Legal Highs Research Articles

Para-Halogenation of Amphetamine and Methcathinone Increases the Mitochondrial Toxicity in Undifferentiated and Differentiated SH-SY5Y Cells.

Halogenation of amphetamines and methcathinones has become a common method to obtain novel psychoactive substances (NPS) also called “legal highs”. The para-halogenated derivatives of amphetamine and methcathinone are available over the internet and have entered the illicit drug market but studies on their potential neurotoxic effects are rare. The primary aim of this study was to explore the neurotoxicity of amphetamine, methcathinone and their para-halogenated derivatives 4-fluoroamphetamine (4-FA), 4-chloroamphetamine (PCA), 4-fluoromethcathinone (4-FMC), and 4-chloromethcathinone (4-CMC) in undifferentiated and differentiated SH-SY5Y cells. We found that 4-FA, PCA, and 4-CMC were cytotoxic (decrease in cellular ATP and plasma membrane damage) for both cell types, whereby differentiated cells were less sensitive. IC50 values for cellular ATP depletion were in the range of 1.4 mM for 4-FA, 0.4 mM for PCA and 1.4 mM for 4-CMC. The rank of cytotoxicity observed for the para-substituents was chloride > fluoride > hydrogen for both amphetamines and cathinones. Each of 4-FA, PCA and 4-CMC decreased the mitochondrial membrane potential in both cell types, and PCA and 4-CMC impaired the function of the electron transport chain of mitochondria in SH-SY5Y cells. 4-FA, PCA, and 4-CMC increased the ROS level and PCA and 4-CMC induced apoptosis by the endogenous pathway. In conclusion, para-halogenation of amphetamine and methcathinone increases their neurotoxic properties due to the impairment of mitochondrial function and induction of apoptosis. Although the cytotoxic concentrations were higher than those needed for pharmacological activity, the current findings may be important regarding the uncontrolled recreational use of these compounds.

Forensic-psychiatric relationships in the context of forensic medical examination of new psychoactive substance-related deaths.

The aim of this study was to find causal relationships between the use of new psychoactive substances (NPS), the so-called "legal highs", and the cause of death, taking into account information indicating the victim's mental disorders before death. The research material includes the results of toxicological analyses of blood samples collected during autopsies of 40 deceased persons whose cause of death was associated with the ingestion of new psychoactive substances (NPS). The blood samples were subjected to solid phase extraction (SPE), and then analyzed by liquid chromatography coupled with electrospray ionization tandem mass spectrometry(LC-ESI-MS-MS) for quantification. Information on individual cases was taken from case files provided by the prosecutor's office for research purposes. As aresult of the analyses, 24 types of NPS were detected, comprising synthetic cathinones - 15 cases, synthetic cannabinoids - 10 cases, synthetic opioids - 13 cases, other - 2 cases. In the study group there were 5 women (12.5%) and 35 men (87.5%). Suicide was demonstrated in 10 cases (25%), while the remaining 30 cases (75%) were accidental deaths, including those in which the actual motivation of drug use could not be determined. Drugs used to treat mental disorders were detected in 6 cases, while analgesics were demonstrated in 5 cases. In the majority of cases, including suicides and accidental deaths where the manner of death was unknown, NPS use and consequent death from various causes are associated with psychiatric disorders.

Family structure vs. risky behaviour and self-efficiency feeling of youth in the period of late adolescence

The purpose of this research was to compare the frequency of the occurrence of risky behaviour and the level of self-efficiency feeling of youth in the period of late adolescence brought up in families of different structure. The structure of a family turned out not to have an important influence on a general feeling of self-efficiency of the examined youth brought up in complete and incomplete families. Risky behaviour which includes missing classes and using intoxicating substances: nicotine, alcohol, drugs, do not differ significantly in the frequency range in the compared groups. However, the quantitative analysis of the results shows more frequent use of legal highs by youth brought up in complete families. The results obtained did not prove the initial assumptions concerning a greater exposure to the possibility of the occurrence of risky behaviour among youth brought up in incomplete families. However, they indicate a higher frequency of occurrence of selected risky behaviour in adolescents from complete families, which prompts further analysis of the factors conditioning this phenomenon. It seems especially important to take into consideration the additional variables which should include: - time devoted to young people by parents fulfilling educational tasks alone or with a partner; - parental attitudes presented by parents.

