H OH OH metabolites Article Usefulness of Oral Fluid for Measurement of Methylone and Its Metabolites: Correlation with Plasma Drug Concentrations and the Effect of Oral Fluid pH Giorgia Sprega 1,† , Alessandro Di Giorgi 1,† , Lourdes Poyatos 2,3,† , Esther Papaseit 2,3 , Clara Pérez-Mañá 2,3 , Anastasio Tini 1 , Simona Pichini 4 , Francesco Paolo Busardò 1, *, Alfredo Fabrizio Lo Faro 1,‡ and Magí Farré 2,3,‡ 1 Department of Excellence-Biomedical Sciences and Public Health, Università Politecnica delle Marche, 60126 Ancona, Italy 2 Department of Clinical Pharmacology, Hospital Universitari Germans Trias i Pujol and Institut de Recerca Germans Trias i Pujol (HUGTiP-IGTP), 08916 Badalona, Spain 3 Department of Pharmacology, Therapeutics and Toxicology and Department of Psychiatry and Forensic Medicine, Universitat Autònoma de Barcelona (UAB), 08193 Cerdanyola del Vallés, Spain 4 National Centre on Addiction and Doping, Istituto Superiore di Sanità, 00161 Rome, Italy * Correspondence: f.p.busardo@univpm.it; Tel.: +39-0712206274 † These authors contributed equally to this work. ‡ These authors contributed equally to this work. Abstract: The aim of this study was to investigate methylone and its metabolites concentration in oral fluid following controlled increasing doses, focusing on the effect of oral fluid pH. Samples were obtained from a clinical trial where twelve healthy volunteers participated after ingestion of 50, 100, 150 and 200 mg of methylone. Concentration of methylone and its metabolites 4-hydroxy-3- methoxy-N-methylcathinone (HMMC) and 3,4-methylenedioxycathinone in oral fluid were measured using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Pharmacokinetic parameters were estimated, and the oral fluid-to-plasma ratio (OF/P) at each time interval was calculated and Citation: Sprega, G.; Di Giorgi, A.; correlated with the oral fluid pH using data from our previous study in plasma. Methylone was Poyatos, L.; Papaseit, E.; Pérez-Mañá, detected at all time intervals after each dose; MDC and HMMC were not detectable after the lowest C.; Tini, A.; Pichini, S.; Busardò, F.P.; dose. Oral fluid concentrations of methylone ranged between 88.3–503.8, 85.5–5002.3, 182.8–13,201.8 Lo Faro, A.F.; Farré, M. Usefulness of and 214.6–22,684.6 ng/mL following 50, 100, 150 and 200 mg doses, respectively, peaked between Oral Fluid for Measurement of 1.5 and 2.0 h, and were followed by a progressive decrease. Oral fluid pH was demonstrated to Methylone and Its Metabolites: Correlation with Plasma Drug be affected by methylone administration. Oral fluid is a valid alternative to plasma for methylone Concentrations and the Effect of Oral determination for clinical and toxicological studies, allowing for a simple, easy and non-invasive Fluid pH. Metabolites 2023, 13, 468. sample collection. https://doi.org/10.3390/ metabo13040468 Keywords: methylone; oral fluid; pharmacokinetics; humans; LC–MS/MS Academic Editor: Márcia Carvalho Received: 17 February 2023 Revised: 7 March 2023 1. Introduction Accepted: 22 March 2023 By the information available as of May 2022, a total of 1127 New Psychoactive Sub- Published: 24 March 2023 stances (NPSs) were reported to the United Nations Office on Drugs and Crime (UN- ODC) [1]. Due to their heterogeneity, there are several classifying systems, one of these providing a classification in four groups: synthetic stimulants, synthetic cannabinoids, Copyright: © 2023 by the authors. synthetic hallucinogens and synthetic depressants [2]. The largest class of synthetic stimu- Licensee MDPI, Basel, Switzerland. lants is represented by synthetic cathinones, compounds chemically related to cathinone, This article is an open access article a psychoactive drug found in the khat plant [3]. Among most incoming abused syn- distributed under the terms and thetic cathinones there is 4-methylmethcathinone (mephedrone) and 3,4-methylenedioxy- conditions of the Creative Commons methcathinone, more commonly known as methylone or MDMC, first identified in 2009 [4]. Attribution (CC BY) license (https:// Its phenethylamine structure is based on that of 3,4-methylenedioxy-methamphetamine creativecommons.org/licenses/by/ (MDMA), with the only difference in the β-ketone group [5]. Both substances inhibit 4.0/). Metabolites 2023, 13, 468. https://doi.org/10.3390/metabo13040468 https://www.mdpi.com/journal/metabolites Metabolites 2023, 13, 468 2 of 12 the neuronal reuptake and enhanced the release of dopamine, norepinephrine and sero- tonin, thus increasing monoamine concentrations in the synaptic cleft [6,7]. Similarly to MDMA, the metabolism of methylone mainly occurs in the liver, where this compound is converted to 3,4-methylenedioxycathinone (MDC) through N-demethylation. However, O- demethylation is another pathway providing 3,4-dihydroxy-N-methylcathinone (HHMC), which is subsequently converted to 4-hydroxy-3-methoxy-N-methylcathinone (HMMC) through O-methylation, the primary metabolite [8,9]. Recently, its acute pharmacological effects after oral controlled administration