International Journal of Molecular Sciences Communication Sweat Testing for the Detection of Methylone after Controlled Administrations in Humans Alessandro Di Giorgi 1,† , Giorgia Sprega 1,† , Lourdes Poyatos 2,3 , Esther Papaseit 2,3 , Clara Pérez-Mañá 2,3 , Annagiulia Di Trana 4 , Maria Rosaria Varì 4, * , Francesco Paolo Busardò 1 , Simona Pichini 4 , Simona Zaami 5 , Alfredo Fabrizio Lo Faro 1,‡ and Magí Farré 2,3,‡ 1 Department of Excellence-Biomedical Sciences and Public Health, Università Politecnica delle Marche, 60121 Ancona, Italy; digiorgiale97@gmail.com (A.D.G.); giorgiasprega1996@gmail.com (G.S.); f.busardo@univpm.it (F.P.B.); fabriziolofaro09@gmail.com (A.F.L.F.) 2 Department of Clinical Pharmacology, Hospital Universitari Germans Trias i Pujol and Institut de Recerca Germans Trias i Pujol (HUGTiP-IGTP), 08916 Badalona, Spain; lpoyatos@igtp.cat (L.P.); epapaseit.germanstrias@gencat.cat (E.P.); cperezm.mn.ics@gencat.cat (C.P.-M.); mfarre.germanstrias@gencat.cat (M.F.) 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; annagiulia.ditrana@iss.it (A.D.T.); simona.pichini@iss.it (S.P.) 5 Department of Anatomical, Histological, Forensic, and Orthopedic Sciences, Università La Sapienza, 00161 Rome, Italy; simona.zaami@uniroma1.it * Correspondence: mariarosaria.vari@iss.it † These authors contributed equally to this work. ‡ These authors also contributed equally to this work. Abstract: The aim of this study was to determine the excretion of methylone and its metabo- lites in sweat following the ingestion of increasing controlled doses of 50, 100, 150 and 200 mg of methylone to twelve healthy volunteers involved in a clinical trial. Methylone and its metabolites 4-hydroxy-3-methoxy-N-methylcathinone (HMMC) and 3,4-methylenedioxycathinone (MDC) were analyzed in sweat patches by liquid chromatography–tandem mass spectrometry. Methylone and Citation: Di Giorgi, A.; Sprega, G.; MDC were detected in sweat at 2 h and reached their highest accumulation (Cmax ) at 24 h after the Poyatos, L.; Papaseit, E.; Pérez- administration of 50, 100, 150 and 200 mg doses. In contrast, HMMC was not detectable at any time Mañá, C.; Di Trana, A.; Varì, M.R.; Busardò, F.P.; Pichini, S.; Zaami, S.; interval after each dose. Sweat proved to be a suitable matrix for methylone and its metabolites’ et al. Sweat Testing for the Detection determination in clinical and toxicological studies, providing a concentration that reveals recent of Methylone after Controlled drug consumption. Administrations in Humans. Int. J. Mol. Sci. 2023, 24, 7395. https:// Keywords: methylone; sweat; LC-MS/MS; pharmacokinetics doi.org/10.3390/ijms24087395 Academic Editor: João Pedro Silva Received: 20 March 2023 1. Introduction Revised: 2 April 2023 Synthetic cathinones constitute the most extensive class of synthetic stimulants, compounds Accepted: 14 April 2023 belonging to the group of New Psychoactive Substances (NPS). Their structure is based on that Published: 17 April 2023 of cathinone, a monoamine alkaloid found in the khat plant with psychoactive effects [1]. One of the most abused compounds of this class is 3,4-methylenedioxy-methcathinone (MDMC), more frequently known as methylone [2]. This drug often appears as a white powder [3]. Copyright: © 2023 by the authors. The structure of methylone differs only from that of 3,4-methylenedioxy-methamphetamine Licensee MDPI, Basel, Switzerland. (MDMA) for the ketone group in the β-position [4]. Consequently, these two compounds This article is an open access article share a similar mechanism of action, which involves the enhancement of monoamine distributed under the terms and release (norepinephrine, dopamine and serotonin) and the inhibition of the dopamine conditions of the Creative Commons transporter (DAT), the serotonin transporter (SERT) and the norepinephrine transporter Attribution (CC BY) license (https:// (NET) [4–6]. creativecommons.org/licenses/by/ As MDMA, methylone is mainly metabolized in the liver. The two major path- 4.0/). ways are N-demethylation, which converts this drug to 3,4-methylenedioxycathinone Int. J. Mol. Sci. 2023, 24, 7395. https://doi.org/10.3390/ijms24087395 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 7395 2 of 9 (MDC), and O-demethylation, which is followed by O-methylation, resulting in 