Solid-phase extraction based on cation-exchange sorbents followed by liquid chromatography high-resolution mass spectrometry to determine synthetic cathinones in urine

Cathinone derivates have become very popular as legal highs and are easily obtained on the market as “bath salts”, getting around the legislation due to their modified structure based on banned substances. The aim of this project is to evaluate two mixed-mode cation-exchange sorbents in solid-phase extraction (SPE) and their efficacy in pretreating urine samples for the determination of synthetic cathinones by liquid chromatography high-resolution mass spectrometry (LC-HRMS). A method for determining a group of eleven trending synthetic cathinones in urine using SPE followed by LC-HRMS using Orbitrap as analyser was developed and validated. In the extraction step, two different cation-exchange sorbents were evaluated and compared, a weak one (Oasis WCX) and a strong one (Oasis MCX). Better results were obtained for Oasis MCX in terms of recoveries and matrix effects (lower, if a clean-up step was applied). Method quantification limits were set at 0.2 ng mL−1 and method detection limits were between 0.04 and 0.16 ng mL−1. The results obtained enabled the studied compounds to be quantified at the usual levels at which they are present in urine samples. Moreover, these limits were lower than the ones found in the literature using similar extraction strategies, which shows that the reported strategy can be a useful tool in forensic and toxicological analyses.

A case of intoxication with a new cathinone derivative α-PiHP - A presentation of concentrations in biological specimens.

Cathinones are currently the second largest and the second most frequently seized group of new psychoactive substances (NPS). One of the most recent synthetic cathinones that has appeared on the 'legal highs' market is alpha-pyrrolidinoisohexanophenone (α-PiHP). An 18-year-old man was found dead in an apartment. The autopsy materials were collected for toxicological analyses. The quantitative analyses were carried out by LC-MS/MS. α-PiHP was detected and quantified in all post-mortem materials except the hair. The determined concentrations of the compound in the blood, urine and bile were 69 ng/mL, 2072 ng/mL, and 341 ng/mL respectively. The concentrations of α-PiHP in solid tissues were in the range of 7-478 ng/g. 4-Chloromethcathinone (4-CMC), N-ethylhexedrone, benzoylecgonine and 3,4-methylenedioxymethamphetamine (MDMA) were also detected in some materials. No cases presenting concentrations of α-PiHP in biological materials have been reported so far. Due to the similarity of structures and the reported dosages, an attempt to compare the concentrations of α-PVP and α-PHP has been made. In the described case, functional death through intoxication of α-PiHP was accepted as the final cause of death. The other detected substances did not contribute to death due to their very likely distant administration. α-PiHP is another new synthetic cathinone that is a danger to the life of users. The described fatal intoxication case presents the concentrations of α-PiHP in post-mortem materials. This data could be valuable for further interpretation of other results from toxicological analyses in cases where the use of α-PiHP is suspected.

Successful use of a novel lux® i-Amylose-1 chiral column for enantioseparation of "legal highs" by HPLC.

Bath salts, fumigations, cleaners and air fresheners, behind these terms substances are hidden, which count as "Legal Highs". These fancy names are used to pretend Legal Highs as harmless compounds, to circumvent legal regulations for marketing as well as to increase the sales. Besides classic illicit drugs of synthetic origin such as amphetamines, cocaine and MDMA, the trade of these compounds, also known as new psychoactive substances (NPS), is not uncommon today. In many countries, NPS are still not subject to drug control. Among them, there are stimulants such as new amphetamine derivatives or cathinones, which possess a chiral centre. Little is known about the fact that the two possible enantiomers may differ in their pharmacological effect. The aim of this study was to test a novel HPLC column for the enantioseparation of a set of 112 NPS coming from different chemical groups and collected by internet purchases during the years 2010-2018. The CSP, namely Lux® 5μm i-Amylose-1, LC Column 250 x 4.6mm, was run in normal phase mode under isocratic conditions, UV detection was performed at 245nm and 230nm, injection volume was 10μl and flow rate was 1ml/min. With a mobile phase consisting of n-hexane/isopropanol/diethylamine (90:10:0.1), herein, 79 NPS were resolved into their enantiomers successfully, for 37 of them baseline resolution was achieved. After increase of lipophily of the mobile phase to 99:1:0.1, another 27 compounds were baseline separated. It was found that all separated NPS are traded as racemic compounds.