of 200 mg in comparison to MDMA 100 mg have been published [10]. Methylone pharmacoki- netics in humans were firstly published in 2022, in comparison to that of MDMA following controlled administration of different doses [4]. For this purpose, a liquid chromatography– tandem mass spectrometry method for the quantification of methylone and HMMC in plasma was developed. The parent drug and its primary metabolite have also been identi- fied in urine samples from consumers [11], whereas methylone alone was determined in hair from chronic users [12]. Oral fluid (OF) is an alternative biological matrix used to assess current drug con- sumption in roadside drug testing, used in place of blood for drug monitoring and in pharmacokinetics studies [13–16]. It allows for a non-invasive, rapid, simple and observed sample collection. Indeed, uncharged basic drugs in blood diffuse across membranes into OF due to the lower pH (6.2–7.4 compared to 7.4 of blood) and ionize, yielding higher OF than blood concentrations [17]. Furthermore, several studies reported the measurement of MDMA and its metabolites in OF as a valuable alternative to plasma determination in clinical and forensic toxicology [18,19]. Concerning methylone, some analytical methods involving gas chromatography–mass spectrometry or liquid chromatography–tandem mass spectrometry have been developed to determine methylone in the OF of cathinone consumers, but no positive samples were found in the analyzed samples [20–24]. Conversely, some other surveys in different parts of the world (United States, Norway, Brazil, Sweden) found OF samples of consumers, reporting a generic use of psychostimulants positive for synthetic cathinones, including methylone [25–28]. Up to now, no investigation studies on methylone and its metabolites’ time course have been reported for OF after controlled administration. The aims of this study were: • To investigate the concentrations of methylone and its metabolites’ MDC and HMMC in OF following controlled administration of different doses to healthy volunteers. • To assess the eventual correlation between OF and methylone plasma concentrations and to determine the effect of the pH of OF on the methylone OF-to-plasma ratio (OF/P). 2. Materials and Methods 2.1. Subjects and Study Design A randomized, cross-over, double-blind, placebo-controlled pilot study was con- ducted on 12 male volunteers at the Hospital Universitari Germans Trias iPujol, Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, in Badalona (Spain). All participants had recreational experiences with psychoactive drugs, such as synthetic cathi- nones, amphetamines, cocaine and MDMA. A general physical examination, a 12-lead electrocardiogram, urinalysis and routine laboratory analyses were performed on each participant. The characteristics of the participants are summarized in Table 1. All subjects declared to be MDMA consumers, with a range of consumption between 5–100 times up to the moment of study, (mean 24). Each volunteer gave written informed consent before the start of the study and was economically remunerated for inconveniences caused by their inclusion in the experiment. The study was conducted in accordance with the Declaration of Helsinki, registered at ClinicalTrials.gov (NCT05488171) and approved by the local Ethical Committee for human research (CEIC-HuGTiP, ref. PI-19-082). Metabolites 2023, 13, 468 3 of 12 Table 1. Characteristics of the participants. Methylone 50 mg Methylone 100 Methylone 150 Methylone 200 Placebo (n = 3) mg (n = 6) mg (n = 5) mg (n = 6) (n = 12) Age (years) 22.3 ± 0.6 (22–23) 22.7 ± 0.8 (22–24) 23.4 ± 0.9 (22–24) 24.0 ± 0.0 (24–24) 23.3 ± 0.9 (22–24) 69.7 ± 14.2 71.0 ± 12.3 71.9 ± 10.0 70.0 ± 3.9 70.2 ± 8.7 Weight (kg) (60.4–86.0) (60.4–87.0) (61.9–87.0) (66.7–76.6) (60.4–87.0) 178.4 ± 3.4 177.0 ± 2.8 176.9 ± 4.8 183.6 ± 9.8 180.4 ± 7.2 Height (cm) (175.0–181.8) (181.8–174.2) (172.8–185.0) (172.8–193.5) (172.8–193.5) 21.9 ± 4.5 22.7 ± 3.9 23.1 ± 3.7 21.0 ± 3.3 21.7 ± 3.5 BMI (kg/m2 ) (18.3–27.0) (18.3–27.9) (19.4–27.9) (18.0–25.7) (18.0–27.9) Subjects were divided into four study groups and underwent three administration sessions, with a washout interval of 5–7 days. Specifically, in each session, single oral doses of 50, 100, 150 and 200 mg methylone or a placebo (dextromaltose) were administered to each participant. Lower doses were administered before higher ones. A good tolerability was observed for all doses. Volunteers were requested to abstain from consumption of any drug of abuse during the period of the study. The abstinence was verified by performing urine drug testing before each session. Specifically, the presence of benzodiazepine, MDMA, morphine, tetrahydrocannabinol, methadone, amphetamine, methamphetamine, cocaine, tricyclic antidepressants and barbiturates was tested using the Drug-Screen Multi 10TD Test [Multi-Line] (Nal Von Minden, Moers, Germany). The Pharmacy Service of Hospital Universitari Germans Trias i Pujol (Badalona, Spain) prepared the placebo and methylone as white soft-gelatin capsules (5 capsules each time, combining capsules with active substances and a placebo to reach the methylone dose), which were administered in a fasting state with 200 mL of tap water. 