Metabolic profile of N-ethylhexedrone, N-ethylpentedrone, and 4-chloromethcathinone in urine samples by UHPLC-QTOF-HRMS.

Forensic laboratories are constantly required to identify new drugs and their metabolites. N-ethylhexedrone (NEH, HEXEN), N-Ethylpentedrone (NEP), and 4-Chloromethcathinone (4-CMC, clephedrone) are synthetic substances structurally related to natural cathinone, alkaloid present in the leaves of the Catha edulis (Khat) plant. These synthetic cathinones (SC) are members of the heterogenous family of new psychoactive substances (NPS) that raised major concerns in scientific and forensic communities over the past years due to their widespread consumption. In this context, we investigated their metabolic profile using of UHPLC-QTOF-HRMS to elucidate the distribution of the parent drug and its metabolites in urine samples over time. Initially, both male and female volunteers were divided into three groups and eight subjects of each group were administered intranasally or orally with one SC (20-40 mg of NEH or NEP intranasal, 100-150 mg of 4-CMC oral). Urine samples were collected at 0-2 and 2-4 or 2-5 h. Urine (50 µL) was diluted 1:2 with acetonitrile/methanol (95:5) and injected into the UHPLC-QTOF-HRMS. Phase-I and phase-II metabolites were identified on the basis of fragmentation patterns and exact masses. Several phase-I and glucuronide-phase-II metabolites were identified in urine samples. Keto group reduction, hydroxylation and dealkylation were the common metabolic pathways identified for all cathinones and the presence of NEH-glucuronide, NEP-glucuronide and 4-CMC-glucuronide was also relevant. Significant is the slower metabolite formation for 4-CMC, which was detected at high concentrations in its original form even 5 h after administration, due to its long half-life and low intrinsic clearance compared to the other SCs. UHPLC-QTOF-HRMS demonstrated a considerable capability to semi-quantify the three synthetic cathinones and identify the target metabolites with high reliability. The introduction of new target compounds improves the efficiency of toxicological screening analysis on real samples and extends the window of detection of the SCs in biological matrices.

GC-MS/MS Determination of Synthetic Cathinones: 4-chloromethcathinone, N-ethyl Pentedrone, and N-ethyl Hexedrone in Oral Fluid and Sweat of Consumers under Controlled Administration: Pilot Study.

This study presents a validated GC-MS/MS method for the detection and quantification of 4-chloromethcathinone or clephedrone (4-CMC), N-ethyl Pentedrone (NEP), and N-ethyl Hexedrone (NEH, also named HEXEN) in oral fluid and sweat and verifies its feasibility in determining human oral fluid concentrations and pharmacokinetics following the administration of 100 mg of 4-CMC orally and 30 mg of NEP and NEH intranasally. A total of 48 oral fluid and 12 sweat samples were collected from six consumers. After the addition of 5 µL of methylone-d3 and 200 µL of 0.5 M ammonium hydrogen carbonate, an L/L extraction was carried out using ethyl acetate. The samples, dried under a nitrogen flow, were then derivatized with pentafluoropropionic anhydride and dried again. One microliter of the sample reconstituted in 50 µL of ethyl acetate was injected into GC-MS/MS. The method was fully validated according to international guidelines. Our results showed how, in oral fluid, the two cathinones taken intranasally were absorbed very rapidly, within the first hour, when compared with the 4-CMC which reached its maximum concentration peak in the first three hours. We observed that these cathinones were excreted in sweat in an amount equivalent to approximately 0.3% of the administered dose for 4-CMC and NEP. The total NEH excreted in sweat 4 h after administration was approximately 0.2% of the administered dose. Our results provide, for the first time, preliminary information about the disposition of these synthetic cathinones in the consumers' oral fluid and sweat after controlled administration.

Challenges in Drug Surveillance: Strengthening the Analysis of New Psychoactive Substances by Harmonizing Drug Checking Services in Proficiency Testing.

