Study On The Stereoselectivity Of Oxidation Metabolism Of Mexiletine And Fenfluramine Enantiomers In Rat Liver Microsome

Study on the stereoselectivity of oxidation metabolism of mexiletine enantiomers in rat liver microsomerac-Mexiletine(Mex) is an orally effective class I b antiarrhythmic agent with a half-life ranging from 6 to 12 hr used to treat ventricular arrhythmias. In vivo experiments have demonstrated it is predominantly metabolized by the liver with <10% excreted as unchanged drug. Mexiletine is a weakly basic drug (pK_α = 8.75) with a narrow therapeutic range. Serum concentrations must remain between 0.8 and 2.0mg/liter, and levels >2.0 mg/liter can cause neurological side effects. Mexiletine possesses a chiral center and is administered as a racemate. It is extensively metabolized in human by various pathways, principally by oxidative processes. It was reported that there are stereoselective differences in serum concentration, protein binding, electrophysiology, urine excretion, and glucuronidation for two enantiomers. In this paper, we describe a stereoselective analytical method for the assay of R-(-)- and S-(+)-mexiletine in rat liver microsomal incubates by capillary GC with flame ionization detector(FID), using N-propylamphetamine as an internal standard, to study the stereoselectivity of oxidation metabolism of mexiletine enantiomers in vitro.Chromatographic Conditions Chromatographic column:HP-l capillary column (15m×0.25mm×0.25μm);carrier gas: N₂, 1kg/cm²;combustion gas: H₂, 0.5 kg/cm²;assistant combustion gas: air, 0.5 kg/cm²;make up gas: N₂, 45ml/min;injector temperature: 250℃;FID temperature: 280℃;oven temperature: 100℃ (2min), 8℃/min to 232℃, 15℃/min to 280℃;injection volume: 1μL.Analytical Procedure Liver microsome was prepared as described by Gibson etal. Microsomal protein was measured by the method of Lowry et al. The pH of 1.0ml of incubation media was adjusted to 12 to 13 with 3 drops of 40% NaOH. lOOul of propylamphetamine(lmg/ml) was added. The mixture was extracted with 2.0 ml chloroform by rotatory shaking for 1 min. The chloroform layer was separated and dried with anhydrous sodium sulfate. 10 u 1 of triethylamine and 40 u 1 of S-( —)-N-(Trifluoroacetyl)- prolylchloride (S-TFPC) were added and the reaction was carried out by gently rocking for 15 min at room temperature. The chloroform layer was washed with 2ml of water. After phase separation, the chloroform was evaporated to dryness under a gentle stream of air at 65 "C. The residue was reconstituted in 40 u 1 of ethyl acetate, and an aliquot of 1 u 1 was analyzed by GC.Results The peak-height ratios of the drug to internal standard(y) were linear with concentration of the drug(x). The linearity of the calibration curves for R-(—)- and S-(+)-mexiletine was in the range of 5.0 to 500 u g/ml. The regression equations of the calibration curves were y= -0.0034+0.0015x (r=0.9969)for R-(-)- Mexiletine and y=-0.0030+0.0017x (r=0.9982) for S-(+)- Mexiletine. The limits of detection (LOD) and quantitation (LOQ) of assay were measured as 1.0 u g/ml ^P 5.0 u g/ml (RSD <17%, n=4) , respectively. The average recoveries of R-(—)- and S-(+)-mexiletine were 93.9% and 93.0%, respectively. The relative standard deviations (RSD) of within-day and between-days were less than 5% and 10%, respectively. The time course and the parameters of enzymatic kinetics of mexiletine enantiomers, Vm and Km, were determined. It is obvious that mexiletine undergoes stereoselective metabolism in rat liver microsome.Study on the stereoselectivity of oxidation metabolism of fenfluramine enantiomers in rat liver microsomeFenfluramine(Fen) is widely used as an appetite suppressant in the treatment of obersity. The chemical structure of fenfluramine contains a stereogenic center, the racemate or dexfenfluramine was used clinically as an anorectic agent, while levofenfluramine is a neuroleptic agent. Only a few methods have been reported for quantifying fenfluramine enantiomers in plasma and urine by high-performance liquid chromatography or gas chromatography have been reported. In this study we describe a reliable enantioselective analytical method for assay of 1- and d-fenfluramine in rat livermicrosomal incubates by GC with FID. We also report an application of this method to study the oxidation metabolism of fenfluramine enantiomers in vitro.Chromatographic Conditions Chromatographic column:HP-l capillary column (15m X 0.25mm X 0.25 U m);carrier gas: N2, lkg/cm2;combustion gas: H2, 0.5kg/cm2;assistant combustion gas: air, 0.5 kg/cm2;make up gas: N2, 45ml/min;injector temperature: 280°C;FID temperature: 280°C;oven temperature-. 100°C (4min), 8°C/min to 280°C(lmin);injection volume: 1 u L.Analytical Procedure Liver microsome was prepared as described by Gibson etal. Microsomal protein was measured by the method of Lowry et al. The pH of 1.0ml of incubation media was adjusted to 12 to 13 with 3 drops of 40% NaOH. 100 u I of propylamphetamine(lmg/ml) was added. The mixture was extracted with 2.0 ml chloroform by rotatory shaking for 1 min. The chloroform layer was separated and dried with anhydrous sodium sulfate. 10 u 1 of triethylamine and 40 u 1 of S-(—)-TFPC were added and the reaction was carried out by gently rocking for 15 min at room temperature. The chloroform layer was washed with 2ml of water. After phase separation , the chloroform was evaporated to dryness under a gentle stream of air at 65 °C. The residue was reconstituted in 40 u 1 of ethyl acetate, and an aliquot of 1 u 1 was analyzed by GC.Results The peak-height ratios of the drug to internal standard(y) were linear with concentration of the' drug(x). The linearity of the calibration curves for 1- and d-fenfiuramine was in the range of 1.0 to 50 u g/ml. The regression equations of the calibration curves were y=— 0.1622+0.4456x (r=0.9998) for 1-fenfluramine and y = -0.3577+0.6952x (r=0.9994) for d-fenfluramine. The limits of detection (LOD) and quantitation (LOQ) of assay were measured as 0.1 H g/ml fP 1.0 u g/ml (RSD< 15%. n=4) , respectively. The average recoveries of 1- and d-fenfluramine were 91.3% and 90.4%, respectively. The relative standard deviations (RSD) of within-day and between-days were less than 5% and 10%, respectively. The time course and the parameters of enzymatic kinetics of fenfluramine enantiomers, Vm and Km, were determined. The results show that fenfluramine undergoes stereoselective metabolism in rat liver microsome.