2.2. Chemicals Methylone, MDC and HMMC were supplied from Cerilliant (Round Rock, TX, USA). The internal standard (IS), methylone-d3 , was purchased from Cayman Chemical (Ann Arbor, MI, USA). Standards were stored at −20 ◦ C until analysis. LC-MS grade water, acetonitrile, methanol, formic acid, ethyl acetate and chloroform were purchased from Carlo Erba (Cornaredo, Italy). In total, 25% purity ammonium hydroxide and 37% pu- rity hydrochloric acid were obtained from Honeywell Fluka™ (Morristown, NJ, USA). SalivetteR tubes with cotton swab were purchased from Sarstedt (Nümbrecht, Germany). 2.3. Oral Fluid Samples Collection OF samples were obtained without any stimulation at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10 and 24 h after drug administration. Samples were collected using standard SalivetteR tubes with a cotton swab and centrifuged. The OF pH was recorded at the time of collection, and the samples were immediately stored at −20 ◦ C until analysis. OF samples from a placebo group that tested negative for methylone were used as drug-free blank samples. Blood samples were obtained at the same time [4]. 2.4. Sample Preparation The OF was allowed to thaw at room temperature. A 100 µL sample was fortified with 10 µL of the 100 ng/mL IS solution. After the addition of 2 µL of 2% NH3 in H2 O (pH 9) solution, a liquid–liquid extraction with 2 mL of chloroform/ethyl acetate 9:1 (v/v) was performed. Samples were roller-mixed for 10 min, centrifuged at 3500 rpm for 5 min and supernatants were separated into clean tubes. A total of 100 µL of 1% HCl in methanol solution (v/v) was added to avoid evaporative losses, and the samples were dried under a gentle nitrogen stream. Samples were dissolved in 100 µL of chromatographic mobile phase A:B (95:5) and transferred to autosampler vials prior to the injection of 1 µL into the HPLC-MS/MS system. Metabolites 2023, 13, 468 4 of 12 The concentration of methylone and its metabolites in OF was determined by using a 1290 Infinity II HPLC coupled to a 6470A triple quadrupole mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) equipped with an electrospray ionization source operat- ing in a positive mode. The separation of the compounds was carried out with a Kinetics® 2.6 µm Phenyl-Hexyl column from Phenomenex® (100 mm × 2.1 mm). The readings of 0.1% formic acid in the water and acetonitrile were mobile phases A and B, respectively, and the flow rate was set to 0.4 mL/min. The elution gradient was set as follows: initial conditions were 5% B, held for 1 min, gradually increased to 50% B within 2.0 min, then increased to 95% B within 4.0 min, finally decreased to 5% B and then held for 6 min. The total run time was 6 min. The autosampler temperature was set to 10 ◦ C, and the column oven temperature was 37 ◦ C. The mass spectrometer operated in the multiple ion monitoring (MRM) acquisition mode, selecting two transitions for each analyte and IS, as validated in our previous study [4]. Validation data are available in the Supplementary Material Table S1. 2.5. pH Measurements of Oral Fluid Samples The pH of the OF samples from the 12 volunteers in the placebo or methylone group was measured at all time intervals using a pH indicator stick (Riedel-de Haën, Hannover, Germany); the range of the pHs was between 6.4–8 (increments of 0.2 pH units). Two observers recorded results, and they were unaware of the treatment conditions. 2.6. Pharmacokinetics and Statistical Analysis The following parameters of the methylone OF concentrations were determined: maximum concentration (Cmax ), time to reach maximum concentration (tmax ) and the area under the concentration–time curve from 0 to 10 h (AUC0–10 ) and from 0 to 24 h (AUC0–24 ). Since what is observed in the OF is a disappearance rather than an elimination, we chose to use a disappearance half-life (t1/2d ) and disappearance constant (Kd ) to describe the considered parameters [29]. AUCs were calculated using the linear trapezoidal rule; the disappearance constant was calculated using the log-linear regression of the three last points with the concentration above the quantification limit. Correlations between the different variables were analyzed using regression analysis. To assess differences in OF pH values between baseline and different times after treatments, we performed an analysis of variance for the repeated measures (for each treatment) and, when significant, a post hoc analysis comparing the baseline with each time point using a Dunnett test. Differences associated with p-values lower than 0.05 were considered statistically significant. 