A Liquid Chromatography Tandem Mass Spectrometry Method for the Simultaneous Estimation of the Dopamine Receptor Antagonist LE300 and Its N-methyl Metabolite in Plasma: Application to a Pharmacokinetic Study

Abstract

LE300 is a novel dopamine receptor antagonist used to treat cocaine addiction. In the current study, a sensitive and fast liquid chromatography–tandem mass spectrometry (LC-MS/MS) has been established and validated for the simultaneous analysis of LE300 and its N-methyl metabolite, MLE300, in rat plasma with an application in a pharmacokinetic study. The chromatographic elution of LE300, MLE300, and Ponatinib (IS, internal standard), was carried out on a 50 mm C18 analytical column (ID: 2.1 mm and particle size: 1.8 μm) maintained at 22 ± 2 °C. The run time was 5 min at a flow rate of 0.3 mL/min. The mobile phase consisted of 42% aqueous solvent (10 mM ammonium formate, pH: 4.2 with formic acid) and 58% organic solvent (acetonitrile). Plasma samples were pretreated using protein precipitation with acetonitrile. The electrospray ionization (ESI) source was used to generate an ion-utilizing positive mode. A multiple reaction monitoring mass analyzer mode was utilized for the quantification of analytes. The linearity of the calibration curves in rat plasma ranged from 1 to 200 ng/mL (r2 = 0.9997) and from 2 to 200 ng/mL (r2 = 0.9984) for LE300 and MLE300, respectively. The lower limits of detection (LLOD) were 0.3 ng/mL and 0.7 ng/mL in rat plasma for LE300 and MLE300, respectively. Accuracy (RE%) ranged from −1.71% to −0.07% and −4.18% to −1.48% (inter-day), and from −3.3% to −1.47% and −4.89% to −2.15% (intra-day) for LE300 and MLE300, respectively. The precision (RSD%) was less than 2.43% and 1.77% for the inter-day, and 2.77% and 1.73% for intra-day of LE300 and MLE300, respectively. These results are in agreement with FDA guidelines. The developed LC-MS/MS method was applied in a pharmacokinetic study in Wistar rats. Tmax and Cmax were 2 h and 151.12 ± 12.5 ng/mL for LE300, and 3 h and 170.4 ± 23.3 ng/mL for MLE300.