BACKGROUND: Drug checking is a proven harm reduction strategy and provides real-time information on the market of new psychoactive substances (NPS). It combines chemical analysis of samples with direct engagement with people who use drugs (PWUD), giving the ability to increase preparedness and responsiveness towards NPS. Next to that, it supports rapid identification of potential unwitting consumption. However, NPS cause a toxicological battle for the researchers, as factors such as the unpredictability and quick shift of the market complicate the detection. METHODS: To evaluate challenges posed towards drug checking services, proficiency testing was set up to evaluate existing analytical techniques and investigate the capability to correctly identify circulating NPS. Twenty blind substances, covering the most common categories of substances, were analyzed according to the existing protocols of the existing drug checking services, including several analytical methods such as gas chromatography-mass spectrometry (GC-MS) and liquid chromatography with diode array detector (LC-DAD). RESULTS: The proficiency test scores range from 80 to 97.5% accuracy. The most common issues and errors are mainly unidentified compounds, presumably due to no up-to-date libraries, and/ or confusion between structural isomers, such as 3- and 4-chloroethcathinone, or structural analogs, such as MIPLA (N-methyl-N-isopropyl lysergamide) and LSD (D-lysergic acid diethylamide). CONCLUSIONS: The participating drug checking services have access to adequate analytical tools to provide feedback to drug users and provide up-to-date information on NPS.

El Chemsex desde los Servicios de Análisis de Sustancias: estudio descriptivo sobre la metanfetamina, mefedrona yotras catinonas sintéticas / Chemsex from Drug Checking Services: Descriptive Study on Methamphetamine, Mephedrone, and Other Synthetic Cathinones

Antecedentes: Los Servicios de Análisis de Sustancias (SAS) han demostrado ser una intervención eficazque permite acceder a población a prácticas de consumo más seguras. Este estudio se centra en la poblaciónusuaria de Chemsex. Material y métodos. Análisis descriptivo de las muestras de sustancias cedidas porpersonas participantes en prácticas de Chemsex, enfocándose en metanfetamina (MET), mefedronay otras catinonas sintéticas. Los datos fueron recopilados por el Servicio de Información, Análisis deSustancias y Asesoramiento sobre Sexualidades (SIASAS) de Chem-Safe (Energy Control). Se examinaronlas características sociodemográficas de las personas usuarias, los contextos de adquisición de las sustanciasy su composición química. Resultados. La población de personas usuarias del SIASAS de Chem-Safe queparticipan en sesiones de Chemsex está compuesta principalmente por hombres de mediana edad. Sehan analizado 15 tipologías de sustancias diferentes con predominancia de MET y catinonas. En la primerade ellas está presente la adulteración por adición únicamente y un predominio de la vía fumada (50%),mientras que en el caso de las catinonas sintéticas se identifica un elevado índice de adulteración porsustitución y un predominio de la vía endovenosa y esnifada (41,67% respectivamente). Conclusiones. Lasprincipales sustancias analizadas desde el SIASAS son la MET y las catinonas sintéticas. Entre ambas existendiferencias tanto en su modo de obtención, como contexto de adquisición, administración y adulteración,lo que comporta un aumento de los riesgos para la salud de las personas usuarias. (AU)

Background: Drug Checking Services (DCS) have proven to be an effective intervention thatallows access to safer consumption practices for the population. This study focuses on the userpopulation engaged in Chemsex practices. Materials and Methods. A descriptive analysis of substance samples provided by individuals participating in Chemsex practices, with a focus on methamphetamine (METH), mephedrone, and other synthetic cathinones. Data were collected bythe Drug Checking and Sexuality Counseling Service (DCSCS) of Chem-Safe (Energy Control).Sociodemographic characteristics of users, substance acquisition contexts, and chemical composition were examined. Results. The Chem-Safe DCSCS user population engaging in Chemsex sessions consists predominantly of middle-aged men. Fifteen different substance typologieswere analyzed, with METH and cathinones predominating. In the case of METH, adulterationthrough addition was found, with a predominance of the smoked route (50%). Synthetic cathinones showed a high rate of adulteration through substitution, with a preference for intravenousand insufflation routes (41.67% each). Conclusions. The main substances analyzed by DCSCS areMETH and synthetic cathinones, revealing differences in acquisition, administration, and adulteration contexts. This contributes to an increased health risk for users. (AU)

Cannabis adulterated with the synthetic cannabinoid receptor agonist MDMB-4en-PINACA and the role of European drug checking services.