Random Forest Processing of Direct Analysis in Real-Time Mass Spectrometric Data Enables Species Identification of Psychoactive Plants from Their Headspace Chemical Signatures

The United Nations Office on Drugs and Crime has designated several “legal highs” as “plants of concern” because of the dangers associated with their increasing recreational abuse. Routine identification of these products is hampered by the difficulty in distinguishing them from innocuous plant materials such as foods, herbs, and spices. It is demonstrated here that several of these products have unique but consistent headspace chemical profiles and that multivariate statistical analysis processing of their chemical signatures can be used to accurately identify the species of plants from which the materials are derived. For this study, the headspace volatiles of several species were analyzed by direct analysis in real-time high-resolution mass spectrometry (DART-HRMS). These species include Althaea officinalis, Calea zacatechichi, Cannabis indica, Cannabis sativa, Echinopsis pachanoi, Lactuca virosa, Leonotis leonurus, Mimosa hositlis, Mitragyna speciosa, Ocimum basilicum, Origanum vulgare, Piper methysticum, Salvia divinorum, Turnera diffusa, and Voacanga africana. The results of the DART-HRMS analysis revealed intraspecies similarities and interspecies differences. Exploratory statistical analysis of the data using principal component analysis and global t-distributed stochastic neighbor embedding showed clustering of like species and separation of different species. This led to the use of supervised random forest (RF), which resulted in a model with 99% accuracy. A conformal predictor based on the RF classifier was created and proved to be valid for a significance level of 8% with an efficiency of 0.1, an observed fuzziness of 0, and an error rate of 0. The variables used for the statistical analysis processing were ranked in terms of the ability to enable clustering and discrimination between species using principal component analysis–variable importance of projection scores and RF variable importance indices. The variables that ranked the highest were then identified as m/z values consistent with molecules previously identified in plant material. This technique therefore shows proof-of-concept for the creation of a database for the detection and identification of plant-based legal highs through headspace analysis.

A16 Next-generation sequencing to detect transmitted drug resistance mutations in Romanian people who inject drugs

Romania has faced an HIV outbreak among people who inject drugs (PWID) since 2011. The introduction of so-called ‘legal highs’ (amphetamine-type stimulants) on the drug market a few years prior contributed substantially to this outbreak. Next-generation sequencing (NGS) provides the possibility to detect drug resistance mutations with higher sensitivity than Sanger sequencing. The aim of this study was to search for transmitted drug resistance (TDR) mutations in strains from PWID recently diagnosed with HIV infection by parallel use of Sanger sequencing and NGS. The study was conducted on strains from 34 PWID diagnosed with HIV infection between 2016 and 2017. Sequencing was performed for the pol (PR, RT, and INT) and env (V2-V3 loop) regions. Sanger sequencing was performed with the commercial ViroseqTMHIV-1 Genotyping system (Abbott Laboratories) and with an in-house protocol for the env gene. NGS was performed in the same genomic regions using Nextera DNA Library Preparation Kit (Illumina) and the Miseq instrument (Illumina). NGS data were processed for error correction, read mapping, and detection of drug resistance mutations with HIV-1 Deepchek analysis software. Geno2pheno algorithm was used for viral tropism prediction and the WHO 2009 list for TDRM analysis. By using NGS, we detected seven cases (20.6%) of TDR in PWID and only two cases (5.8%) with standard sequencing. The TDR mutations detected by NGS were K103N, K101EN, Y181C, T215S in RT gene, I54V and M46L in PR, and none in INT. Two NNRTI mutations (K103N and K101EN) were detected in the same sample. Most of the TDR identified were present in the minority population (between 1% and 2% of the total reads) explaining the higher sensitivity of NGS method compared with standard sequencing. No significant differences were observed between these two methods when tropism prediction was analyzed. The majority of the viruses circulating in this group were R5-tropic. All strains showed more resistance mutations when analyzed by deep sequencing than by Sanger sequencing and more than previously observed in other risk groups. NGS proved to be a sensitive tool to detect TDR in newly infected PWID.