3,4- dihydroxy-N-methylcathinone (HHMC) and 4-hydroxy-3-methyoxy-N-methylcathinone (HMMC), respectively. The pharmacological effects of methylone in humans resemble those of MDMA, including altered perception (such as changes in light, different bodily sensations), empathy, increased sociability, stimulation and euphoria [7]. Similar to other synthetic cathinones, methylone use has been shown to be related to consumer fatalities [1]. In this context, several cases of death are reported in the literature. A 23-year-old man was transported to the hospital with a body temperature of 41 ◦ C and aggressive behavior; his death occurred after 3 h. Since high concentrations of methylone were detected in his biological matrices, the cause of death was attributed to this drug [8]. In another case, methylone was ingested during a party with friends [9]. Sweat is an alternative matrix useful for drug testing and monitoring; it is generally collected by applying an adhesive patch to different parts of the body (back, shoulders and chest). The exact mechanism of excretion of drugs in sweat is not still fully explained, but there are several proposed mechanisms, which include passive diffusion from the blood into the sweat glands. Specifically, since sweat is more acidic than blood (pH 6.3 and 7.4, respectively), basic drugs diffuse into sweat through a concentration gradient [10]. An alternative mechanism involves the diffusion of drugs through the stratum corneum to the skin surface, dissolving in sweat [11]. Moreover, the reabsorption of MDMA from the skin was reported [12], and this may result in the continued presence of drugs on the skin surface also within time windows not covered by other biological matrices such as blood or urine. Several analytical methods for the determination of methylone in other matrices have been reported in the literature, such as in plasma and serum [2], urine [13], oral fluid [14] and hair [15]. However, to the best of our knowledge, no research in sweat has been yet published, nor pharmacokinetic studies involving this matrix. The aims of this study were as follows: - to develop and validate an LC-MS/MS method for the determination of methylone and its metabolites, MDC and HMMC, in sweat; - to investigate the excretion of methylone, MDC and HMMC in sweat after the ingestion of increasing controlled doses (50, 100, 150 and 200 mg) to 12 healthy volunteers involved in a clinical trial. 2. Results Figure 1 shows methylone’s mean concentration–time profile for each administered dose (50, 100, 150 and 200 mg). The drug was detected in sweat at 2 h after each dose at different concentrations (34.3, 96.0, 52.8 and 115.0 ng/patch following 50, 100, 150 and 200 mg doses, respectively). It has to be underlined that sweat concentrations are not temporal fractions, but reflect the accumulation of the parent drug and metabolites. In this context, an interesting concentration–time profile was observed after each dose. In particular, the administration of 50 mg methylone presented a first peak value at 4 h (mean: 40.8 ng/patch), with no further release, leading to a mean concentration of 6.7, 12.3 and 9.0 ng/patch at 6, 8 and 10 h post-administration, respectively. Finally, methylone reached the mean value of 254.2 ng/patch at 24 h, representing drug accumulation in 24 h. Inter-subject variability was large, with a coefficient of variation, CV (%), ranging from 19% (at 8 and 10 h) to 60% (at 24 h). The administration of 100 mg methylone showed progressive drug excretion resulting in mean sweat concentrations of 96.0, 154.6, 398.9 and 651.9 ng/patch at 2, 4, 6 and 8 h post- administration. Then, concentrations rapidly increased to 813.7 ng/patch at 10 h. Similarly to what occurred with the 50 mg dose, the highest drug accumulation was observed at 24 h (mean: 1346.8 ng/patch, 5.3-fold higher than that obtained after 50 mg methylone). However, this subject showed the highest CV (%) values, ranging from 85% (at 6 h) to 201% (at 2 h). Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 3 Int. J. Mol. Sci. 2023, 24, 7395 3 of 9 Methylone 50 mg Methylone 100 mg 500 2000 Concentration (ng/patch) Concentration (ng/patch) 400 1500 300 1000 200 500 100 0 0 0 2 4 6 8 10 24 0 2 4 6 8 10 24 Time (h) Time (h) Methylone 150 mg Methylone 200 mg 2500 5000 Concentration (ng/patch) Concentration (ng/patch) 2000 4000 1500 3000 1000 2000 500 1000 0 0 0 2 4 6 8 10 24 0 2 4 6 8 10 24 Time (h) Time (h) Figure 1. Mean concentration–time profiles of methylone contained in sweat patches following Figure increasing controlled 1. Mean concentration–time administrations (50, 100, 150 andprofiles 200 mg).of methylone contained in sweat patches follo increasing controlled administrations (50, 100, 150 and 200 mg). Following 150 mg methylone dose administration, sweat concentrations remained constant between 4, 6Inand this context, 8 h, an interesting with values concentration–time of 487.9, 474.6 profilerespectively. and 466.3 ng/patch, was observed after Then, an increasedose. In particular, to 676.1 ng/patchthe wasadministration observed at 10ofh,50and mgthemethylone presented a first highest accumulation at peak va 4 h (mean: 24 h (1655.7 ng/patch). 40.8 ng/patch), Inter-subject with no variability further was large,release, between leading 36% (atto 24 a mean concentration h) and 67% o (at 6 h). 12.3 and 9.0 ng/patch at 6, 8 and 10 h post-administration, respectively. Finally, meth A different reached excretion theprofile mean value of 254.2 ng/patch was observed after theatadministration 24 h, representing drug of the 200accumulation mg h. Inter-subject variability was large, with a coefficient dose. A concentration of 115.0 ng/patch methylone was detected at 2 h, and then of variation, CV (%), the ranging 19% (at 8 and 10 h) to 60% (at 24 h). concentration rapidly increased to 1486.6 at 4 h. Between 4 and 6 h post-treatment, the concentration slightlyThe administration increased of 100 mg to 1727.7 ng/patch, methylone followed showedrapid by a further progressive increase todrug excr resulting in mean sweat concentrations of 96.0, 154.6, 2784.6 at 8 h. At 10 h, methylone showed a slight decrease to 2324.6 ng/patch. Similarly to398.9 and 651.9 ng/patch at 2 and 8 h post-administration. Then, concentrations rapidly each administration, the highest drug accumulation in sweat was observed at 24 h, with a increased to 813.7 ng/pat 10 h. Similarly mean value of 3530.6 ng/patch.toInter-subject what occurred with the was variability 50 mg dose,than lower the that highest drug accumulation observed for lower doses, with observed values thatat 24ranged h (mean: 1346.821% between ng/patch, and 51%. 5.3-fold higher than that obtained after 5 methylone). Sweat specimens were alsoHowever, analyzed this for subject showedmetabolites. methylone the highest CV HMMC,(%) values, ranging from the major (at 6 h) to 201% (at 2 h). plasma metabolite, was not detected after each administered dose and the only metabolite detected was MDC (Figure Following 150 mg 2). This methylone compound wasdose administration, detected at 2 h with sweat valuesconcentrations of 8.3, 8.2, rema constant between 4, 6 and 8 h, with values of 487.9, 474.6 8.7 and 8.8 ng/patch after the administration of 50, 100, 150 and 200 mg methylone. Then, and 466.3 ng/patch, respect Then, an increase to 676.1 ng/patch was observed at 10 h, and the highest accumulati a slight increase was observed between 2 and 8 h following the lowest dose, prior to a 24 h (1655.7 ng/patch). Inter-subject variability was large, between 36% (at 24 h) and decrease at 10 h. Conversely, a more rapid increase was noticed between 2 and 6 h after the (at 6 h). 100 mg dose, followed by constant values between 6 and 10 h. A slight progressive increase A different excretion profile was observed after the administration of the 20 was observed between 2 and 10 h when 150 mg methylone was administered. A different dose. A concentration of 115.0 ng/patch methylone was detected at 2 h, and the profile was noticed after the 200 mg dose. MDC sweat concentrations rapidly increased concentration rapidly increased to 1486.6 at 4 h. Between 4 and 6 h post-treatmen between 2 and 8 h and remained constant between 8 and 10 h. For each administered dose, concentration slightly increased to 1727.7 ng/patch, followed by a further rapid inc a rapid increase was observed at 10 h until reaching the highest metabolite accumulation of to 2784.6 at 8 h. At 10 h, methylone showed a slight decrease to 2324.6 ng/patch. Sim 15.0, 16.6, 23.6 and 24.5 ng/patch at 24 h after the 50, 100, 150 and 200 mg doses, respectively. to