Sensitive liquid chromatographic–tandem mass spectrometric method for the determination of fluoxetine and its primary active metabolite norfluoxetine in human plasma

A sensitive method for the simultaneous determination of fluoxetine and its major active metabolite norfluoxetine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The samples were extracted from alkalised plasma with hexane–isoamyl alcohol (98:2, v/v) followed by back-extraction into formic acid (2%). Chromatography was performed on a Phenomenex® Luna C18 (2) 5 μm, 150×2 mm column with a mobile phase consisting of acetonitrile–0.02% formic acid (340:660, v/v) at a flow-rate of 0.35 ml/min. Detection was achieved by a Perkin-Elmer Sciex API 2000 mass spectrometer (LC–MS–MS) set at unit resolution in the multiple reaction monitoring mode. TurboIonSpray ionisation was used for ion production. The mean recoveries for fluoxetine and norfluoxetine were 98 and 97%, respectively, with a lower limit of quantification set at 0.15 ng/ml for the analyte and its metabolite. This assay method makes use of the increased sensitivity and selectivity of mass spectrometric (MS–MS) detection to allow for a more rapid (extraction and chromatography) and sensitive method for the simultaneous determination of fluoxetine and norfluoxetine in human plasma than has previously been described.