Selective Analysis of Dopamine Receptor Antagonist LE300 and its N-Methyl Metabolite in Mouse Sera at the Trace Level by HPLC–Fluorescence Detection

Abstract

A highly selective, sensitive, and reliable high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of a novel type of dopamine receptor antagonist LE300 and its N-methyl metabolite in mouse sera. LE300, its N-methyl metabolite, and verapamil (an internal standard) were detected using excitation and emission wavelengths of 275 and 340 nm, respectively. HPLC analysis using a deproteinization procedure was performed by injecting an aliquot of the supernatant into the chromatographic system. Chromatographic separation was achieved on a reversed-phase Spherisorb Cyano (CN) column with a mobile phase consisting of acetonitrile:50 mM phosphate buffer pH 3.5 (70:30, v/v) pumped at a flow rate of 1.0 mL min⁻¹. Regression analyses showed excellent linearity (r = 0.999) for concentrations of LE300 ranging from 4 to 500 ng mL⁻¹ and for concentrations of its N-methyl metabolite of 6–600 ng mL⁻¹. The HPLC-FLD method had limits of detection of 1.6 ng mL⁻¹ for LE300 and 2.4 ng mL⁻¹ for its N-methyl metabolite in mouse sera. The precision results, expressed as the intraday and interday relative standard deviation (RSD) values, ranged from 0.65 to 2.85 % (repeatability) and from 0.37 to 2.62 % (intermediate precision) for LE300 and its N-methyl metabolite, respectively; these values are in line with ICH guidelines. The assay was successfully applied in a pharmacokinetic study. The mean values of T _max and C _max were 2 h and 25.03 ± 5.60 ng mL⁻¹ for LE300 and 3 h and 19.92 ± 2.88 ng mL⁻¹ for its N-methyl metabolite, respectively.