each administration, the highest drug accumulation in sweat was observed at 24 h, A graph comparing the administered methylone doses and the accumulation of the parent drug and its metabolite at 24 h is shown in Figure 3. increase was observed between 2 and 10 h when 150 mg methylone was administered. A different profile was noticed after the 200 mg dose. MDC sweat concentrations rapidly increased between 2 and 8 h and remained constant between 8 and 10 h. For each administered dose, a rapid increase was observed at 10 h until reaching the highest Int. J. Mol. Sci. 2023, 24, 7395 metabolite accumulation of 15.0, 16.6, 23.6 and 24.5 ng/patch at 24 h after the 50, 100, 150 4 of 9 and 200 mg doses, respectively. MDC 50 mg MDC 100 mg 20 20 Concentration (ng/patch) Concentration (ng/patch) 15 15 10 10 5 5 0 0 0 2 4 6 8 10 24 0 2 4 6 8 10 24 Time (h) Time (h) MDC 150 mg MDC 200 mg 30 30 Concentration (ng/patch) Concentration (ng/patch) 20 20 10 10 0 0 0 2 4 6 8 10 24 0 2 4 6 8 10 24 Time (h) Time (h) Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 5 Figure 2. Concentration–time profile of metabolite MDC contained in sweat patches following Figure 2. Concentration–time profile of metabolite MDC contained in sweat patches following in- increased controlled methylone administrations (50, 100, 150 and 200 mg). creased controlled methylone administrations (50, 100, 150 and 200 mg). Excretion of methylone in sweat at 24 h Excretion of MDC in sweat at 24 h A graph comparing the administered methylone 5000 30 doses and the accumulation of the parent drug and its metabolite at 24 h is shown in Figure 3. Concentration (ng/patch) Concentration (ng/patch) 4000 20 3000 2000 10 1000 0 0 g g g g g g g g m m m m m m m m 0 0 50 0 0 50 0 0 15 15 10 20 10 20 g g Methylone administered doses Methylone administered doses Figure Figure 3. The 24 3. The of h excretion 24 methylone h excretionandof methylone and MDC MDC in sweat in sweat following following increased increased contr controlled methylone administrations (50, 100, methylone administrations (50, 100, 150 and 200 mg). 150 and 200 mg). 3. Discussion 3. Discussion Forsweat For the first time, the first time, sweat patches patches were used were used to monitor to monitor methylone methylone excretion excretion in hu in human sweatdrug sweat for 24 h after for 24 h after drug administration. administration. Drugs are mainly Drugs are mainly into incorporated incorporated this matrixinto this m through passivethrough passive diffusion, due todiffusion, due to a gradient a concentration concentration gradient that allows thethat allows of diffusion thethe diffusion o free fraction offree the fraction drug from of the drug to plasma from plasma sweat to sweat [10,16]. [10,16].sweat Moreover, Moreover, has asweat pH ofhas6.3a pH of 6 in normal conditions; thus, it is more normal conditions; thus,acidic thanacidic it is more bloodthan [17].blood [17]. Methylone Methylone is a basicisdrug a basic drug with a pKa around a pK7.96; consequently, a around this drug should 7.96; consequently, convert this drug to its should ionized convert toform and its ionized form accumulate in accumulate sweat [10,17]. In this[10,17]. in sweat context,Inlipophilicity this context,islipophilicity another physical–chemical is another physical–chem parameter that parameter can suggest thatthe presence of drugs can suggest theinpresence this matrix. of Indeed, drugs inparent this compounds matrix. Indeed, pa crossing membranes compounds can be detectable crossing at higher membranes canconcentrations be detectable than their concentrations at higher more polar than metabolites [12,18,19]. more polar metabolites [12,18,19]. Similarly to oral fluid, the Henderson–Hasselbach equation allows the calculatio the excretion of a basic drug also in sweat, using the pH of blood and sweat, and the of the drug [10]. In particular, the theoretical sweat/plasma ratio for methylone shou around 11.2. However, we could not verify this theoretical calculation since, in our st we applied sweat patches that did not allow the calculation of the volume of colle sweat. Moreover, the loss of water during wear could lead to an effect on these meas Int. J. Mol. Sci. 2023, 24, 7395 5 of 9 Similarly to oral fluid, the Henderson–Hasselbach equation allows the