Simultaneous quantification of selective serotonin reuptake inhibitors and metabolites in human plasma by liquid chromatography-electrospray mass spectrometry for therapeutic drug monitoring

A simple and sensitive liquid chromatography-electrospray ionization mass spectrometry method was developed for the simultaneous quantification in human plasma of all selective serotonin reuptake inhibitors (citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline) and their main active metabolites (desmethyl-citalopram and norfluoxetine). A stable isotope-labeled internal standard was used for each analyte to compensate for the global method variability, including extraction and ionization variations. After sample (250 mu l) pre-treatment with acetonitrile (500 mu l) to precipitate proteins, a fast solid-phase extraction procedure was performed using mixed mode Oasis MCX 96-well plate. Chromatographic separation was achieved in less than 9.0 min on a XBridge C18 column (2.1 x 100 mm; 3.5 mu m) using a gradient of ammonium acetate (pH 8.1: 50 mM) and acetonitrile as mobile phase at a flow rate of 0.3 ml/min. The method was fully validated according to Societe Francaise des Sciences et Techniques Pharmaceutiques protocols and the latest Food and Drug Administration guidelines. Six point calibration curves were used to cover a large concentration range of 1-500 ng/ml for citalopram, desmethyl-citalopram, paroxetine and sertraline, 1-1000 ng/ml for fluoxetine and fluvoxamine, and 2-1000 ng/ml for norfluoxetine. Good quantitative performances were achieved in terms of trueness (84.2-109.6%), repeatability (0.9-14.6%) and intermediate precision (1.8-18.0%) in the entire assay range including the lower limit of quantification. Internal standard-normalized matrix effects were lower than 13%. The accuracy profiles (total error) were mainly included in the acceptance limits of +/- 30% for biological samples. The method was successfully applied for routine therapeutic drug monitoring of more than 1600 patient plasma samples over 9 months. The beta-expectation tolerance intervals determined during the validation phase were coherent with the results of quality control samples analyzed during routine use. This method is therefore precise and suitable both for therapeutic drug monitoring and pharmacokinetic studies in most clinical laboratories. (C) 2012 Elsevier B.V. All rights reserved.