Methylone and MDMA Pharmacokinetics Following Controlled Administration in Humans

Synthetic cathinones are novel psychoactive substances structurally related to cathinone and include 3,4-methylenedioxy-N-alkylates that resemble MDMA in structure and effects. Methylone (3,4-methylenedioxy-methcathinone) differs from MDMA by a ketone at the β position and shares pharmacological actions with MDMA, including inhibition of monoamine reuptake and interactions with human monoamine transporters. Despite these similarities, human pharmacokinetic data for methylone are scarce, and animal work has given mixed indications about linearity of methylone disposition; by contrast, oral MDMA pharmacokinetics are known to be non-linear, in part due to metabolic self-inhibition. Poyatos and colleagues set out to characterise, for the first time in humans, the plasma pharmacokinetics of methylone and its primary metabolite HMMC after controlled oral administration across a 50–200 mg dose range, and to determine whether methylone exhibits linear or non-linear kinetics. A secondary aim was to develop and validate a single LC-MS/MS assay able to quantify methylone, MDMA and their principal metabolites in human plasma following controlled dosing.

The study was a randomised, cross-over, double-blind, placebo-controlled human trial conducted in 12 male recreational stimulant users at a single clinical centre in Spain. Participants (mean age 23 years, mean weight 70.2 kg) underwent three experimental sessions each, with sessions separated by 5–7 days. In each session subjects received single oral doses drawn from the set of methylone (50, 100, 150 or 200 mg), MDMA (100 mg) or placebo (dextromaltose); for blinding participants and staff received five capsules per session so the active dose was concealed. Blood was sampled pre-dose and at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10 and 24 h after dosing; plasma was separated and stored at -20 °C until analysis. The extracted text does not clearly state which participants received which specific dose combinations, and the authors note that not all participants received every dose. A new high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and fully validated according to OSAC guidance for simultaneous quantification of methylone, MDMA and selected metabolites (for methylone: HMMC; for MDMA: HMMA, HMA and MDA). Sample preparation used liquid–liquid extraction (chloroform:ethyl acetate 9:1) with deuterated internal standards, acidified methanol addition, evaporation and reconstitution prior to injection. Chromatography employed a phenyl-hexyl column with a 6-min gradient and detection used electrospray ionisation in positive mode with scheduled multiple reaction monitoring; method parameters (transitions, collision energies, etc.) were optimised using MassHunter software. Validation experiments assessed linearity (calibration model: linear least-squares with 1/x2 weighting; R2 ≥ 0.99), limits of detection and quantification, accuracy and imprecision at low/medium/high QC levels, carryover, matrix effects, recovery, dilution integrity, and analyte stability (room temperature, 4 °C, -20 °C and after three freeze/thaw cycles). Pharmacokinetic analysis was non-compartmental and performed using WinNonlin, with graphical work done in GraphPad Prism 9. Standard pharmacokinetic parameters derived included Cmax (maximum concentration), Tmax (time to Cmax), AUC (area under the concentration–time curve) and T1/2 (elimination half-life).

