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

• Published in: Chromatographia Vol. 78; no. 9-10; pp. 655 - 661
• Main Authors: Hefnawy, Mohamed, Alanazi, Amer, Abounassif, Mohammed, Mohammed, Mostafa, Al-Swaidan, Ibrahim, Attia, Sabry, Mostafa, Gamal, El-Subbagh, Hussein, Lehmann, Jochen
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.05.2015; Springer Berlin Heidelberg
• Subjects: Analytical Chemistry; antagonists; Chemistry; Chemistry and Materials Science; Chromatography; detection limit; dopamine receptors; guidelines; high performance liquid chromatography; Laboratory Medicine; metabolites; mice; Original; pharmacokinetics; Pharmacy; phosphates; Proteomics; regression analysis; verapamil; wavelengths; Trace analysis; Pharmacokinetics; LE300; HPLC; Fluorescence detection
• ISSN: 0009-5893; 1612-1112
• DOI: 10.1007/s10337-015-2879-x

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⁻¹and 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⁻¹for LE300 and 2.4 ng mL⁻¹for 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 Tₘₐₓand Cₘₐₓwere 2 h and 25.03 ± 5.60 ng mL⁻¹for LE300 and 3 h and 19.92 ± 2.88 ng mL⁻¹for its N-methyl metabolite, respectively.