calculation of the excretion of a basic drug also in sweat, using the pH of blood and sweat, and the pKa of the drug [10]. In particular, the theoretical sweat/plasma ratio for methylone should be around 11.2. However, we could not verify this theoretical calculation since, in our study, we applied sweat patches that did not allow the calculation of the volume of collected sweat. Moreover, the loss of water during wear could lead to an effect on these measures. Methylone was detectable in sweat at 2 h after the administration of all doses, cor- responding to its peak concentration in plasma and oral fluid, and reached the highest concentration in sweat at 24 h, corresponding to the lowest methylone concentrations in plasma and oral fluid [2,20]. As expected, the main compound detected in sweat was the parent drug; the metabolite MDC was found at lower concentrations, while HMMC was not detected at any time interval. This is probably due to the more hydrophilic nature of the metabolites compared to methylone. Moreover, the presence of a secondary amine and a hydroxyl group lead HMMC to be more polar than MDC, which has a primary amine but also a dioxolane heterocycle. This is in agreement with a previous study on the sweat testing of MDMA following controlled administration. Indeed, its primary hydrophilic metabolite HMMA was found only in traces in the sweat patches of administered volun- teers [12]. A constant MDC concentration was observed between 8 and 10 h following the administration of 100, 150 and 200 mg methylone doses. We can hypothesize that the resting period may affect the perspiration rate and the accumulation of the drug in sweat. Several studies demonstrated that the concentrations of drugs in sweat can decrease due to skin reabsorption, as observed for diazepam, MBDB, MDMA and cocaine [21–23]. This phenomenon could have explained the decrease in methylone concentrations noticed in some cases in this research. However, a confirmation study on skin reabsorption was not carried out. Overall, the sweat patch allowed us to monitor methylone accumulation in sweat during the 24 h after administration. Since the local ethical committee’s permission was provided for a 24 h study, we could not investigate methylone excretion in sweat over a longer period of time. However, based on a previous study on MDMA, which was detectable up to a week after a single administration [24], we can hypothesize that this drug can be excreted in sweat for more than 24 h. Methylone concentrations at 24 h and the ratio between the area of the patch and the body surface area were used to approximate the total amount of methylone excreted in sweat. The mean value of methylone excreted during 24 h post-administration was 0.31, 3.85, 2.02 and 4.3 mg following 50, 100, 150 and 200 mg doses, respectively. These values are equivalent to 0.62%, 3.85%, 1.35% and 2.15% of 50, 100, 150 and 200 mg administered, respectively, considering equal sweating at each body location. The large inter-subject variability observed in this study can be justified by the dif- ferent perspiration rates and volumes of sweat between the individuals who received the same methylone dose. These differences were reported also for MDMA in a previous research study [12]. Moreover, the pharmacological activity of methylone regarding body temperature regulation contributed to the enhanced inter-subject variability. Another factor that contributed to the inter-subject variability was the location of the patch. In this context, a study comparing cocaine and metabolite concentrations in sweat patches applied on the back and on the shoulders found eight-fold higher levels on the back than on the shoulders [17]. For this reason, the sweat patches in this study were applied on the back, to determine the highest drug concentrations in sweat. 