belongs to same category drug so, it was selected as an internal standard for the quantification of fluoxetine. The protein precipitation (PPT) method was used to extract analyte from 250 μl aliquot of human plasma. Chromatographic separation was achieved on BDS Hypersil C18 (50 x 4.6 mm, 5 μm) column at in 4.0 min run time using isocratic mobile phase consisting of acetonitrile and mixture of ammonium acetate containing 0.15% formic acid (55:45 % v/v) at a flow rate of 0.5 ml/min. The ionization was carried out through Electron spray ionization (ESI) operating in positive ion mode and detection was via multiple reaction monitoring (MRM) acquisition mode using the respective m/z 310.1→44.2 for fluoxetine and 278.1→233.1 for IS. The method was validated to be linear over the concentration range 0.25 to 40.00 ng/ml. Results: This LC-MS/MS method was found to be accurate and precise with intra-batch and inter-batch accuracy (% Nominal) of ±15 % and precision of <15 % and the method was successfully applied in analyzing human plasma samples of fluoxetine.