“Air fresheners” as legal highs

“Air fresheners” as legal highs

Substance Use and Prevention Programs in Berlin’s Party Scene: Results of the SuPrA-Study

Background: Berlin is internationally known for its nightlife. In a nation-wide and Europe-wide comparison, the use of legal and illegal substances is comparatively higher in Berlin than in other similar cities. However, few data exist about the drug use in the party scene. Objective: This study aims to assess the sociodemographic characteristics of Berlin’s party scene and its patterns of substance use as well as expectations towards prevention in order to derive appropriate preventive measures. Methods: Using questionnaires, both online (n = 674) and in the field (n = 203), a total of 877 people of the Berlin party scene were interviewed. The questionnaires ascertained the demographic information of the participants and patterns of substance use in the scene. It also collected the demand for consulting services and personal assessments on the usefulness of prospective and existing prevention programs and offers. Results: The study participants were 29 years old (SD 7.5); 43% were female. Alcohol is the most common substance in the party scene, followed by cannabis, MDMA/Ecstasy, amphetamine, cocaine, and ketamine. In this particular cohort, methamphetamine and “legal highs” did not play a major role. The most demanded preventive measure was more education about drugs and the so called drug-checking. Conclusions: Prevention in this area is both needed and requested, and an expansion of the existing programs (e.g., by so far politically controversial drug-checking) should be considered.

Functional evaluation of carboxy metabolites of synthetic cannabinoid receptor agonists featuring scaffolds based on L-valine or L-tert-leucine.

Indole- and indazole-based synthetic cannabinoid receptor agonists (SCRAs), featuring valine or tert-leucine substituents, are commonly abused new psychoactive substances (NPS). A major metabolic pathway for these SCRAs is hydrolysis of the terminal amide or methylester functionalities. Although these hydrolysis products were already detected as main ingredients in some "legal highs," these metabolites are often poorly characterized. Here, we report a systematic investigation of the activity of 7 common hydrolysis metabolites of 15 SCRAs featuring scaffolds based on L-valine or L-tert-leucine in direct comparison to their parent compounds. An activity-based cannabinoid receptor 1 (CB1 ) bio-assay was used for activity profiling of SCRAs and their metabolites in a stable HEK293T cell system. The recruitment of β-arrestin2 to the activated CB1 (each fused to one part of a split Nanoluciferase) was provoked by adding the (putative) SCRAs. Luminescence of the functionally complemented luciferase was monitored by a 96-well plate-reader. The major hydrolysis metabolites of 5F-AB-PINACA, ADB-CHMICA, ADB-CHMINACA, ADB-FUBICA, and their methyl- and ethylester derivatives showed no detectable CB1 activation at concentrations up to 1μM. On the other hand, metabolites of 5F-ADB-PINACA, AB-CHMINACA, and ADB-FUBINACA did retain activity, although significantly reduced as compared to the parent compounds (EC50 values >100nM). Activity-based characterization of SCRAs and their metabolites at CB1 may not only allow a better insight into the complex interplay between SCRAs and their metabolites in intoxications, but may also allow application of the concept of "activity equivalents" present in biological fluids or, alternatively, in confiscated materials.

Pharmacological characterizations of the 'legal high' fluorolintane and isomers

1,2-Diarylethylamines represent a class of molecules that have shown potential in the treatment of pain, epilepsy, neurodegenerative disease and depression. Examples include lefetamine, remacemide, and lanicemine. Recently, several 1,2-diarylethylamines including the dissociatives diphenidine, methoxphenidine and ephenidine as well as the opioid MT-45, have appeared as ‘research chemicals’ or ‘legal highs’. Due to their recent emergence little is known about their pharmacology. One of these, 1-[1-(2-fluorophenyl)-2-phenylethyl]pyrrolidine (fluorolintane, 2-F-DPPy), is available for purchase with purported dissociative effects intended to resemble phencyclidine (PCP) and ketamine. To better understand this emerging class, pharmacological investigations were undertaken for the first time on fluorolintane and its five aryl-fluorine-substituted isomers. In vitro binding studies revealed high affinity for N-methyl-D-aspartate (NMDA) receptors with fluorolintane (Ki = 87.92 nM) with lesser affinities for related compounds. Additional affinities were seen for all compounds at several sites including norepinephrine (NET), serotonin (SERT) and dopamine (DAT) transporters, and sigma receptors. Notably high affinities at DAT were observed, which were in most cases greater than NMDA receptor affinities. Additional functional and behavioral experiments show fluorolintane inhibited NMDA receptor-induced field excitatory postsynaptic potentials in rat hippocampal slices and inhibited long-term potentiation induced by theta-burst stimulation in rat hippocampal slices with potencies consistent with its NMDA receptor antagonism. Finally fluorolintane inhibited prepulse inhibition in rats, a measure of sensorimotor gating, with a median effective dose (ED50) of 13.3 mg/kg. These findings are consistent with anecdotal reports of dissociative effects of fluorolintane in humans.

