A rapid HPLC-DAD method for the toxicological analysis of fluoxetine and norfluoxetine in human plasma

Fluoxetine (d,l-N-methyl-3-phenyl-3-[(alpha,alpha,alpha,-trifluoro-p-tolyl)oxy]propyl-amine) is the parent drug of the SSRI (selective serotonin reuptake inhibitor) antidepressant class. After administration, fluoxetine is subject to hepatic metabolism by cytochrome P450 enzymes, whose main product is the demethylated metabolite norfluoxetine, which has comparable pharmacological activity. In the past few years, our research group has developed sensitive methods based on HPLC with fluorimetric detection for the therapeutic drug monitoring of fluoxetine and norfluoxetine in human plasma. Recently, however, we have been required to determine very high fluoxetine levels in patients who took an overdose of the drug and we have found that the previously developed methods are unsuitable. For this reason, an alternative, rapid and feasible HPLC procedure with diode array detection (DAD) has been developed. The mobile phase is composed of an acetonitrile / aqueous tetramethyl-ammonium perchlorate mixture, while the stationary phase is a C8 reversed phase column; quantitation of the analyte peaks is carried out at 230 nm. The application of the method to plasma samples requires a sample pre-treatment step, which is carried out by means of solid-phase extraction, loading 250 µL of plasma on Oasis HLB cartridges and eluting the analytes with methanol. This procedure gives satisfactory extraction yield values, as well as good sample purification from matrix interference. The method seems to be suitable for the analysis of fluoxetine and its metabolite in human plasma for toxicological purposes.