4. Materials and Methods 4.1. Subjects and Study Design In total, 14 male volunteers were included in a cross-over, randomized, double-blind and placebo-controlled study at the Hospital Universitari Germans Trias i Pujol, Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, in Badalona (Spain). All volunteers were recreational users of drugs such as MDMA, amphetamines, synthetic Int. J. Mol. Sci. 2023, 24, 7395 6 of 9 cathinones and cocaine. Each participant received routine laboratory tests, a general physical examination, urinalysis and a 12-lead electrocardiogram. Table 1 presents the characteristic of the participants. All volunteers claimed to have consumed MDMA in the range of 5–100 times within their lives, with a mean value of 24 times. All subjects gave written informed consent before their inclusion in the research and were economically compensated for the inconveniences caused by their participation in the study. The study was carried out in accordance with the Declaration of Helsinki, approved by the local ethical committee for human research (CEIC-HuGTiP, ref. PI-19-082). Table 1. Characteristics of the participants. Methylone 50 mg (n = 3) Methylone 100 mg (n = 5) Methylone 150 mg (n = 4) Methylone 200 mg (n = 7) Placebo (n = 14) 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) Weight (kg) 69.7 ± 14.2 (60.4–86.0) 71.0 ± 12.3 (60.4–87.0) 71.9 ± 10.0 (61.9–87.0) 70.0 ± 3.9 (66.7–76.6) 70.2 ± 8.7 (60.4–87.0) Height (cm) 178.4 ± 3.4 (175.0–181.8) 177.0 ± 2.8 (181.8–174.2) 176.9 ± 4.8 (172.8–185.0) 183.6 ± 9.8 (172.8–193.5) 180.4 ± 7.2 (172.8–193.5) BMI (kg/m2 ) 21.9 ± 4.5 (18.3–27.0) 22.7 ± 3.9 (18.3–27.9) 23.1 ± 3.7 (19.4–27.9) 21.0 ± 3.3 (18.0–25.7) 21.7 ± 3.5 (18.0–27.9) Volunteers were divided into four groups and underwent three administration ses- sions, divided by a washout time of 5–7 days. Each participant was administered single oral doses of the placebo (dextromaltose), 50, 100, 150 or 200 mg methylone in each session. The selected methylone doses were based on previous observational studies reporting methy- lone and mephedrone’s effects. The World Health Organization reported 60–120 mg methy- lone doses as “threshold”, 100–150 mg as “light”, 100–250 mg as “common”, 160–270 mg as “strong” and >250 mg as “very strong” [25]. In a previous observational study, oral doses of 100 mg (n = 1), 150 mg (n = 2; 1 male and 1 female), 200 mg (n = 4; 2 male and 2 female) and 300 mg (n = 1; male) were well tolerated [26]. Moreover, in a controlled trial with mephedrone, we carried out a dose-finding pilot study with doses of 50, 100, 150 and 200 mg mephedrone and 100 mg MDMA. The highest methylone dose produced similar effects to that observed following the administration of 100 mg MDMA [27,28]. Based on the above-reported studies, we decided to administer well-tolerated methylone doses and the one (200 mg) producing effects similar to those obtained with 100 mg MDMA. The administration of higher methylone doses was performed after lower ones. More- over, good tolerability was noticed after each dose. Subjects were required not to consume any drug of abuse during the period of the study. Nonetheless, a urine drug test preceded each experimental session to confirm their abstinence. In particular, the drugs screened were barbiturate, cocaine, amphetamine, benzodiazepine, morphine, MDMA, methadone, methamphetamine, tetrahydrocannabinol and tricyclic antidepressants (Drug-Screen Multi 10TD Test [Multi-Line], Nal von Minden, Germany). For each class of compounds tested, all participants tested negative. Soft gelatin capsules of placebo and methylone were prepared by the Pharmacy Service of Hospital Universitari Germans Trias i Pujol and administered with 200 mL of tap water in a fasting state. 4.2. Chemicals Methylone, MDC and HMMC were supplied by Cerilliant (Round Rock, TX, USA). 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, methanol, acetonitrile, formic acid and ethyl acetate were obtained from Carlo Erba (Cornaredo, Italy). Ammonium hydroxide (25% purity) and hydrochloric acid (37% purity) were supplied by Honeywell Fluka™ (Morristown, NJ, USA). 4.3. Sweat Sample Collection Six patches were located on the back of each volunteer after the skin was cleaned with a 70% 2-propanol cotton swab and removed at 2, 4, 6, 8, 10 and 24 h after methylone/placebo administration. This was performed by pulling the adhesive edge, taking care not to touch or contaminate the absorbent part. Then, the patch was labeled prior to storage in Int. J. Mol. Sci. 2023, 24, 7395 7 of 9 plastic bags at −20 ◦ C until analysis. Placebo sweat patches, which all tested negative for methylone by LC-MS/MS, were used as drug-free blank samples. 