The risk of emerging new psychoactive substances: The first fatal 3-MeO-PCP intoxication in The Netherlands

Structural analogs of classic drugs, also called designer drugs, are a booming market due to the easy accessibility on the internet and their legal status. One of those ‘legal highs’ is an analog of phencyclidine, namely 3-methoxyphencyclidine (3-MeO-PCP). Very few fatalities have been reported where 3-MeO-PCP contributed to the death of an individual. We present the first fatal case in the Netherlands and one of the few worldwide. Postmortem biological samples and the presumed abused unknown substance, sold as ant poison, were obtained. 3-MeO-PCP was detected, and the resulting concentration was 152 μg/l in whole blood. The presumed taken unknown sample was identified as 3-MeO-PCP and thus linked to the victim. The cause of death was a combination of 3-MeO-PCP, amphetamine, and alcohol. Improved diagnostic skills are necessary to face these emerging novel psychoactive substances also in light of public health and social risks.

Functional evaluation of metabolites of synthetic cannabinoid receptor agonists

Synthetic cannabinoid receptor agonists (SCRAs) are commonly abused new psychoactive substances (NPS). The human body extensive metabolizes these SCRAs, to facilitate clearance and excretion. In certain cases, bio-activation of these compounds and/or generation of active metabolites has been reported, which might contribute to toxicological effects. Moreover, these metabolites have also already been detected as main ingredients in some “legal highs”, but their toxicological profile remains poorly characterized. The activity of seven common hydrolysis metabolites of fifteen SCRAs, featuring scaffolds based on valine or tert-leucine, was systematically investigated in direct comparison to their parent compound. An activity-based cannabinoid receptor 1 (CB1) bio-assay was used for activity profiling of SCRAs and their metabolites in a stable HEK293 T cell system. The recruitment of β-arrestin 2 to the activated CB1 (each fused to one part of a split Nanoluciferase) was provoked by adding the (putative) SCRAs. Luminescence of the functionally complemented luciferase was monitored by a 96-well plate-reader. The major hydrolysis metabolites of 5F-AB-PINACA, ADB-CHMICA, ADB-CHMINACA, ADB-FUBICA, and their methyl- and ethylester derivatives showed no detectable CB1 activation at concentrations up to 1 μM. On the other hand, metabolites of 5F-ADB-PINACA, AB-CHMINACA, ADB-FUBINACA did retain activity, although significantly reduced as compared to the parent compounds (EC 50 values > 150 nM). The present study provides critical and missing data related to the potential toxicological characteristics of common hydrolysis metabolites of 15 SCRAs featuring scaffolds based on L-valine and L- tert -leucine. Results indicate overall severely impaired activity of these metabolites at CB 1 . These data highlight that the hydrolysis metabolites of closely related SCRAs can possess markedly distinct pharmacological characteristics, ranging from no activity detected to EC 50 values around 150 nM. The bioassay can serve as a tool to determine the activity of SCRAs and their metabolites at CB1. The assay may allow a better insight into the complex interplay between SCRAs and their metabolites in intoxications involving SCRAs.

A validated method for the quantification of mitragynine in sixteen commercially available Kratom (Mitragyna speciosa) products

The recent rise in the recreational use of plant-based “legal highs” has prompted the development of methods for the identification of the bulk material, and quantification of their psychoactive components. One of these plants is Mitragyna speciosa, commonly referred to as Kratom. While traditional use of this plant was primarily for medicinal purposes, there has been a rise in its recreational use, and as a self-prescribed medication for opioid withdrawal. Although Kratom contains many alkaloids, mitragynine and 7-hydroxymitragynine are unique psychoactive biomarkers of the species, and are responsible for its psychoactive effects. A rapid validated method for the quantification of mitragynine in Kratom plant materials by direct analysis in real time-high-resolution mass spectrometry (DART-HRMS) is presented. It has a linear range of 5–100 μg mL−1, and a lower limit of quantification of 5 μg mL−1. The protocol was applied to determination of the mitragynine content of 16 commercially available Kratom plant products purchased online. The mitragynine amounts in these materials ranged from 2.76 to 20.05 mg g−1 of dried plant material. The utilization of DART-HRMS affords a mechanism not only for the preliminary identification of bulk plant material as being M. speciosa-derived (with no sample preparation required), but also provides the opportunity to quantify its psychoactive components using the same technique.

