High-performance liquid chromatographic method for the disposition of mazindol and its metabolite 2-(2-aminoethyl)-3-(p-chlorophenyl)-3-hydroxyphthalimidine in mouse brain and plasma

A high-performance liquid chromatographic method was developed for the analysis of the appetite suppressant mazindol and its metabolite 2-(2-aminoethyl)-3-(p-chlorophenyl)-3-hydroxyphthalimidine (Met) in mouse brain and plasma. The two compounds were quantified by measuring Met after two different sample pretreatments. For mazindol determination, the treatment involved the hydrolysis of mazindol to Met, by incubating the sample at 80 °C for 15 min at pH 10.6 followed by liquid-liquid extraction procedure while for the determination of Met, the hydrolysis step was omitted. The obtained Met was analyzed by HPLC after its derivatization with the fluorescent reagent 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). The separation was performed on an ODS column with mobile phase consisted of a mixture of acetonitrile-methanol-0.1 M acetic acid (46:4:50, v/v/v) containing tetrahydrofuran (6%). The effluent was monitored at excitation and emission wavelengths of 330 and 445 nm, respectively. Calibration curves of mazindol and Met ranged from 0.1 to 25 ng/ml and from 0.5 to 250 ng/g in spiked mouse plasma and brain tissue, respectively. The method is highly sensitive with the limits of detection for Met on column of 2.8 and 3.5 fmol in plasma and brain, respectively, at a signal-to-noise ratio of 3. The intra- and inter-day precisions were less than 4.5 and 9.7%, in plasma and less than 8.8 and 7.2% in brain, respectively. The developed method was applied for the monitoring of mazindol and Met levels in mouse plasma and brain tissue regions after single intraperitoneal administration of mazindol, 0.5 mg/kg.