A validated, sensitive HPLC‐MS/MS method for quantification of cis‐para‐methyl‐4‐methylaminorex (cis‐4,4'‐DMAR) in rat and human plasma: application to pharmacokinetic studies in rats

Abstract

4,4'-DMAR is an analogue of the known psychostimulants 4-methylaminorex and aminorex. In the light of reports of deaths associated with its abuse, and the easy access from Internet vendors, the EU Council recently decided on control measures across member states. Here we describe a validated method for measuring plasma levels of cis-4,4'-DMAR, crucial for preclinical studies and analysis in human plasma. Chromatographic separation was done by gradient elution on a Kinetex C18 column with 0.1% formic acid in water and 0.1% formic acid in acetonitrile at 0.2 mL/min. Detection was by positive electrospray ionization (ESI+) in multiple reaction monitoring mode monitoring the quantifier transitions m/z 191.4 → m/z 148.3 for cis-4,4'-DMAR and m/z 259.3 → m/z 194.2 for carbamazepine (internal standard). Protein precipitation with 1% of formic acid in acetonitrile was used in cis-4,4'-DMAR extraction from plasma; recovery was high (>93%) with a negligible matrix effect. This method provides an accurate, precise, and sensitive method for cis-4,4'-DMAR quantification in human and rat plasma, following European Medicine Agency guidelines for bioanalytical method validation. Pharmacokinetic studies were conducted in rats. After an intravenous dose of 1 mg/kg, plasma levels declined rapidly (≥80% in 4 h), followed by a slow elimination phase (t of 5.14 ± 0.65 h). Absorption was rapid after intraperitoneal injection (t = 15 min) with a rapid decline thereafter; C and AUC showed dose-proportionality over the dose range 1-10 mg/kg. This method was successfully applied to investigate pharmacokinetic properties in rats and could be used to quantify cis-4,4'-DMAR levels in human plasma. Copyright © 2016 John Wiley & Sons, Ltd.

Brain Disposition of cis-para-Methyl-4-Methylaminorex (cis-4,4′-DMAR) and Its Potential Metabolites after Acute and Chronic Treatment in Rats: Correlation with Central Behavioral Effects

“…There was a rapid distribution phase lasting about 1 to 2 hours, followed by a slow elimination phase with a terminal halflife (t 1/2 ) of about 5 hours. The bioavailability after intraperitoneal injection was about 20% (Lucchetti et al, 2016), lower than the analog 4-methylaminorex (Meririnne et al, 2004).…”

“…The powder contained 4,49-DMAR in the cis form with purity . 99%, as previously confirmed (Lucchetti et al, 2016). On delivery, cis-4,49-DMAR powder was stored at 4°C in dry conditions.…”

“…cis-4,49-DMAR stock solutions and working solutions for standard points (0.025, 0.05, 0.1, 0.5, 1, 5, and 10 mg/ml) and quality controls (QCs) (0.025, 0.075, 3.0, and 7.5 mg/ml) were prepared in MeOH as previously described (Lucchetti et al, 2016). A stock solution of carbamazepine was also prepared in MeOH at a concentration of 1 mg/ml and diluted to 5 mg/ml in the same organic solvent as needed.…”

“…A recently validated method was used (Lucchetti et al, 2016). Briefly, plasma and brain samples were thawed at room temperature and processed in parallel with two replicates of QC samples at three concentrations (7.5, 300, and 750 ng/ml for plasma or 75, 3000, and 7500 ng/g for brain) and with freshly prepared calibration curves linear in the range of 2.5-1000 ng/ml for plasma samples or 25-10,000 ng/g for brain samples.…”

“…We recently validated a high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) method for quantification of cis-4,49-DMAR in rat and human plasma and applied it for preliminary evaluation of the PK profile after a single intravenous injection in rats (Lucchetti et al, 2016). There was a rapid distribution phase lasting about 1 to 2 hours, followed by a slow elimination phase with a terminal halflife (t 1/2 ) of about 5 hours.…”