Simultaneous determination of sibutramine and its N-desmethyl metabolites in human plasma by liquid chromatography–electrospray ionization–mass spectrometry: Method and clinical applications Original Research Article

A sensitive liquid chromatography–electrospray ionization–mass spectrometry (LC–ESI–MS) method for simultaneous determination of sibutramine and its two active N-desmethyl metabolites in human plasma using phenoprolamine hydrochloride as the internal standard (IS) was established. After being made alkaline with saturated sodium bicarbonate, plasma samples were extracted with cyclohexane and separated by HPLC on a reversed phase C18 column with a mobile phase of 10 mM ammonium acetate buffer (adjusted the pH to 3.5 with acetic acid)–methanol (25:75 (v/v)). Analytes were determined using electrospray ionization in a single quadrupole mass spectrometer. LC–ESI–MS was performed in the selected-ion monitoring (SIM) mode using target ions at m/z: 280 for sibutramine, m/z: 266 for N-mono-desmethylsibutramine (metabolite 1), m/z: 252 for N-di-desmethylsibutramine (metabolite 2) and m/z: 344 for the IS. Calibration curves were linear over the ranges of 0.05–20 μg l−1 for sibutramine, 0.02–20 μg l−1 for metabolite 1 and 0.1–30 μg l−1 for metabolite 2. The intra-assay variability values were less than 7.6% for sibutramine, 8.9% for metabolite 1 and 5.5% for metabolite 2. The inter-assay variability values were less than 11.8% for sibutramine, 12.7% for metabolite 1 and 9.4% for metabolite 2. The mean plasma extraction recoveries of sibutramine, metabolites 1 and 2 were 90.2, 90.9 and 91.0%, respectively. The method has been successfully applied to study the pharmacokinetics of sibutramine and its two metabolites in healthy male Chinese volunteers.