BACKGROUND: European drug checking services exchange information on drug trends within the Trans European Drug Information (TEDI) network, allowing monitoring and coordination of responses. Starting in Spring 2020, several services detected the synthetic cannabinoid receptor agonist MDMB-4en-PINACA in adulterated low-THC cannabis products. METHODS: Cannabis products suspected of adulteration were analyzed for the presence of MDMB-4en-PINACA by 9 services in 8 countries within the TEDI network. If available, phytocannabinoid analysis was also performed. RESULTS: 1142 samples sold as cannabis in herbal, resin and e-liquid form were analyzed, of which 270 were found to contain MDMB-4en-PINACA. All cannabis samples contained low THC (<1%), except the e-liquids which contained no phytocannabinoids. Three serious health incidents requiring hospitalization after use of an adulterated cannabis sample were reported. CONCLUSION: Adulteration of cannabis with synthetic cannabinoid receptor agonists is a new phenomenon that carries risk for people who use it. Given that cannabis consumers are not a usual target group for drug checking services, services and associated harm reduction interventions could be reconfigured to include them.

Source identification of amphetamine-like stimulants in Spanish wastewater through enantiomeric profiling.

Amphetamine (AMP), methamphetamine (MAMP) and 3,4-methylenedioxymethamphetamine (MDMA) occur in wastewater not only as a result of illicit consumption, but also, in some cases, from prescription drug use or by direct drug disposal into the sewage system. Enantiomeric profiling of these chiral drugs could give more insight into the origin of their occurrence. In this manuscript, a new analytical methodology for the enantiomeric analysis of amphetamine-like substances in wastewater has been developed. The method consists of a solid-phase extraction (SPE) followed by liquid chromatography-triple quadrupole-tandem mass spectrometry (LC-MS/MS), which showed low quantification limits in the 2.4-5.5 ng L-1 range. The LC-MS/MS method was first applied to characterize a total of 38 solid street drug samples anonymously provided by consumers. The results of these analysis showed that AMP and MDMA trafficked into Spain are synthesized as racemate, while MAMP is exclusively produced as the S(+)-enantiomer. Then, the analytical method was employed to analyse urban wastewater samples collected from the wastewater treatment plants (WWTPs) of five different cities in 2018 and 2019. Consumption estimated through normalized population loads in wastewater showed an increased pattern of AMP use in the Basque Country. Furthermore, the enantiomeric profiling of wastewater samples was contrasted to lisdexamfetamine (LIS) and selegiline (SEL) prescription figures, two pharmaceuticals which metabolize to S(+)-AMP, and to R(-)-AMP and R(-)-MAMP, respectively. From this analysis, and considering uncertainties derived from metabolism and adherence to treatment, it was concluded that LIS is a relevant source of AMP in those cases with low wastewater loads, i.e. up to a maximum of 60% of AMP detected in wastewater in some samples could originate from LIS prescription, while SEL does not represent a significant source of AMP nor MAMP. Finally, removal efficiencies could be evaluated for the WWTP (serving ca. 860,000 inhabitants) with higher AMP influent concentrations. The removal of AMP was satisfactory with rates higher than 99%, whereas MDMA showed an average removal of approximately 60%, accompanied by an enrichment of R(-)-MDMA.

Acute Pharmacological Effects and Oral Fluid Concentrations of the Synthetic Cannabinoids JWH-122 and JWH-210 in Humans After Self-Administration: An Observational Study.

Synthetic cannabinoids (SCs) are a group of new psychoactive drugs used recreationally with potential health risks. They are monitored by the EU Early Warning System since 2010 due to severe adverse effects on consumers. JWH-122 and JWH-210 are naphthoylindole SCs and potent cannabinoid receptor CB1 and CB2 agonists. Information about the effects of SCs usually is available from intoxication cases and surveys, and few studies on humans after controlled administration or observational/naturalistic studies using standardized measures of cardiovascular and subjective effects are available. The aim of this study was to evaluate the acute pharmacological effects of JWH-122 and JWH-210 recreational consumption in a 4 h observational study and assess their disposition in oral fluid (OF). Sixteen volunteers self-administered 1 mg dose of JWH-122 (n = 8) or 2.25 mg mean dose of JWH-210 (range 2-3 mg, n = 8) by inhalation (smoking). Physiological parameters including blood pressure (systolic and diastolic), heart rate (HR), and cutaneous temperature were measured. A set of visual analog scales, the 49-item short-form version of the Addiction Research Center Inventory (ARCI), and the Evaluation of the Subjective Effects of Substances with Abuse Potential (VESSPA-SSE) were used for the evaluation of subjective effects. OF was collected at baseline and at 10, 20, and 40 min and 1, 2, 3, and 4 h after self-administration. Statistically significant increases in systolic blood pressure (SBP), diastolic blood pressure (DBP), and HR were observed after JWH-122 self-administration but not after JWH-210 self-administration. JWH-210 self-administration produced significant changes in subjective drug effects, similar to those induced by THC (intensity, high, good effects, and hunger). The subjective effects following JWH-122 consumption were minimal. The maximal effects were mostly observed 20 min after intake. JWH-122 and JWH 210 OF concentration reached a peak 20 min after administration and could not be detected after 3 h. The results demonstrated a different pattern of effects of these two SCs. Due to the limitations of our observational study, further research with a larger sample and controlled studies are needed to better define the acute pharmacological effect and health risk profile of JWH-122 and JWH-210.