3. Results 3.1. Concentration–Time Profile and Pharmacokinetic of Methylone, MDC and HMMC in Oral Fluid The time courses of methylone and its metabolites in OF after oral administration of 50, 100, 150 and 200 mg drugs are shown in Figure 1. The highest concentration of parent compounds in OF was observed at 2 h after each administered dose. Specifically, the means of the methylone Cmax were reported as 547.8, 5002.3, 13,383.6 and 20,464.9 ng/mL following the 50-, 100-, 150- and 200-mg doses, respectively. After the absorption phase, OF concentrations decreased to mean values of 88 (50 mg dose), 215.7 (100 mg dose), 199.6 (150 mg dose) and 1159.1 ng/mL (200 mg dose) at 24 h after administration. The pharmacokinetic parameters for methylone in OF are presented in Table 2. 10 was higher than that of HMMC, with values representing 5.4%, 7.9% and 8.7% of the methylone AUC0–10 following the 100-, 150- and 200-mg doses, respectively. A similar pro- file was obtained comparing AUC0–24 concentrations. Figures 2 and 3 show the correlation between the methylone dose and the values of Cmax as well as correlation between the dose and AUC0–24, respectively. In both cases, a linear correlation value was observed when considering doses between 100 to 200 mg (r2 = 0.6183 and r2 = 0.5908, respectively), despite Metabolites 2023, 13, 468 an elevated intersubject variability and the small number of participants. The dose of 50 5 of 12 mg was excluded from the calculation due to the low number of participants administered with this dose (n = 3). Methylone oral fluid concentration HMMC oral fluid concentration 20,000 2000 Methylone 50 mg (n=3) Methylone 100 mg (n=6) Methylone 100 mg (n=6) Methylone 150 mg (n=5) Concentration (ng/ml) 1500 Concentration (ng/ml) 15,000 Methylone 150 mg (n=5) Methylone 200 mg (n=6) Methylone 200 mg (n=6) 10,000 1000 5,000 500 0 0 0 1 2 3 4 6 8 10 24 0 1 2 3 4 6 8 10 24 Time (h) Time (h) MDC oral fluid concentration 1500 Methylone 100 mg (n=6) Methylone 150 mg (n=5) Concentration (ng/ml) 1000 Methylone 200 mg (n=6) 500 0 0 1 2 3 4 6 8 10 24 Time (h) Figure 1. Concentration–time profiles of methylone and its metabolites in OF following controlled 1. Concentration–time Figureadministration profilesdoses. of different methylone of methylone and its metabolites in OF following controlled administration of different methylone doses. Metabolites MDC and HMMC were not detected following the administration of 50 mg of methylone. Conversely, after the administration of 100-, 150- and 200-mg doses, HMMC reached the Cmax between 1.5 and 2.0 h and showed AUC0–10 values representing 4.6%, 3.2% and 7.2% of the methylone AUC0–10 concentrations, respectively. This pattern was similar for AUC0–24 .The same metabolite reached Cmax in plasma between 0.9 and 1.5 h [4]. The highest MDC concentration was reached at 3 h after the administration of the 100 and 150 mg doses, while it was reached at 4 h after the 200 mg dose. The MDC AUC0–10 was higher than that of HMMC, with values representing 5.4%, 7.9% and 8.7% of the methy- lone AUC0–10 following the 100-, 150- and 200-mg doses, respectively. A similar profile was obtained comparing AUC0–24 concentrations. Figures 2 and 3 show the correlation between the methylone dose and the values of Cmax as well as correlation between the dose and AUC0–24 , respectively. In both cases, a linear correlation value was observed when considering doses between 100 to 200 mg (r2 = 0.6183 and r2 = 0.5908, respectively), despite an elevated intersubject variability and the small number of participants. The dose of 50 Metabolites 2023, 13, x FOR PEER REVIEW mg13 6 of was excluded from the calculation due to the low number of participants administered with this dose (n = 3). Dose - Cmax r2= 0.6183 40,000 p= 0.0002 30,000 Cmax (ng/mL) 20,000 10,000 0 100 150 200 Dose (mg) Figure2.2.Relationship Figure Relationship between between administered administered methylone methylone dosesdoses (100, (100, 150200 150 and and 200and mg) mg) and attained attained Cmax . Cmax. Metabolites 2023, 13, 468 6 of 12 Table 2. Pharmacokinetic parameters for methylone and its metabolites in OF. Values are presented as mean + standard deviation (SD) and coefficient of variation (CV) %. Methylone HMMC MDC 50 mg 100 mg 150 mg 200 mg 100 mg 150 mg 200 mg 100 mg 150 mg 200 mg (Mean ± SD, CV%) (Mean ± SD, CV%) (Mean ± SD, CV%) (Mean ± SD, CV%) (Mean ± SD, CV%) (Mean ± SD, CV%) (Mean ± SD, CV%) (Mean ± SD, CV%) (Mean ± SD, CV%) (Mean ± SD, CV%) (n = 3) (n = 6) (n = 5) (n = 6) AUC0–10 (ng/mL × h) 2009.3 ± 425.2, 21% 16,724.1 ± 4179.4, 25% 34,454.6 ± 12,481.2, 36% 72,247.7 ± 33,659.2, 46% 778.5 ± 159.1, 20% 1118.0 ± 86.7, 8% 5179.0 ± 583.5, 11% 898.8 ± 95.0, 11% 2735.4 ± 441.3, 16% 6283.7 ± 1491.2, 24% AUC0–24 (ng/mL × h) 3350.0 ± 378.7, 11% 21,353.9 ± 5079.8, 24% 39,993.2 ± 14,995.3, 37% 96,628.3 ± 43,532.8, 45% 1023.1 ± 198.2, 19% 1307.3 ± 91.7, 7% 6821.7 ± 727.1, 11% 1017.5 ± 117.6, 12% 4086.4 ± 1104.4, 27% 7652.2 ± 1611.1, 21% tmax (h) 1.5 2.0 2.0 1.5 1.75 2.0 2.0 2.0 4.0 4.0 226.2 ± 3.6, Cmax (ng/mL) 547.8 ± 84.8, 15% 5002.3 ± 1192.7, 24% 13,383.6 ± 5379.8, 40% 20,464.9 ± 7979.5, 39% 236.8 ± 45.3, 19% 473.3 ± 56.4, 11% 1370.6 ± 420.6, 31% 474.4 ± 55.9, 12% 1430.5 ± 383.2, 27% 2% Kd (h−1 ) 0.053 ± 0.022, 42% 0.116 ± 0.02, 