In a first controlled human study, oral methylone (50–200 mg) showed rapid, dose‑proportional (linear) pharmacokinetics with Cmax and AUC increasing proportionally, Tmax ≈1.5–2 h and t1/2 ≈6–7 h. A validated LC–MS/MS assay quantified methylone, MDMA and metabolites and found the primary metabolite HMMC had much lower exposure (Cmax ~10–14× and AUC ~21–29× lower) and faster kinetics than the parent drug.
The aim of this study is to define, for the first time, human methylone and HMMC plasma pharmacokinetics following controlled administration of 50–200 mg methylone to 12 male volunteers. A new LC-MS/MS method was validated to quantify methylone, MDMA, and their metabolites in plasma. The study was a randomized, cross-over, double-blinded and placebo-controlled study, with a total of 468 plasma samples collected. First, 10 µL of MDMA-d5, MDA-d5 and methylone-d3 internal standards were added to 100 µL of plasma. Two mL of chloroform and ethyl acetate 9:1 (v/v) were then added, mixed well and centrifuged. The supernatant was fortified with 0.1 mL acidified methanol and evaporated under nitrogen. Samples were reconstituted with a mobile phase and injected into the LC-MS/MS instrument. The method was fully validated according to OSAC guidelines (USA). Methylone plasma concentrations increased in a dose-proportional manner, as demonstrated by the increasing maximum concentration (Cmax) and area under the curve of concentrations (AUC). Methylone Cmax values were reported as 153, 304, 355 and 604 ng/mL, AUC0–24 values were reported as 1042.8, 2441.2, 3524.4 and 5067.9 h·ng/mL and T1/2 values as 5.8, 6.4, 6.9 and 6.4 h following the 50, 100, 150 and 200 mg doses, respectively. Methylone exhibited rapid kinetics with a Tmax of 1.5 h for the 50 mg dose and 2 h approximately after all the other doses. HMMC exhibited faster kinetics compared to methylone, with a Cmax value that was 10–14-fold lower and an AUC0–24 value that was 21–29-fold lower. Methylone pharmacokinetics was linear across 50–200 mg oral doses in humans, unlike the previously described non-linear oral MDMA pharmacokinetics. An LC-MS/MS method for the quantification of methylone, MDMA and their metabolites in human plasma was achieved. Methylone exhibited linear pharmacokinetics in humans with oral doses of 50–200 mg.
Papers cited by this study that are also in Blossom
Papaseit, E., Pérez-Mañá, C., Mateus, J. A. et al. · Neuropsychopharmacology (2016)
Vizeli, P., Schmid, Y., Prestin, K. et al. · European Neuropsychopharmacology (2017)
Papers in Blossom that reference this study
Ramaekers, J. G., Kuypers, K. P. C., Vollenweider, F. X. · Molecular Psychiatry (2026)
Jones, A., Warner-Schmidt, J., Kwak, H. et al. · JAMA Psychiatry (2026)
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.
The best fit calibration model was a linear least-squares regression model with 1/×2 weighting, as confirmed by Mandel test coefficients. All coefficient of determination results were greater than or equal to 0.99. Linearity, calibration results, LOD and LOQ are shown in Table. LOQ was set as the lowest non-zero calibrator for each analyte (Figure). There was no carryover observed with any of the analytes. Accuracy and imprecision were calculated at the following three QC concentrations (n = 5): 7.5, 150 and 350 ng/mL for MDMA and methylone; 1.5, 20 and 80 ng/mL for HMMC, MDA, HMMA and HMA. Bias was <20% of the target. The ANOVA approach defined by OSAC guidelines determined the overall within-and between-run imprecision. All CV values were less than 20%, as shown in Table. Abbreviations: HMMC, 4-hydroxy-3methoxymethcathinone; MDMA, 3,4-methylendioxymethamphetamine; MDA, 3,4-methylendioxyamphetamine; HMA, 4-hydroxy-3methoxyamphetamine; HMMA, 4-hydroxy-3methoxymethamphetamine; LOD, limit of detection; LOQ, limit of quantification; L, low-quality control; M, medium-quality control; H, high-quality control; CV, coefficient of variation. Accuracy and imprecision were calculated at the following three QC concentrations (n = 5): 7.5, 150 and 350 ng/mL for MDMA and methylone; 1.5, 20 and 80 ng/mL for HMMC, MDA, HMMA and HMA. Bias was <20% of the target. The ANOVA approach defined by OSAC guidelines determined the overall within-and between-run imprecision. All CV values were less than 20%, as shown in Table. There were no interfering peaks in any of the biological matrix pools analyzed. Dilution integrity was evaluated by extracting plasma samples with concentrations two times the ULOQ and diluting the samples 2-, 5-, 10-and 20-fold in blank plasma. Concentrations of replicates (n = 5) for the diluted samples were within ±20% of the target for all compounds. All analytes were stable at room temperature for 24 h, when refrigerated (4 • C) for 24 h, after three freeze/thaw cycles, 24 h post-extraction in the autosampler (+10 • C) and when stored at -20 • C up to 4 months after QC sample preparation (concentration differences less than 20% with respect to time zero response). Matrix effects were evaluated at low (1.5 and 7.5 ng/mL), mid (20 and 150 ng/mL) and high (80 and 350 ng/mL) concentrations. The post-extraction addition method determined ionization suppression (negative value) or ionization enhancement (positive value) within ±20% for all analytes.