A Comparison of Acute Pharmacological Effects of Methylone and MDMA Administration in Humans and Oral Fluid Concentrations as Biomarkers of Exposure.

Considered the ß-keto analogue of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy), 3,4-Methylenedioxymethcathinone (methylone) is a synthetic cathinone. Over the years, methylone has been used as a substitute for conventional psychostimulants, such as MDMA. To date, little is known about the human pharmacology of methylone; the only available information has been provided by surveys or published intoxication reports. In the present observational-naturalistic study, we evaluate the acute subjective and physiological effects of methylone after oral self-administration in comparison to MDMA in healthy poly-drug users. Fourteen participants (10 males, 4 females) selected their single oral doses of methylone from 100 to 300 mg (n = 8, mean dose 187.5 mg) or MDMA from 75 to 100 mg (n = 6, mean dose 87.5 mg) based on their experience. Study variables were assessed at 0, 1, 2, and 4 h (h) and included vital signs (non-invasive blood pressure, heart rate, cutaneous temperature) and subjective effects using visual analogue scales (VAS), the 49-item Addiction Research Centre Inventory (ARCI) short form, and the Evaluation of the Subjective Effects of Substances with Abuse Potential (VESSPA-SSE) questionnaire. Additionally, oral fluid concentrations of methylone and MDMA were determined. Acute pharmacological effects produced by methylone followed the prototypical psychostimulant and empathogenic profile associated with MDMA, although they were less intense. Methylone concentrations in oral fluid can be considered a useful biomarker to detect acute exposure in oral fluid. Oral fluid concentrations of MDMA and methylone peaked at 2 h and concentrations of MDMA were in the range of those previously described in controlled studies. Our results demonstrate that the potential abuse liability of methylone is similar to that of MDMA in recreational subjects.

Acute Pharmacological Effects and Oral Fluid Biomarkers of the Synthetic Cannabinoid UR-144 and THC in Recreational Users.

Synthetic cannabinoids (SCs) are one of the most frequent classes of new psychoactive substances monitored by the EU Early Warning System and World Health Organization. UR-144 is a SC with a relative low affinity for the CB1 receptor with respect to that for the CB2 receptor. As with other cannabinoid receptor agonists, it has been monitored by the EU Early Warning System since 2012 for severe adverse effects on consumers. Since data for UR-144 human pharmacology are very limited, an observational study was carried out to evaluate its acute pharmacological effects following its administration using a cannabis joint as term of comparison. Disposition of UR-144 and delta-9-tetrahydrocannibinol (THC) was investigated in oral fluid. Sixteen volunteers smoked a joint prepared with tobacco and 1 or 1.5 mg dose of UR-144 (n = 8) or cannabis flowering tops containing 10 or 20 mg THC (n = 8). Physiological variables including systolic and diastolic blood pressure, heart rate and cutaneous temperature were measured. A set of Visual Analog Scales (VAS), the Addiction Research Centre Inventory (ARCI)-49-item short form version and the Evaluation of the Subjective Effects of Substances with Abuse Potential (VESSPA-SSE) were administered to evaluate subjective effects. Oral fluid was collected at baseline, 10, 20, 40 min and 1, 2, 3 and 4 h after smoking, for UR-144 or THC concentration monitoring. Results showed significant statistical increases in both systolic and diastolic blood pressure and heart rate after both UR-144 and cannabis smoking. Both substances produced an increase in VAS related to stimulant-like and high effects, but scores were significantly higher after cannabis administration. No hallucinogenic effects were observed. Maximal oral fluid UR-144 and THC concentrations appeared at 20 and 10 min after smoking, respectively. The presence of UR-144 in oral fluid constitutes a non-invasive biomarker of SC consumption. The results of this observational study provide valuable preliminary data of the pharmacological effects of UR-144, showing a similar profile of cardiovascular effects in comparison with THC but lower intensity of subjective effects. Our results have to be confirmed by research in a larger sample to extensively clarify pharmacological effects and the health risk profile of UR-144.