Sacharomyces cerevisiae as a screening tool to assess cathinones’ toxicity

Introduction: Novel psychoactive substances (NPS) are sold on Internet under the appearance of herbal blends or bath salts as an alternative to cannabis, khat and ephedra. Synthetic cathinones are artificial mimetics of khat and like methamphetamine are indirect agonists of dopamine, serotonin and noradrenaline receptors [1]. Ninety-seven fatal cases of intoxication by ingestion of mephedrone (4-methylmethcathinone), one of the types of synthetic cathinone derivatives, were reported on literature since 2010 [2]. New synthetic cathinones emerges in the street market and internet on a regular base and the properties of this substances change regularly, due to modification on the molecules, to avoid legislation. This makes almost impossible to characterize synthetic cathinones toxicological profiles on a short time scale, mostly due to the typical time-consuming experimental assays. In here we propose the use of Saccharomyces cerevisiae, which has been proven an invaluable model to study the fundamental molecular mechanisms involved in several human diseases [3], growth curves in the presence of synthetic cathinones to get an indicative measure of its toxicity. Materials and methods: BY4741 yeast cells were diluted to a standardized OD600 nm =2×10−3 (∼1.67×10-5 cells/mL) in YNB medium (Yeast Nitrogen Base w/o Amino Acids 0.67%, glucose 2%, required amino acids) and grown at 30 °C. Buphedrone was acquired from the best source available. The cytotoxic potential of synthetic cathinones was assessed by monitoring the growth of Saccharomyces cerevisiae BY4741 in the presence and absence of substances. Yeast growth curves (O.D.600nm versus growth time) were compared. Results: Figure 1 shows that yeast growth in the presence of 75 mM cat is lower when compared to the growth curve in the absence of the substance. These preliminary results points that cathinones inhibits yeast growth. Figure 1. Growth curves results in Saccharomyces cerevisiae BY4741 in the presence of 75 mM of buphedrone. Discussion and conclusions: Saccharomyces cerevisiae has been used previously to test the toxicity of pharmacological drugs. Our results show that yeast is sensitive to cathinones suggesting that this system has potential to assess the possible toxicity of these legal highs. This rapid and inexpensive methodology may allow high throughput screening of cathinones’ toxicity, which might help the legislators to schedule NPS avoiding expected public health problems.

Fatal intoxication with new synthetic cannabinoids AMB-FUBINACA and EMB-FUBINACA

Introduction: Synthetic cannabinoids are currently the largest group of new psychoactive substances. Those that have been subjected to legal control are replaced by newer uncontrolled substances, which causes constant and dynamic changes to the drug market. Some of the most recent synthetic cannabinoids that have appeared on the “legal highs” market are AMB-FUBINACA and EMB-FUBINACA.Case history: A 27-year-old man was found dead on a bed in an apartment. At autopsy, congestion of internal organs, pulmonary oedema and left-sided pleural adhesions were found. The medical examiner concluded that the man died due to acute respiratory failure. The autopsy materials (blood, urine, liver, kidney, stomach, intestine, lung and brain) were collected for further toxicological analyses.Methods: The synthetic cannabinoids AMB-FUBINACA and EMB-FUBINACA were isolated from autopsy materials by precipitation with acetonitrile. The quantitative analyses were carried out by LC-MS/MS.Results: AMB-FUBINACA and EMB-FUBINACA were detected and quantified in all post-mortem materials except the blood. The determined concentrations of these compounds in solid tissues were in the range of 0.2–0.9 ng/g and 0.2–3.5 ng/g. The highest concentrations of AMB-FUBINACA and EMB-FUBINACA were revealed in the stomach content (5.8 and 36.2 ng/mL, respectively).Discussion: The presented case demonstrates that even in cases of fatalities, it is possible that the parent substance will not be present in the blood, while being present in other autopsy materials. The determined concentrations of the compounds may indicate oral administration of synthetic cannabinoids. It can also be assumed that AMB-FUBINACA and EMB-FUBINACA probably contributed to death.Conclusion: The presented case shows that synthetic cannabinoids can be undetected in the blood of even seriously or fatally intoxicated people. This situation means that the analysis of only blood samples may not confirm poisoning. The presented case also suggests that AMB-FUBINACA and EMB-FUBINACA use is dangerous to health and may lead to fatal intoxication.