The analytical method was fully validated over five consecutive days for the simultaneous quantification of methylone, MDMA and their metabolites and later applied to authentic samples. Plasma samples were collected from twelve male participants after administration of controlled doses of methylone and/or MDMA. Overall, this rapid and simple HPLC-MS/MS method enabled robust and sensitive quantification of methylone, MDMA and their metabolites, with good precision, accuracy, and efficiency. All analytes were stable at room temperature, 4 • C and -20 • C for 24 h, and also after three freeze/thaw cycles, demonstrating the applicability of the validated method for routine analysis. As expected, methylone concentrations were dose-proportional, at least at the doses administered from 50 to 200 mg. Methylone concentrations were higher than MDMA concentrations at the same 100 mg dose; however, not all participants received all doses (n = 3). Regarding metabolites, at a 200 mg methylone dose, HMMC concentrations were approximately 20-times lower that the parent drug concentrations, HMMA concentrations were 16-fold lower than the parent drug, while MDA concentrations were 3-times lower than MDMA after the 100 mg dose. Previous studies reported that methylone is extensively metabolized in a manner similar to its structural analog MDMA, as illustrated by the formation of O-demethylated metabolites (such as HMMC). The pharmacokinetics of MDMA were studied in both humans and animal models, and it was widely demonstrated that MDMA concentrations exhibit non-linear behavior, due to the inhibition of its own metabolism. By contrast, Elmore et al. found non-linear pharmacokinetics in rats after subcutaneous administration of methylone at doses of 3, 6 and 12 mg/kg. Unlike its structural analog, linear pharmacokinetics were observed for methylone and its more abundant metabolite (HMMC) in humans, as shown here. It was also verified that plasma concentrations of methylone and its metabolite displayed more rapid kinetics when compared with MDMA. Methylone is a common drug of abuse, but to date, only a few studies have addressed its pharmacokinetics and metabolism in animal models. For the first time, we investigated the pharmacokinetics of methylone and HMMC in humans and documented linear pharmacokinetics based on normalized C max and AUC values. However, the observed variance is most likely related to individual variability and different participants who received the 50, 100, 150, 200 mg doses of methylone. Comparison of the normalized AUC 0-10 and AUC 0-24 values obtained in this study (14.5, 15.9, 14.9, 16.6 and 20.8, 24.4, 23.5, 25.3, respectively) with those obtained by de la Torre et al. for MDMA (9.15, 17.8, 18.5, 21 and 34.5) further supports the thesis of methylone linear pharmacokinetics. The elimination half-life of methylone was 6 h, considerably higher than other cathinones, such as mephedrone (2,3 h) or cathinone (4 h), but considerably less than other phenethylamines, such as MDMA (around 8 h) or amphetamine-methamphetamine (12 h). A limitation of the present study is that only one methylone metabolite, HMMC, was measured. Unfortunately, nor MDC or HHMC could be determined due to the unavailability of pure chemical standards at the time of the study. There are few published reports on methylone intoxication, but given the pharmacological similarity between methylone and MDMA reported in an observational study, there most likely are toxicological effects. The toxicological consequences of methylone non-linear pharmacokinetics in animals are poorly understood. In humans, the enzyme CYP2D6 is inhibited by MDMA, and in animals, methylone results in the same inhibition. Our results, in the range of 50-200 mg, showed linear pharmacokinetics and do not support CYP2D6 inhibition. Furthermore, substrates of the enzyme (MDMA and MDA) could be responsible for pharmacokinetic interactions, leading to acute toxic and neurotoxic effects. Furthermore, it has been described that the disposition of MDMA is mainly affected by the CYP2D6 polymorphism. However, this influence may be less relevant due to the effect of the self-inhibition mechanism of MDMA on this enzyme and the contribution of other isoenzymes of cytochrome P450 in the metabolism process. When assessing this effect on pharmacokinetics, it was observed that poor metabolizers achieved higher maximum concentrations of MDMA (+15%) and MDA (+50%), but lower concentrations of HMMA (-50-70%) compared to extensive metabolizers. These differences may have implications in pharmacological effects and toxicity, since poor metabolizers experience higher and faster increases in blood pressure and subjective effects. In addition, higher concentrations of HHMA could lead to the increased formation of some quinones, which are involved in the mechanisms of serotoninergic neurotoxicity. Similarly, a potential influence of enzyme polymorphisms on the toxicity of the reactive intermediate methylone metabolite HHMC can be hypothesized. However, it is still unknown if these effects also occur after methylone administration and further studies to verify this hypothesis are needed. Considering the few methylone deaths reported in the literature and the fact that methylone exhibits linear pharmacokinetics, it is possible that dose-response proportionality protects against overdose. These results must be confirmed in large samples of subjects and doses in future studies.
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Schmidt, E. F., Warner-Schmidt, J., Stogniew, M. et al. · Frontiers in Neuroscience (2024)
Poyatos, L., Pérez-Mañá, C., Hladun, O. et al. · Frontiers in Pharmacology (2023)
Warner-Schmidt, J., Pittenger, C., Stogniew, M. et al. · Frontiers in Psychiatry (2023)