17% 0.147 ± 0.01, 7% 0.108 ± 0.02, 18% 0.115 ± 0.01, 9% 0.142 ± 0.01, 7% 0.106 ± 0.02, 19% 0.156 ± 0.02, 13% 0.115 ± 0.05, 43% 0.17 ± 0.03, 18% t1/2d (h) 14.6 ± 6.2, 43% 6.1 ± 1.0, 16% 4.7 ± 0.4, 8% 6.6 ± 1.5, 22% 6.1 ± 0.5, 8% 4.9 ± 0.4, 9% 6.7 ± 1.0, 15% 4.5 ± 0.6, 13% 7.6 ± 4.6, 60% 4.3 ± 0.9, 22% Abbreviations: AUC, area under the curve; HMMC, 4-hydroxy-3-methoxymethcathinone; Cmax , maximum concentration; Kd , disappearance constant; t1/2 d , disappearance half-life; tmax , time to reach maximum concentration, presented as median value; MDC, 3,4-methylenedioxycathinone. Metabolites 2023, 13, x FOR PEER REVIEW 8 of 13 Metabolites 2023, 13, x FOR PEER REVIEW 8 of 13 Metabolites 2023, 13, 468 7 of 12 Dose - AUC0-24 Dose - AUC0-24 r2= 0.5908 200,000 r2=0.0003 p= 0.5908 200,000 p= 0.0003 150,000 (ng/mL·h) 150,000 (ng/mL·h) AUC0-24 100,000 100,000 AUC0-24 50,000 50,000 0 0 100 150 200 100 150 Dose (mg) 200 Dose (mg) Figure 3. Relationship between administered methylone doses (100, 150 and 200 mg) and obtained Figure AUC 3. Relationship between administered methylone doses (100, 150 and 200 mg) and obtained 0–24. AUC0–24 AUC 0–24. . 3.2. Measurement of pH in Oral Fluid Samples 3.2. Measurement of 3.2. Measurement of pH pH in in Oral Oral Fluid Fluid Samples Samples Figure 4 shows the 24 h time profile of the OF pH following the administration of Figure Figure 4 shows the 4 showsdoses 24 h the 24and time h time profile profile of of the the OF pH pH following OFsamples following the the administration administration of of different methylone considering placebo as baselines. different methylone doses and considering placebo samples as baselines. different methylone doses and considering placebo samples as baselines. 0.2 0.2 0.0 Methylone 50 mg (n=3) 0.0 –-0.2 Methylone 50 mg (n=3) Methylone 100 mg (n=6) –-0.2 Methylone 100 mg (n=6) Methylone 150 mg (n=5) Methylone 150 mg (n=5) pHpH –-0.6 –-0.6 Methylone 200 mg (n=6) Salivary Methylone 200 mg (n=6) Placebo (n=12) Salivary –-1.0 Placebo (n=12) –-1.0 –-1.4 –-1.4 –-1.8 –-1.8 0 1 2 3 6 4 8 10 24 0 1 2 3 6Time (h)48 10 24 Time (h) Figure Figure 4. 4. Time Time profiles profiles of of OF OF pH pH following following controlled controlled administration administration of of different different methylone methylonedoses. doses. Figure 4. Time indicate Filled profiles of OF pH following controlled administration of different methylone doses. Filled symbols symbols indicate significant significant differences in that differences in that time time point with point with respect respect to each to each dose baseline. dose baseline. Filled symbols indicate significant differences in that time point with respect to each dose baseline. OF OF pH decreased in individuals treated with methylone methylone when when compared compared with with that from OF the pH decreased placebo group.inIn individuals the In particular, particular, treated pH with methylone that pH pattern pattern matched when compared matched that of ofmethylone with that methylonepharma- pharma- from the placebo group. In particular, the pH pattern matched that of cokinetics in OF, showing a minimal decrease for the lowest 50 mg dose, while significant methylone pharma- cokinetics were decreases decreases in OF,observed were showingwith observed a minimal with decrease increasing increasing doses. doses.forMinimum the lowestpH Minimum 50 mg pH dose, values values werewhile were significant obtained obtained be- be- decreases tween tween 1 were and 2 observed h, with corresponding increasing to the t doses. for Minimum each pH administered values corresponding the tmax for each administered dose. Finally, the max dose. were obtained Finally, the be- pH tween 1 and returned 2 h, corresponding to pretreatment values onlyto the tmax case in the for each of theadministered dose. Finally, 50-mg dose, whereas for thethe pH other returned to pretreatment values only in the case of the drug doses, it kept on showing values lower than the pre-treatment ones.50-mg dose, whereas for the other drugFigure doses, 5it shows Figure kept onthe showing values correlation lower than between the pre-treatment the doses of methylone ones. and the maximum Figure 5 shows effect on the pH, showing effect the correlation showing aa dose between dose response response with the doses with aalinear ofcorrelation(r(r2and methylone linearcorrelation 2= the maximum = 0.6073). Higher effect on the doses correlatedpH, correlated to showing to aa higher a dose higherreduction response reductionininthe thepHwith a linear pHvalues, correlation values,asasalso alsoshown shown (r2 = 0.6073). Higher inin thethe time timecourse of course doses pH correlated changes by to time a higher and dose reduction (see Figure of pH changes by time and dose (see Figure 4). in the 4). pH values, as also shown in the time course of pH changes by time and dose (see Figure 4). Metabolites Metabolites 2023, 2023, 13, x468 FOR PEER REVIEW 9 8ofof 13 12 Metabolites 2023, 13, x FOR PEER REVIEW 9 of 13 Dose vs pH Dose vs pH Emax Emax 0.0 0.0 2 r2r==0.6073 0.6073 –-0.5 pp<<0.0001 0.0001 –-0.5 pH Emax pH Emax –-1.0 –-1.0 –-1.5 –-1.5 –-2.0 –-2.0 00 50 50 100 100 150 150 200 200 Dose (mg) Dose (mg) Figure 5. Correlation between methylone doses and maximum effects on pH (Emax). Figure Figure 5. Correlation between 5. Correlation between methylone methylone doses doses and and maximum maximum effects effects on pH (Emax). 