Sensitive and Selective Liquid-Chromatographic Assay of Fluoxetine and Norfluoxetine in Plasma with Fluorescence Detection After Precolumn Derivatization

Abstract

Abstract We determined fluoxetine (Prozac) and its major metabolite norfluoxetine in plasma by liquid chromatography with fluorescence detection. After liquid-liquid extraction from 1 mL of plasma, the extract was derivatized at room temperature with dansyl chloride, and the highly fluorescent derivatives were chromatographed with a reversed-phase C18 column and a mobile phase of phosphate buffer and acetonitrile. Dansylated fluoxetine, norfluoxetine, and the internal standard were eluted in less than 14 min with no interference from endogenous material. The calibration curve was linear over the concentration range 25-800 micrograms/L with inter- and intra-assay imprecision (CV) of less than 10%. Validity of the assay was checked by comparing results for 110 patients' samples with those by a liquid-chromatographic method with ultraviolet detection (r = 0.993 for fluoxetine, 0.957 for norfluoxetine). The identity of the dansylated derivatives was verified by positive chemical ionization mass spectroscopy. The lower limit of detection was approximately 3 micrograms/L. Because no major antidepressant, neuroleptic, or respective drug metabolites interfere with the quantification of fluoxetine and norfluoxetine, this is a useful procedure for pharmacokinetic studies and in clinical settings.