The embodiment of wastewater data for the estimation of illicit drug consumption in Spain.

Data obtained from wastewater analysis can provide rapid and complementary insights in illicit drug consumption at community level. Within Europe, Spain is an important country of transit of both cocaine and cannabis. The quantity of seized drugs and prevalence of their use rank Spain at the top of Europe. Hence, the implementation of a wastewater monitoring program at national level would help to get better understanding of spatial differences and trends in use of illicit drugs. In this study, a national wastewater campaign was performed for the first time to get more insight on the consumption of illicit drugs within Spain. The 13 Spanish cities monitored cover approximately 6 million inhabitants (12.8% of the Spanish population). Untreated wastewater samples were analyzed for urinary biomarkers of amphetamine, methamphetamine, MDMA, cocaine, and cannabis. In addition, weekend samples were monitored for 17 new psychoactive substances. Cannabis and cocaine are the most consumed drugs in Spain, but geographical variations showed, for instance, comparatively higher levels of methamphetamine in Barcelona and amphetamine in Bilbao, with about 1-fold higher consumption of these two substances in such metropolitan areas. For amphetamine, an enantiomeric profiling was performed in order to assure the results were due to consumption and not to illegal dumping of production residues. Furthermore, different correction factors for the excretion of cannabis were used to compare consumption estimations. All wastewater results were compared with previously reported data, national seizure data and general population survey data, were a reasonable agreement was found. Daily and yearly drug consumption were extrapolated to the entire Spanish population with due precautions because of the uncertainty associated. These data was further used to estimate the retail drug market, where for instance cocaine illicit consumption alone was calculated to contribute to 0.2-0.5% of the Spanish gross domestic product (ca. 3000-6000 million Euro/year).

Acute Pharmacological Effects of Oral and Intranasal Mephedrone: An Observational Study in Humans.

Mephedrone (4-methylmethcathinone) is a synthetic cathinone with psychostimulant properties which remains one of the most popular new psychoactive substances (NPS). It is frequently used orally and/or intranasally. To date, no studies have evaluated the acute effects and pharmacokinetics after self-administration of mephedrone orally (ingestion) and intranasally (insufflation) in naturalistic conditions. An observational study was conducted to assess and compare the acute pharmacological effects, as well as the oral fluid (saliva) concentrations of mephedrone self-administered orally and intranasally. Ten healthy experienced drug users (4 females and 6 males) self-administered a single dose of mephedrone, orally (n = 5, 100-200 mg; mean 150 mg) or intranasally (n = 5, 50-100 mg, mean 70 mg). Vital signs (blood pressure, heart rate, and cutaneous temperature) were measured at baseline (0), 1, 2, and 4 h after self-administration. Each participant completed subjective effects questionnaires: A set of Visual Analogue Scales (VAS), the 49-item Addiction Research Centre Inventory (ARCI), and Evaluation of the Subjective Effects of Substances with Abuse Potential (VESSPA-SSE) at baseline, 1, 2, and 4 h after self-administration. Oral fluid and urine were collected during 4 h. Both routes of mephedrone self-administration enhanced ratings of euphoria and well-being effects and increased cardiovascular effects in humans. Although it was at times assessed that the oral route produced greater and larger effects than the intranasal one, concentrations of mephedrone in oral fluid and also the total amount of mephedrone and metabolites in urine showed that concentrations of mephedrone are considerably higher when self-administered intranasally in comparison to orally. Controlled clinical trials are needed to confirm our observational results.

Determination of the Synthetic Cannabinoids JWH-122, JWH-210, UR-144 in Oral Fluid of Consumers by GC-MS and Quantification of Parent Compounds and Metabolites by UHPLC-MS/MS.