The validated LC-MS/MS assay met the acceptance criteria reported: calibration curves had coefficients of determination ≥ 0.99, bias at QC levels was <20% and coefficients of variation were <20% across runs. No significant carryover or interfering peaks were observed; matrix effects were within ±20% and analytes were stable under the tested storage and handling conditions (including up to 4 months at -20 °C). Plasma pharmacokinetics for methylone and its metabolite HMMC were characterised after oral methylone doses of 50, 100, 150 and 200 mg. Methylone showed rapid absorption with Tmax ≈1.5 h for the 50 mg dose and ≈2 h for the higher doses. Reported methylone Cmax values were 153.0 ng/mL (50 mg), 304.0 ng/mL (100 mg), 355.0 ng/mL (150 mg) and 604.0 ng/mL (200 mg). Corresponding AUC0-10 values were 729.2, 1596.2, 2245.6 and 3329.1 h·ng/mL and AUC0-24 values were 1042.8, 2241.2, 3524.4 and 5067.9 h·ng/mL for the four doses. Twenty-four–hour concentrations (Clast) were 8.0, 18.8, 38.6 and 47.8 ng/mL, yielding terminal half-lives (T1/2) of 5.8, 6.4, 6.9 and 6.4 h for the 50, 100, 150 and 200 mg doses, respectively. HMMC concentrations were substantially lower than parent drug levels: Cmax values were 10–14-fold lower than methylone, AUC0-10 values 19–24-fold lower, and AUC0-24 values 21–29-fold lower. HMMC Cmax occurred between 0.9 and 1.5 h and Clast at 24 h ranged between 0.4 and 1.3 ng/mL across doses. The authors report that, across doses, HMMC concentrations were approximately 20 times lower than methylone concentrations. For reference, pharmacokinetics after a 100 mg oral MDMA dose were also reported: MDMA Cmax was 66.1 ng/mL at 2.0 h, AUC0-10 was 468.2 h·ng/mL and AUC0-24 was 888.6 h·ng/mL. MDMA metabolites showed the following metrics: MDA Cmax 21.9 ng/mL at 4.0 h (AUC0-10 135.9; AUC0-24 290.8), HMMA Cmax 85.6 ng/mL at 3 h (AUC0-24 294.1; T1/2 5.4 h; Clast 1.8 ng/mL at 24 h) and HMA Cmax 20.8 ng/mL at 3 h (AUC0-24 71.0; T1/2 5.3 h; Clast 0.7 ng/mL at 24 h). The reported relative abundance for MDMA and metabolites was MDMA > HMMA > MDA > HMA. The investigators characterise methylone pharmacokinetics as dose-proportional across the 50–200 mg oral range. They present normalised dose–response metrics (Cmax-based proportionality values of 3.1, 3.0, 2.4 and 3.0 for the 50, 100, 150 and 200 mg doses, respectively; and AUC0-24 values of 20.8, 24.4, 23.5 and 25.3) consistent with linear kinetics. By contrast, previously published MDMA data show non-linear increases in AUC with dose.

The investigators report that the newly developed HPLC-MS/MS method was robust, sensitive and suitable for routine analysis of methylone, MDMA and selected metabolites in human plasma, meeting validation criteria for precision, accuracy, stability and lack of interference. Plasma sampling and analysis from twelve male participants after controlled administration provided the dataset used for pharmacokinetic characterisation. Methylone displayed rapid absorption and elimination with an apparent T1/2 of roughly 6 h and dose-proportional increases in Cmax and AUC across 50–200 mg oral doses. The authors note that methylone concentrations at the 100 mg dose were higher than corresponding MDMA concentrations in their dataset, but also acknowledge that not all participants received every dose (n = 3 in some dosing cells), which complicates direct comparisons. HMMC levels were markedly lower than parent drug concentrations and showed earlier peak times compared with methylone. Interpreting these findings against existing literature, the authors contrast methylone’s linear pharmacokinetics in this human sample with the well-described non-linearity of MDMA in humans and with animal reports suggesting possible non-linear methylone kinetics. They report that, within the 50–200 mg dose range studied, observations do not support CYP2D6-mediated self-inhibition sufficient to produce non-linearity for methylone, though they acknowledge enzyme polymorphisms and interactions could affect disposition and toxicity for MDMA and potentially for methylone. The authors highlight mechanistic uncertainties, noting that poor CYP2D6 metaboliser status affects MDMA concentrations and metabolite patterns and that analogous effects for methylone (including formation of reactive intermediates) remain hypothetical. Key limitations acknowledged by the authors include a small sample size, uneven allocation of participants to specific dose levels (so not all participants received all doses), and the measurement of only one methylone metabolite (HMMC) owing to lack of available reference standards for other methylone metabolites such as MDC or HHMC. The authors recommend larger studies with broader metabolite panels and varied dosing to confirm linearity, to characterise interindividual variability and to investigate potential toxicological implications.

The study reports the first validated analytical method for simultaneous quantification of methylone, MDMA and principal metabolites in human plasma and presents the first human pharmacokinetic data for methylone and its metabolite HMMC after controlled oral dosing. Methylone exhibited linear, dose-related increases in plasma concentrations across 50–200 mg and an elimination half-life of approximately 6–7 h. The authors conclude that further studies with larger samples, additional metabolites and broader dose ranges are needed to fully understand the pharmacokinetics and potential health impacts of synthetic cathinones; they state the validated assay will be useful for future investigations and toxicological casework.