4.4. Sample Preparation and Analyte Measurement Each patch was fortified with 0.5 mL of 200 ng/mL of IS solution and allowed to dry at room temperature for 30 min. The outer adhesive part of the patch was removed, and the absorbent part was cut into small pieces and placed in a glass tube with 2 mL of methanol for 1 h. Then, the methanolic solution was transferred to another glass tube. Finally, 100 µL was transferred to an autosampler vial, prior to the injection of 1 µL into the HPLC-MS/MS system. Methylone and its metabolites’ concentrations in sweat were determined through a 1290 Infinity II HPLC coupled to a 6470A triple-quadrupole mass spectrometer (Agilent Technologies, Palo Alto, CA, USA), with an electrospray ionization source set in positive mode (ESI+). The separation of the compounds was obtained through a Kinetics® 2.6 µm Phenyl-Hexyl 100 × 2.1 mm column (Phenomenex® ). Then, 0.1% formic acid in water and acetonitrile were used as mobile phases A and B, respectively, and the flow rate was set to 0.4 mL/min. The elution gradient consisted of an initial condition of 5% B, held for 1.0 min, gradually increased to 50% B within 2.0 min, then increased to 95% B within 4.0 min, and finally decreased to 5% B and held for 6.0 min. The total run time was 6 min. The autosampler and column oven temperatures were set to 10 ◦ C and 37 ◦ C, respectively. The mass spectrometer operated in multiple reaction monitoring (MRM) acquisition mode, selecting two transitions for each analyte and IS, as validated in our previous study. Validation parameters are presented in Supplementary Material Table S1. Using the above-reported analytical method, the limits of detection were 1.5, 0.2 and 0.2 ng/patch and the limits of quantification were 5.0, 0.5 and 0.5 ng/patch for methylone, MDC and HMMC, respectively. 5. Conclusions The analytical method proved to be suitable for the detection of these compounds in sweat, with good accuracy, precision, sensitivity and linearity. Sweat represented an unconventional matrix, allowing the detection of methylone and its metabolite MDC at a few hours and at 24 h after the administration of 50, 100, 150 and 200 mg doses. HMMC was not detectable at any time interval. Thus, as expected, the parent drug was the principal analyte detected in sweat samples. However, investigators should take into consideration that the concentration of a drug in sweat can be affected by several factors, such as the different perspiration rates, the volume of sweat and the pharmacological activity of methylone regarding the body temperature, resulting in high inter-subject variability. Sweat is a suitable matrix for methylone and its metabolites’ determination in clinical and toxicological studies, providing a concentration that reveals recent drug consumption. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms24087395/s1. 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, A.D.G., L.P., G.S., A.F.L.F., E.P., A.D.T., M.F., M.R.V., F.P.B., S.P., S.Z. and C.P.-M.; data curation, A.D.G., L.P., G.S., A.F.L.F., E.P., A.D.T., M.F., M.R.V., F.P.B., S.P., S.Z. 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., A.F.L.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 study 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; a predoctoral PFIS fellowship, grant number FI18/00179; and Plataformas ISCIII de apoyo a la I + D + I en Biomedicina y Ciencias de la Salud, grant number PT20/00018. It was also partially funded by the project “Implementation of the identification and Int. J. Mol. Sci. 2023, 24, 7395 8 of 9 study of the effects of NPS: Development of a multicenter research to enhance the database of the 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’s 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. PI-19-082). Informed Consent Statement: Written informed consent to publish this paper has been obtained from the study participants. Data Availability Statement: Data are contained within the article. Acknowledgments: The authors thank S. Di Carlo, L. Martucci, A. Bacosi, M. Sciotti and C. Fraioli for their technical assistance. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Zaami, S.; Giorgetti, R.; Pichini, S.; Pantano, F.; Marinelli, E.; Busardò, F.P. Synthetic Cathinones Related Fatalities: An Update. Eur. Rev. Med. Pharmacol. Sci. 2018, 22, 268–274. 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