The consumption of synthetic cannabinoids (SCs) has significantly increased in the last decade and the analysis of SCs and their metabolites in human specimens is gaining interest in clinical and forensic toxicology. A pilot study has been carried out using a combination of an initial last generation gas chromatography-mass spectrometry (GC-MS) screening method for the determination of JWH-122, JWH-210, UR-144) in oral fluid (OF) of consumers and an ultra-high performance liquid chromatography high resolution mass spectrometry (UHPLC-HRMS) confirmatory method for the quantification of the parent compounds and their metabolites in the same biological matrix. OF samples were simply liquid-liquid extracted before injecting in both chromatographic systems. The developed methods have been successfully validated and were linear from limit of quantification (LOQ) to 50 ng/mL OF. Recovery of analytes was always higher than 70% and matrix effect always lower than 15% whereas intra-assay and inter-assay precision and accuracy were always better than 16%. After smoking 1 mg JWH-122 or UR-144 and 3 mg JWH-210, maximum concentration of 4.00-3.14 ng/mL JWH-122, 8.10-7.30 ng/mL JWH-210 ng/mL and 7.40 and 6.81 ng/mL UR-144 were measured by GC-MS and UHPLC-HRMS respectively at 20 min after inhalation. Metabolites of JWH 122 and 210 were quantified in OF by UHPLC-HRMS, while that of UR144 was only detectable in traces. Our results provide for the first time information about disposition of these SCs and their metabolites in consumers OF. Last generation GC-MS has proven useful tool to identify and quantify parent SCs whereas UHPLC-HRMS also confirmed the presence of SCs metabolites in the OF of SCs consumers.

Understanding the pharmacokinetics of synthetic cathinones: Evaluation of the blood-brain barrier permeability of 13 related compounds in rats.

Synthetic cathinones are the second most commonly seized new psychoactive substance family in Europe. These compounds have been related to several intoxication cases, including fatalities. Although the pharmacological effects, metabolism, and pharmacokinetics of cathinones have been studied, there is little information about the permeability of these compounds through the blood-brain barrier (BBB). This is an important parameter to understand the behavior and potency of cathinones. In this work, 13 selected cathinones have been analyzed in telencephalon tissue from Sprague-Dawley rats intraperitoneally dosed at 3 mg/kg. Our results revealed a direct relationship between compound polarity and BBB permeability, with higher permeability for the more polar cathinones. The chemical moieties present in the cathinone had an important impact on the BBB permeability, with lengthening of the α-alkyl chain or functionalization of the aromatic ring with alkyl moieties resulting in lower concentration in telencephalon tissue. Our data suggest that transport of cathinones is a carrier-mediated process, similar to cocaine transport across the BBB.

Acute Effects of 2C-E in Humans: An Observational Study.

2,5-Dimethoxy-4-ethylphenethylamine (2C-E) is psychedelic phenylethylamine, with a chemical structure similar to mescaline, used as new psychoactive substance (NPS). It inhibits norepinephrine and serotonin uptake and, more relevant, acts as a partial agonist of the serotonin 2A (5-HT2 A), 2B (5-HT2 B), and (5-HT2 C) receptors. Consumers have reported that 2C-E induces mild-moderate psychedelic effects, but its pharmacology in humans, including pharmacological effects and pharmacokinetics, have not yet studied. To assess the acute effects of 2C-E on physiological and subjective effects and evaluate its pharmacokinetics, an observational study was carried-out. Ten recreational users of psychedelics self-administered a single oral dose of 2C-E (6.5, 8, 10, 15, or 25 mg). Blood pressure and heart rate were evaluated at baseline, 2, 4, and 6 h post-administration. Three rating scales were administered to evaluate subjective effects: a set of Visual Analog Scales (VAS), the 49-item short form version of the Addiction Research Centre Inventory (ARCI), and the Evaluation of the Subjective Effects of Substances with Abuse Potential (VESSPA-SSE) at baseline, 2, 4, and 6 h after self-administration. To assess 2C-E concentrations oral fluid (saliva) was collected during 6 h. 2C-E induced primarily alterations in perceptions, hallucinations, and euphoric-mood. Saliva maximal concentrations were achieved 2 h after self-administration. Administration of oral 2C-E at recreational doses produces a group of psychedelic-like effects such to 2C-B and other serotonin-acting drugs.

LC-MS/MS method for the quantification of new psychoactive substances and evaluation of their urinary detection in humans for doping control analysis.