3.3.OF/plasma 3.3. OF/plasma Ratio Ratio for for Methylone Methylone 3.3. OF/plasma Ratio for Methylone The time The time course time course course of of different methylone different methylone doses in OF and plasma isisreported ininFigure 6. The Methylone reached of the different highest methylonedoses concentration doses in in inOF both OF and andplasma matrices plasma at 2 h isreported after reported inFigure administration Figure6. Methylone reached the highest concentration in both matrices at 2 h after administration of 6.ofMethylone different reached doses, the highest concentration in bothhigher matrices at those 2 h after administration different doses, butbut thetheOFOF values values were were significantly significantly than higher than those in in plasma. plasma. Spe- Specifi- ofcifically, different doses, the main but ratios the OF betweenvalues the were OF and significantly plasma C higher than those in plasma. Spe- max were 3.6, 16.4, 37.7 and 33.9 after cally, the main ratios between the OF and plasma Cmax were 3.6, 16.4, 37.7 and 33.9 after cifically, the main ratios theadministration administration 50,between of 50, 100, the OF and plasma Cmax were 3.6, 16.4, 37.7 and 33.9 after the of 100, 150 150 and and200 200mg mgmethylone, methylone,respectively. respectively. the administration of 50, 100, 150 and 200 mg methylone, respectively. Oral fluid Methylone 50 mg Plasma Oral fluid Methylone 100 mg Plasma 800 Oral fluid Methylone 50 mg 200 Plasma 6000 Oral fluid Methylone 100 mg 300 Plasma Oral fluid (n=3) Oral fluid (n=6) 800 200 6000 5000 300 Plasma Oral fluid(n=3) (n=3) Plasma Oral(n=6) fluid (n=6) Concentration (ng/ml) Concentration (ng/ml) Concentration (ng/ml) (ng/ml) 600 150 Plasma (n=3) 5000 4000 200 Plasma (n=6) Concentration (ng/ml) Concentration (ng/ml) Concentration (ng/ml) (ng/ml) 600 150 Concentration 400 100 4000 3000 200 Concentration 400 100 3000 2000 100 200 50 1000 2000 100 200 50 0 0 0 1000 0 0 1 2 3 4 6 8 10 24 0 1 2 3 4 6 8 10 24 0 Time (h) 0 0 Time (h) 0 0 1 2 3 4 6 8 10 24 0 1 2 3 4 6 8 10 24 Time (h) 150 mg Methylone Time Methylone (h)mg 200 Oral fluid Plasma Oral fluid Plasma 20,000 400 20,000 800 Methylone 150 mg Oral fluid (n=5) Methylone 200 mg Oral fluid (n=6) Oral fluid Plasma Oral fluid Plasma Plasma (n=5) Plasma (n=6) 20,000 400 20,000 600 800 Concentration (ng/ml) Concentration (ng/ml) Concentration (ng/ml) (ng/ml) (ng/ml) 15,000 300 15,000 Oral fluid (n=5) Oral fluid (n=6) Plasma (n=5) Plasma (n=6) Concentration Concentration (ng/ml) Concentration (ng/ml) Concentration (ng/ml) 15,000 10,000 300 200 15,000 10,000 400 600 Concentration 10,0005,000 200 100 10,000 5,000 200 400 5,000 0 0 100 0 5,000 0 200 0 1 2 3 4 6 8 10 24 0 1 2 3 4 6 8 10 24 Time (h) Time (h) 0 0 0 0 0 Figure6.6. 0 1 2 3 4 24 Time profile of different methylone 1 2 3 4 6 8 Time (h) 10 Figure Time profile of different methyloneconcentrations 6 in 8OF and concentrations plasma. in OF10and Time (h) 24 plasma. Accordingly, FigureAccordingly, after 6. Time profileafter increasing of different the theadministered methylone increasing methylone concentrations administered in OF anddoses, methylone thethe plasma. doses, ratio thethe ratio 24 h24 h OF/P ratio OF/P ratio exhibited exhibitedmean meanmaximum maximum values of 20.7, values 46.046.0 of 20.7, andand 31.8 31.8 at 2 hat(corresponding to 2 h (corresponding the t ) after 100, 150 and 200 mg methylone, respectively, while after 50 mg to the tmax ) after 100, 150 and 200 mg methylone, respectively, while after 50 mg dose24the Accordingly, max after increasing the administered methylone doses, the dose ratio the the h highest OF/P ratio highest OF/P OF/P was exhibited11.8 at mean was 11.8 24 h (Figure maximum at 24 7). The h (Figurevalues p-value 7). Theofp-value was 20.7, 46.0 <0.0001 wasand for 31.8 at <0.0001 each 2h for administration. (corresponding each to administration. the tmax) after 100, 150 and 200 mg methylone, respectively, while after 50 mg dose the highest OF/P was 11.8 at 24 h (Figure 7). The p-value was <0.0001 for each administration. Metabolites 2023,13, Metabolites2023, 13,468 x FOR PEER REVIEW 109 of 12 13 Methylone 60 Methylone 50 mg (n=3) Methylone 100 mg (n=6) 40 Methylone 150 mg (n=5) OF/P ratio Methylone 200 mg (n=6) 20 0 0 1 2 3 4 6 8 10 24 Time (h) Figure7. Figure 7. Time Timeprofile profileof ofmethylone methyloneOF/P OF/P ratio. 4. 