Fluorometric determination of dl-fenfluramine, dl-norfenfluramine and phentermine in plasma by achiral and chiral high-performance liquid chromatography

High-performance liquid chromatography (HPLC) with fluorescence detection has been developed for the simultaneous determination of sympathomimetic amines including ephedrine, norephedrine, 2-phenylethylamine, 4-bromo-2,5-dimethoxyphenylethylamine, phentermine (Phen) and dl-fenfluramine (Fen) in spiked human plasma. Furthermore, an enantioselective HPLC method for the separation of d-Fen (dexfenfluramine) and l-Fen (levofenfluramine) in addition to their active metabolites d- and l-norfenfluramine (Norf) is described. The detection was achieved at emission wavelength of 430 nm with excitation wavelength of 325 nm for both methods. The analytes were extracted from plasma (100 μl) at pH 10.6 with ethyl acetate using fluoxetine as the internal standard. The extracts were evaporated and derivatized with the fluorescence reagent 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride in the presence of carbonate buffer (pH 9.0). A gradient separation was achieved on a C₁₈ column for the achiral separation or on a Chiralcel OD-R column for the chiral separation. The methods were fully validated, and shown to have excellent linearity, sensitivity and precision. The chiral method has been applied for the determination of d- and l-enantiomers of Fen and Norf, in addition to Phen in rat plasma after an intraperitoneal administration of dl-Fen and Phen, simultaneously.