Simultaneous determination of fluoxetine and its major active metabolite norfluoxetine in human plasma by LC-MS/MS using supported liquid extraction.
A rapid, sensitive and selective bioanalytical method was developed for the simultaneous determination of fluoxetine and its primary metabolite norfluoxetine in human plasma. Sample preparation was based on supported liquid extraction (SLE) using methyl tert-butyl ether to extract the analytes from human plasma. Chromatography was performed on a Synergi 4 μ polar-RP column using a fast gradient. The ionization was optimized using ESI (+) and selectivity was achieved by tandem mass spectrometric analysis using MRM functions, m/z 310 → 44 for fluoxetine, m/z 296 → 134 for norfluoxetine and m/z 315 → 44 for fluoxetine-d5 (internal standard). The method is linear over the range of 0.05-20 ng/mL (using a human plasma sample volume of 0.1 mL) with a coefficient determination of greater than 0.999. The method is accurate and precise with intra-batch and inter-batch accuracy (%bias) of < ± 15% and precision (%CV) of <15% for both analytes. A run time of 4 min means a high throughput of samples can be achieved. To our knowledge, this method appears to be the most sensitive one reported so far for the quantitation of fluoxetine and norfluoxetine and can be used for routine therapeutic drug monitoring or pharmacokinetic studies.