4. Discussion Discussion For For the the first first time time ever, ever,this thisstudy studyshowed showedthe thepattern pattern of ofthe theOFOFconcentration–time concentration–time profile profile of methylone and its metabolites HMMC and MDC following increasing of methylone and its metabolites HMMC and MDC following increasing parent parent drug doses. drug doses. As Asexpected, expected,methylone methylone appeared appeared in OF at concentrations in OF at concentrations remarkable remarkablehigherhigher than thosethan in plasma those after each in plasma afteradministered each administereddose. As already dose. demonstrated As already demonstratedfor MDMA [19], this for MDMA is [19], probably due to the passive diffusion between plasma and OF. this is probably due to the passive diffusion between plasma and OF. Indeed, methylone Indeed, methylone is a basic drug (pKa 7.96), and the lower OF pH leads uncharged methylone in blood to diffuse across is a basic drug (pKa 7.96), and the lower OF pH leads uncharged methylone in blood to membranes into OF and ionize, accumulating in this matrix (ion trapping phenomenon). diffuse across membranes into OF and ionize, accumulating in this matrix (ion trapping The theoretical OF/P ratio for methylone should be 3.3, as calculated with the phenomenon). Henderson–Hasselbach equation [30]. In our study, the mean OF/P ratio was 3.8, 20.7, The theoretical OF/P ratio for methylone should be 3.3, as calculated with the Hen- 46.0 and 24.1 at tmax following 50, 100, 150 and 200 mg of methylone. The difference between derson–Hasselbach equation [30]. In our study, the mean OF/P ratio was 3.8, 20.7, 46.0 and the theoretical and experimental OF/P ratio values are attributable to the pH decrease, which 24.1 at tmax following 50, 100, 150 and 200 mg of methylone. The difference between the matches with the pharmacokinetic time profile of methylone; indeed, methylone OF con- theoretical and experimental OF/P ratio values are attributable to the pH decrease, which centrations increases with the decrease in pH. It should be noted that the OF/P ratio at tmax matches with the pharmacokinetic time profile of methylone; indeed, methylone OF con- following the 50 mg dose was the only experimental value similar to that obtained theoreti- centrations increases with the decrease in pH. It should be noted that the OF/P ratio at tmax cally. In support of this thesis, no significant changes in the pH were observed after 50 mg of following the methylone. Then,50 mg dose increased the ratio was the only experimental exponentially value for the 100similar and 150tomg that obtained doses, showing theo-a retically. In support of this thesis, no significant changes in the pH nonlinear pattern; finally, it decreases, and after 200 mg it was similar to that obtained afterwere observed after 50 mg mg 100 of methylone. of methylone. Then, Thethe ratio increased mechanism exponentially of action for the 100toand may also contribute 150 mg doses, an increase in the showing OF a nonlinear concentrations. pattern; finally, Methylone and MDMA it decreases, and after 200 act on serotonergic mg it was similarleading neurotransmission, to that obtained to after 100 and vasoconstriction mg of methylone. changes The mechanism in hemodynamics [31],ofthus action the may also contribute production of OF may to be an increase in the OF concentrations. Methylone and reduced, resulting in a concentration of compounds into this matrix. MDMA act on serotonergic neurotrans- mission, leading OF In addition, to vasoconstriction and changes contains a considerable amountin hemodynamics [31], thus the of inorganic compounds, produc- including tion of OF may be reduced, resulting in a concentration of−compounds water and strong and weak ions (Na+, K+, Mg+, Ca2+, Cl , bicarbonate and phosphate), into this matrix. whichInmay addition, function OF ascontains buffering a considerable agents. We do amount not knowof inorganic compounds, the relevance including of the inorganic water and strong and weak ions (Na+, K+, Mg+, Ca2+, Cl −, bicarbonate and phosphate), compound in our study. We tried to standardize all the conditions in order to reduce the which may relevance of function as buffering intrinsic/extrinsic agents. factors. In We ourdo not know subjects, onlythe taprelevance water was of administered the inorganic and separated from the OF sampling. Chewing gum, alcoholic beverages ortocoffee compound in our study. We tried to standardize all the conditions in order reduce the were relevance of intrinsic/extrinsic factors. In our subjects, only tap not allowed during experimental sessions. In addition, subjects presented similar baseline water was administered and before pHs separateddrugfrom the OF sampling. administration. Chewing gum, Since methylone shows alcoholic sympatheticbeverages action,orthe coffee were changes not in allowed the pH could during be theexperimental sessions. In addition, due the norepinephrine action onsubjects salivary presented glands. similar baseline pHs Thebefore drug administration. concentrations of methyloneSince methylone in the OF of shows sympathetic the present studyaction, agree the withchanges those in the pH in observed could be the due a previous the norepinephrine observational study, whereactionsubjects on salivary