Validation of a new HPLC-MS/MS method for quantification of 4,4'-DMAR in human and rat plasma: application to pharmacokinetic studies in rat

Introduction: 4,4′-DMAR is a para-methyl analog of the known psychostimulants 4-methylaminorex and aminorex. In light of the reports of deaths associated with its abuse, and the easy accessibility by Internet vendors, the European Council recently decided control measures across Member States. Here we describe a validated method for measuring plasmatic levels of 4,4′-DMAR, crucial either for preclinical studies and analysis in human blood. Methods: Chromatography was performed by gradient elution on a Kinetex C18 column; MS detection was achieved by ESI positive ionization in MRM mode. The HPLC-MS/MS methods was validated following EMA guidelines in both human and rat plasma. Pharmacokinetic studies were conducted in rats. Results: Calibration curve was linear in the range 2.5-1000 ng/mL (r2 always > 0.993). The method was accurate (bias always ≤ 11.5%) and reproducible (% CV always ≤ 13.1). The recovery was high (> 93%) with a negligible matrix effect. Analytes were stable under all tested conditions. After acute intravenous treatment of rats with 1 mg/kg, plasma levels declined rapidly (≥ 80% in 4 hours), followed by a slow elimination phase (t1/2 of 5.14 ± 0.65 h). Rapid absorption was found after intraperitoneal administration (tmax = 15 min) and a rapid decline thereafter; Cmax and AUC0-240min showed dose-proportionality over the dose range 1-10 mg/kg. Conclusions: This method provides an accurate, precise, and sensitive method for 4,4’-DMAR quantification in plasma and was successfully applied to investigate pharmacokinetic properties in rats. Moreover the method could be applied to quantify 4,4’-DMAR levels in human plasma.