Novel Drugs of Abuse.

Novel drugs of abuse are synthetic illicit drugs, or analogues of known illicit drugs, that can be more potent. Novel drugs of abuse are often labeled as designer drugs, research chemicals, legal highs, or psychoactive substances. They are often sold as designated legal or nondrug products, such as incense, plant food, or bath salts, with labeling such as "Not for Human Consumption" or "For Use in Research Only." The prevalence of use of novel drugs of abuse is difficult to determine because specific drugs, compounds, and availability of these drugs are constantly evolving. Changes in chemical structures lead to heterogeneity in physiologic response and clinical symptoms, even within the same category of drug. Pediatricians and emergency medicine physicians should be knowledgeable about novel drugs of abuse and their resulting symptoms for prevention and identification of their use.

Forensic Electrochemistry: The Electroanalytical Sensing of Mephedrone Metabolites

The constant and persistent synthesis and abuse of new psychoactive substances have sparked the requirement for rapid, on-site, sensitive analytical protocols for their sensing and quantification. Mephedrone (4-MMC) is currently one of the most popular legal highs among recreational drug abusers and imposes a serious public health problem. In this paper, the electrochemical sensing of two metabolites of 4-MMC, namely, nor-mephedrone (4-methylcathinone, 4-MC) and dihydromephedrone (4-methylephedrine, 4-MMC-R), utilizing screen-printed graphite electrodes is performed. The accessible linear ranges by cyclic voltammetry were found to correspond to 40–300 μg mL–1 for 4-MC in both phosphate buffer solution (PBS, pH 7.0) and spiked diluted human urine, whereas in the case of 4-MMC-R, the linearity ranges are 15–300 μg mL–1 (PBS, pH 3.0) and 25–300 μg mL–1 (spiked diluted human urine). To maximize the assay sensitivity, differential pulse voltammetry (DPV) was performed toward the sensing of 4-MC, which exhibited a linear response over the range 10–250 and 10–300 μg mL–1 in PBS pH 7.0 and spiked diluted human urine, respectively. However, 4-MMC-R demonstrated slightly higher sensitivity over the range 5–300 μg mL–1 in both PBS pH 3.0 and spiked diluted human urine. Using DPV, the limits of detection (3σ) were calculated and found to correspond to ca. 3.97 and 3.64 μg mL–1 for 4-MC and 4-MMC-R (PBS, pH 7.0 and 3.0), respectively, and ca. 6.34 and 3.87 μg mL–1 for 4-MC and 4-MMC-R (spiked diluted human urine), respectively. The potential interference of adulterants’ metabolites commonly found in NPS street samples was also explored (at both pH 7.0 and 3.0). The electrochemical approach reported herein provides a novel laboratory tool for the identification and quantification of synthetic cathinone metabolites and has potential for the basis of a portable analytical sensor for their fast, cheap, reliable, and accessible determination in the field.

Inflammation and oxidative stress are key mediators in AKB48-induced neurotoxicity in vitro

Synthetic cannabinoids were introduced into market in early 2000s; since these “legal highs” are dramatically popular among youth, it becomes a deadly problem. Synthetic cannabinoids have high affinity to cannabinoid receptors; leading to various clinical symptoms. AKB48 (Apinaca) has been classified as a third-generation synthetic cannabinoid for the first time in 2014.The toxicity profile of AKB48 is unclear due to little information that mainly obtained from clinical and forensic cases; however, it is believed to be similar with other psychoactive substances. Thus, we aimed to investigate the possible toxicity mechanisms of AKB48 in SH-SY5Y (human bone marrow neuroblastoma) cell line. IC50 value of AKB48 was calculated as 160.91 μM by MTT assay. AKB48 treatment enhanced (≥1.2-fold) the fluorescence intensity indicating increased reactive oxygen species production; however, glutathione levels did not changed in the range of 25–200 μM exposure concentrations. Cannabinoid type-1 receptor (CB1) expression was increased ≥15-fold in the range of 25–50 μM of AKB48, while cannabinoid type-2 receptor (CB2) did not expressed in SH-SY5Y cells. Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α) were up-regulated with a dose-dependent manner, and the profiles were almost identical; however, mitogen-activated protein kinase 8 (MAPK 8) was only upregulated with 25 μM of AKB48 and nuclear factor kappa B (NF-ĸB) did not change. Our results should raise the concerns about the safety associated with synthetic cannabinoids uses.