glands. self-administered different dosesThe concentrations of methylone of methylone (100 mg, 150 mg, 200 in mgthe and OF of 300themg; present n = 8)study agree with and provided OFthose 1, 2 andob- 4served h afterinadministration a previous observational [32]. study, where subjects self-administered different doses of methylone (100 mg, 150 mg, 200 mg and 300 mg; n = 8) and provided OF 1, 2 and 4 h after administration [32]. Metabolites 2023, 13, 468 10 of 12 The exceeding theoretical values were also observed in other studies with metham- phetamine after a different route of administration, such as the intravenous route [33]; this allowed us to exclude the hypothesis of buccal contamination for the first 2 h following oral administration. Moreover, similar to our previous study [19], juice and snacks given to the volunteers at 3 h after the administration should have eliminated this contamination also in the following hours. Overall, the pharmacokinetic profiles showed similar patterns. However, some in- terindividual variations were noticed, especially for methylone, where CV% increased with increasing doses. As MDMA, methylone was reported to be metabolized by CYP2D6 [34]; its genetic polymorphism may be the cause of high interindividual variations observed in methylone time-course profiles. Furthermore, several xenobiotics are substrates of this enzyme, leading to an inhibition or an induction [35,36]. OF t1/2d was comparable for methylone and HMMC following 100, 150 and 200 mg doses; conversely, MDC showed a different profile in regard to the clearance rate, especially compared to methylone and the HMMC pharmacokinetic profile. In fact, its elimination rate was more rapid following 100 and 200 mg doses, whereas it was slower when the 150 mg methylone dose was administered. This parameter suggests a prolonged effect compared to other synthetic cathinones such as mephedrone (2.12 h) [37]; furthermore, several studies demonstrated that its metabolite MDC can cross the blood–brain barrier in rats, producing a significant increase in brain extracellular dopamine and serotonin concentrations [9]. Therefore, the pharmacological activity of MDC may enhance the psychoactive effects of methylone. However, concentrations were much lower than its parent drug. Conversely, a study demonstrated that HMMC does not cross the blood–brain barrier in rats, thus it may be inactive [8]. 5. Conclusions The analytical method demonstrated to be suitable for methylone and metabolite detection in OF, with good precision, accuracy and efficiency. This matrix represents an alternative to plasma for clinical and toxicological purposes, allowing us to detect a recent drug assumption and make a simple, rapid and non-invasive collection. In our study, methylone was detectable at a high concentration (> 80 ng/mL) at 24 h also, but it should be noted that the high administered doses and controlled conditions may have enlarged the detection window. This study confirmed the matching pharmacokinetic pattern of methylone and MDMA, already suggested by the structure similarities. In particular, both compounds exhibited an experimental OF/P ratio much higher than that theoretically calculated through the Henderson–Hasselbach equation. This was evident for higher methylone doses (100, 150 and 200 mg), whereas the two ratios were comparable at the lowest administered dose (50 mg). Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/metabo13040468/s1, Table S1: Method validation parameters. Author Contributions: Conceptualization, M.F., S.P. and F.P.B.; methodology, A.D.G., G.S. and L.P.; validation, A.D.G. and G.S.; investigation, L.P., A.D.G., A.F.L.F., E.P., A.T., M.F., F.P.B., S.P. and C.P.-M.; data curation, L.P., A.D.G., A.F.L.F., E.P., A.T., M.F., F.P.B., S.P. and C.P.-M.; writing—original draft preparation, A.D.G., G.S., L.P. and A.F.L.F.; writing—review and editing, S.P., M.F., F.P.B., L.P., A.D.G. and G.S.; supervision, S.P., F.P.B. and M.F.; funding acquisition, M.F. and S.P. All authors have read and agreed to the published version of the manuscript. Funding: This investigation was partially supported by grants from the Instituto de Salud Carlos III (ISCIII, Fondo de Investigación en Salud (FIS)-Fondo Europeo de Desarrollo Regional (FEDER), grant numbers PI17/01962 and PI20/00879; predoctoral PFIS fellowship grant number FI18/00179; Plataformas ISCIII de apoyo a la I + D + I en Biomedicina y Ciencias de la Salud grant number PT20/00018). It is also partially funded by the project “Implementation of the identification and study of the effects of NPS: Development of a multicenter research to enhance the database of the Metabolites 2023, 13, 468 11 of 12 National drug Addiction Observatory and the Early Warning System” and by the project “National Early Warning System on Drugs (SNAP)” following the Italian Department of Antidrug policies. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the local human research ethics committee CEI-HUGTiP (ref. 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