Synthetic cathinones are a class of new psychoactive substances (NPS) with structural and pharmacological similarity to amphetamines. Methylone, a synthetic cathinone NPS, is the β-keto analog of MDMA. There are no controlled administration data for methylone in humans. The aim of this research was development of an LC-MS/MS method to simultaneously quantify methylone, MDMA and their metabolites 4-hydroxy-3-methoxy-N-methylcathinone (HMMC), 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymeth-amphetamine (HMMA) and 4-hydroxy-3-methoxyamphetamine (HMA) in human plasma and application of the method to the first controlled oral methylone administration study.

Briefly, 10μL 100ng/mL internal standard mixture (methylone-d3, MDA-d5 and MDMA-d5) and 2μL NH2 2% in H2O (pH 9) were added to 100μL plasma, tubes were stirred in a roller mixer for 10min and centrifuged at 3500rpm for 5min. Supernatants were collected into clean tubes, 100μL acidic methanol (1% HCl) was added to prevent evaporative losses, and samples dried under nitrogen for approximately 30min. Samples were reconstituted in 100μL mobile phase A:B (95:5) (0.1% formic acid in water:acetonitrile) and transferred into autosampler vials for LC–MS/MS analysis. Analytes separation was obtained on a UHPLC 1290 Infinity II (Agilent Technologies Italia S.p.a.) coupled to a mass spectrometer (6470A Triple Quadrupole LC-MS) equipped with an electrospray ionization source operating in positive-ion mode (ESI+).

Limits of quantification (LLOQ) were 5 (methylone) to 500 (MDMA) μg/L and 0.5 to 50μg/L for their metabolites. A randomized double blinded study was conducted in 12 male volunteers (aged 18–45years old; weight ranging from 50 to 90kg) and divided into 3 sessions, with single oral doses of 50, 100, 150 or 200mg methylone, 100mg MDMA or placebo in each session. Plasma samples were collected before and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10 and 24h after dosing.

The analytical method was validated over five successive days in plasma following the most recent criteria for bioanalytical method development and validation. The method was linear for all analytes over the calibration range. Intra- and inter-assay accuracies were ±15%, while intra- and inter-assay precision were ±10%. Methylone plasma concentrations increased in a dose-related manner. Methylone Cmax were 138, 243, 320 and 530μg/L following the 50, 100, 150 and 200mg doses, respectively, while corresponding area under the curve (AUC) were 1065, 2486, 3511 and 5489min ×μg/L. Methylone Tmax were 1h for the lowest 50mg dose and 2h for the remaining doses. The last detected methylone concentrations (Clast) at 24h were 10.5, 18.8, 36.4 and 51.8μg/L (for 50, 100, 150 and 200mg respectively). For methylone's HMMC metabolite, Cmax were 6.6, 16.9, 28.3, and 32.7μg/L, while AUC were 36.9, 90.1, 129 and 173min ×μg/L. HMMC exhibited rapid kinetics with Tmax of 1–1.25h for all doses. As for Clast, HMMC concentrations were 0.5, 0.4, 1.3 and 0.8μg/L (for 50, 100, 150 and 200mg, respectively) 24h after drug administration.

Methylone is a popular drug of abuse, yet no studies addressed its metabolism and pharmacokinetics in humans. A validated LC-MS/MS method for simultaneous quantification of methylone, MDMA and their metabolites (HMMC, MDA, HMMA and HMA) in human plasma is presented. Considering the pharmacokinetic study for methylone and HMMC, concentrations following the 100mg dose were approximately doubled compared to the 50mg dose, and after the 200mg dose were about four times higher than the lowest dose (within ±20%), suggesting linear pharmacokinetics for both analytes.