The constant legal adaptation of new psychoactive substances (NPS), challenges their evaluation in different fields. In sports, NPS are prohibited in competition with a reporting limit (RL) of 50 ng/mL for the parent compound or a metabolite. However, there is a lack of comprehensive methodologies and excretion studies for monitoring NPS. This work aims to develop an analytical methodology for the NPS quantification and to evaluate the suitability of monitoring the urinary parent stimulants after NPS misuse. A method for the quantification of 14 common NPS was developed and validated. The method was found to be linear in the range 1-1000 ng/mL, and was shown to be accurate and precise. A lowest limit of quantification (LLOQ) of 1 ng/mL was established for all analytes except for benzylpiperazine (5 ng/mL). The method was able to confirm the identity of the analytes at the LLOQ for most NPS. The methodology was applied to the quantification of the parent compound in urine samples collected from an observational study where several healthy volunteers (n ≥ 6 per drug) ingested active doses of mephedrone (MEPH), methylone (MDMC), 2,5-dimetoxy-4-ethylphenetylamine (2C-E), or 6-(2-aminopropyl)benzofuran (6-APB). It was observed that for MDMC and 6-APB, the quantification of the urinary parent drug at the current RL is a proper strategy for detecting their misuse. However, this strategy seems to be insufficient for evaluating MEPH and 2C-E misuse. Monitoring the most abundant metabolite of MEPH (4'-carboxy-MEPH) and the reduction of the RL to 10 ng/mL for the 2C-E evaluation are proposed.

Unexpected identification and characterization of a cathinone precursor in the new psychoactive substance market: 3',4'-methylenedioxy-2,2-dibromobutyrophenone.

3',4'-methylenedioxy-2,2-dibromobutyrophenone has been identified and fully characterized in a sample obtained from an anonymous consumer acquired as ketamine through the Internet market. The substance has been deeply characterized by using standard and high performance analytical techniques such as: attenuated total reflectance-infrared spectroscopy, gas chromatography-mass spectrometry, high-resolution mass spectrometry, elemental analysis, and nuclear magnetic resonance, including 1H, 13C, distortionless enhancement by polarization transfer, two dimensional homonuclear 1H-1H correlation spectroscopy, and 1H-13C heteronuclear single-quantum correlation spectra. 3',4'-methylenedioxy-2,2-dibromobutyrophenone is a precursor or intermediate in the synthesis of several synthetic cathinone derivatives, such as pentylone and methylenedioxy pyrovalerone. It is expected that 3',4'-methylenedioxy-2,2-dibromobutyrophenone does not act as psychoactive substance through disruption nor dysregulation of central and peripheral nervous systems, due to the absence of the characteristic amine group of cathinone derivatives. Although it cannot be considered a trend in new psychoactive substances consumption, the presence in the market and the unknown toxicity of this substance makes it a relevant fact.

Acute Pharmacological Effects of 2C-B in Humans: An Observational Study.

2,5-dimethoxy-4-bromophenethylamine (2C-B) is a psychedelic phenylethylamine derivative, structurally similar to mescaline. It is a serotonin 5-hydroxytryptamine-2A (5-HT2A), 5-hydroxytryptamine-2B (5-HT2B), and 5-hydroxytryptamine-2C (5-HT2C) receptor partial agonist used recreationally as a new psychoactive substance. It has been reported that 2C-B induces mild psychedelic effects, although its acute pharmacological effects and pharmacokinetics have not yet been fully studied in humans. An observational study was conducted to assess the acute subjective and physiological effects, as well as pharmacokinetics of 2C-B. Sixteen healthy, experienced drug users self-administered an oral dose of 2C-B (10, 15, or 20 mg). Vital signs (blood pressure and heart rate) were measured at baseline 1, 2, 3, 4, and 6 hours (h). Each participant completed subjective effects using three rating scales: the visual analog scale (VAS), the Addiction Research Centre Inventory (ARCI), and the Evaluation of the Subjective Effects of Substances with Abuse Potential (VESSPA-SSE) at baseline, 2-3 and 6 h after self-administration (maximum effects along 6 h), and the Hallucinogenic Rating Scale (maximum effects along 6 h). Oral fluid (saliva) was collected to assess 2C-B and cortisol concentrations during 24 h. Acute administration of 2C-B increased blood pressure and heart rate. Scores of scales related to euphoria increased (high, liking, and stimulated), and changes in perceptions (distances, colors, shapes, and lights) and different body feelings/surrounding were produced. Mild hallucinating effects were described in five subjects. Maximum concentrations of 2C-B and cortisol were reached at 1 and 3 h after self-administration, respectively. Oral 2C-B at recreational doses induces a constellation of psychedelic/psychostimulant-like effects similar to those associated with serotonin-acting drugs.

Something New about Something Old: A 10-Year Follow-Up on Classical and New Psychoactive Tryptamines and Results of Analysis.

New psychoactive tryptamines may be a public health risk since they intend to mimic the hallucinogenic effects of regulated psychoactive drugs. Few studies describe uses and clinical effects of unregulated new psychoactive tryptamines. This study aims (1) to explore the presence of tryptamines classified as NPS among the substances delivered for analysis to a harm-reduction organization; (2) to describe the substances found in the samples after analysis; and (3) to compare analytical results of regulated vs. non-regulated tryptamines. Samples delivered and analyzed by gas chromatography-mass spectrometry from 2006 to 2015 were included. A descriptive study of results was conducted. From 25,296 samples that were delivered, 436 were tryptamines; from these 232 (53.21%) were non-regulated. The most delivered non-regulated tryptamine was 4-AcO-DMT. A search of the PubMed database in July 2016 revealed that no studies in humans have ever been carried out with 4-AcO-DMT. Unregulated tryptamines likely contained one unadulterated substance (p ≤ 0.001). The number of samples submitted which contained tryptamines increased during the course of the study, with significant differences in client expectations vs. analysis results between the controlled and uncontrolled groups. There is a need for further research in order to prevent the potential health risks associated with their use.

Patterns of use and toxicity of new para-halogenated substituted cathinones: 4-CMC (clephedrone), 4-CEC (4-chloroethcatinone) and 4-BMC (brephedrone).

OBJECTIVE: This paper aims to present results of the analysis of clephedrone (4-CMC), 4-chloroethcathinone (4-CEC), and brephedrone (4-BMC) on recreational drug markets and a systematic review of all the available information concerning these substances. MATERIAL AND METHODS: Samples collected by the drug checking service of the Spanish harm reduction NGO-Energy Control were analyzed and systematic research was conducted. Between June 2014 and October 2016, 1,471 samples with at least one NPS were analyzed, 397 of which contained cathinones. RESULTS: Clephedrone was found in 29 samples, brephedrone in 8, and both were present in 2 samples. 4-Chloroethcathinone was detected in 5 samples. Eleven out of the 47 purchased samples (23.4%) were tested to contain the substance the user expected. Samples received were mainly sold as 3-MMC, MDMA, ketamine, and other cathinones. No literature on the effects or toxicity of these substances was found; the only information available was on internet fora. On many posts, users exhibit concerns about potential toxicity and side effects of using these substances. CONCLUSION: Since the emergence of these substances could prove to be the next step to the cat-and-mouse game existing between drug producers and legislation, further clinical and epidemiological research should be carried out in order to build evidence to support policy for public health issues.

The hidden web and the fentanyl problem: Detection of ocfentanil as an adulterant in heroin.

BACKGROUND: The popularization of anonymous markets such as Silk Road is challenging current drug policy and may provide a new context for old issues, such as adulteration of heroin with fentanyl derivatives. The aims of this paper are to report the presence of ocfentanil, a novel, potent, non-controlled fentanyl analog, in samples sold as heroin in the hidden web, and to summarize the effects reported by users. METHODS: In 2015, four samples allegedly bought as heroin in cryptomarkets of the hidden web were sent to Energy Control for analysis. Energy Control is a Spanish harm reduction NGO that offers anonymous drug checking with the purpose of adapting counselling to the specific substances present in the drug and monitor the drug market. Identification was performed by GC/MS and LC/MS/MS. We contacted the submitters of the samples and performed an Internet search to retrieve additional information. RESULTS: One sample contained ocfentanil, caffeine and heroin. Three samples contained the aforementioned substances plus paracetamol. Two out of the four contacted users reported distinct short acting, opioid-like effects. No fora discussion could be found about the effects of ocfentanil, neither web pages nor individuals advertising the substance. CONCLUSION: We report the presence of a new substance detected in the hidden web as an adulterant of heroin, ocfentanil. It has short acting opioid-like effects, roughly the same potency as fentanyl, and can be injected, snorted or smoked. Severe side effects have been associated with its use, including one death. No discussion about this substance could be found in the Internet, which suggests this substance has not been sold as such. Available data about purities of drugs purchased in cryptomarkets suggest that adulteration is not a severe problem and this agrees with users' perceptions. However, this study suggests that adulteration is a real threat not only at the street level, but also for users that buy substances in cryptomarkets, and suggest the need for harm reduction initiatives in this setting.