Comparative Assessment of the Addictive Potential of Synthetic Cathinones by Zebrafish Conditioned Place Preference (CPP) Paradigm.

Synthetic cathinones have gained increasing popularity in the illicit drug market, yet their abuse potential remains poorly understood. In this study, zebrafish were used to compare the addictive potential of three cathinone analogs, namely pentylone, eutylone, and N-ethylpentylone (NEP). The zebrafish received various doses (0 to 60 mg/kg) of the cathinone analogs by oral gavage over two sessions per day for two consecutive days to induce conditioned place preference (CPP). Pentylone, eutylone, and NEP dose-dependently induced CPP, with NEP showing significantly higher CPP than pentylone and eutylone at the dose of 20 mg/kg. The fish that received 60 mg/kg of cathinones underwent extinction, followed by reinstatement triggered by drug priming. NEP required six sessions to meet the criteria of extinction, followed by eutylone, which required four sessions, and pentylone, which required three sessions. Furthermore, NEP and eutylone at a dose of 40 mg/kg could reinstate the extinguished CPP, while 60 mg/kg of pentylone was necessary for CPP reinstatement. The persistence of susceptibility to reinstatement was also assessed at 7 and 14 days after the initial reinstatement. The CPP induced by all three cathinone analogs could be reinstated 7 days after the initial reinstatement, whereas only CPP induced by NEP, but not pentylone and eutylone, could be reinstated again after 14 days. Considering the potency to induce CPP, resistance to extinction, and the propensity for reinstatement, the abuse liability rank order of the cathinone analogs might be as follows: NEP > eutylone > pentylone. These findings suggest that the zebrafish CPP paradigm can serve as a viable model for assessing the relative abuse liability of substances.

Identification, synthesis and quantification of eutylone consumption markers in a chemsex context.

Eutylone is a cathinone-derived synthetic amphetamine scheduled by the World Health Organization and European Monitoring Centre for Drugs and Drug Addiction since 2022 due to its growing consumption. We report here an eutylone intoxication involving a 38-year-old man and a 29-year-old woman in a chemsex context. A bag containing a white crystalline powder labelled as a research product was found in their vehicle. Nuclear magnetic resonance and liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analyses identified the powder as eutylone and confirmed purity superior to 99%. LC-HRMS data analysis using molecular networking allowed to propose new eutylone metabolites in blood samples in a graphical manner. We described 16 phase I (e.g. hydroxylated or demethylated) and phase II metabolites (glucuroconjugates and sulfoconjugates). The same metabolites were found both in male and female blood samples. Toxicological analyses measured eutylone concentration in blood samples at 1374 ng/mL and 1536 ng/mL for the man and the woman, respectively. A keto-reduced metabolite (m/z 238.144) was synthesized to permit its quantification at 67 ng/mL and 54 ng/mL in male and female blood samples, respectively. Overall, the identification of these metabolites will increase the knowledge of potential drug consumption markers and allow to implement mass spectrometry databases to better monitor future drug abuse or consumption.

Effects of Serial Polydrug Use on the Rewarding and Aversive Effects of the Novel Synthetic Cathinone Eutylone.

BACKGROUND: As individual synthetic cathinones become scheduled and regulated by the Drug Enforcement Administration (DEA), new ones regularly are produced and distributed. One such compound is eutylone, a novel third-generation synthetic cathinone whose affective properties (and abuse potential) are largely unknown. The following experiments begin to characterize these effects and how they may be impacted by drug history (a factor affecting reward/aversion for other drugs of abuse). METHODS: Eutylone was assessed for its ability to induce conditioned taste avoidance (CTA; aversive effect) and conditioned place preference (CPP; rewarding effect) and their relationship (Experiment 1). Following this, the effects of exposure to cocaine or 3,4-methylenedioxymethamphetamine [MDMA] on eutylone's affective properties were investigated (Experiment 2). RESULTS: Eutylone produced dose-dependent CTA and CPP (Experiment 1), and these endpoints were unrelated. Pre-exposure to cocaine and MDMA differentially impacted taste avoidance induced by eutylone (MDMA > cocaine) and did not impact eutylone-induced place preference. CONCLUSIONS: These data indicate that eutylone, like other synthetic cathinones, has co-occurring, independent rewarding and aversive effects that may contribute to its abuse potential and that these effects are differentially impacted by drug history. Although these studies begin the characterization of eutylone, future studies should examine the impact of other factors on eutylone's affective properties and its eventual reinforcing effects (i.e., intravenous self-administration [IVSA]) to predict its use and abuse liability.

Quantification of new psychoactive substances in Australian wastewater utilising direct injection liquid chromatography coupled to tandem mass spectrometry.

The dynamic new psychoactive substances (NPS) market presents a great challenge for public health officers, law enforcement and analytical and forensic chemists. Wastewater analysis is a complementary tool in the ongoing surveillance of these compounds but the low doses, somewhat unknown metabolism and the different chemical classes have made analytical methods difficult to develop. The current study presents a direct injection method for the quantification of 32 NPS. These include a range of classes: phenethylamines (25C-NBOMe and 4-fluoroamphetamine), synthetic cathinones (3-methylmethcathinone, butylone, dibutylone, eutylone, methcathinone, N-ethylheptedrone, N-ethylhexedrone, N-ethylpentylone and pentylone), synthetic cannabinoid receptor agonists (5F-EMB-PICA, 5F-MDMB-PICA, 5F-MDMB-PINACA, AMB FUBINACA, MDMB-4en-PINACA, cumyl pegaclone and cumyl-5F-pegaclone), opioids (2-methyl AP-237, AP-238, brorphine, isotonitazene, metonitazene and protonitazene), benzodiazepines (clonazolam, etizolam, flualprazolam and flubromazolam), plant-based NPS (7-hydroxymitragynine and mitragynine) and dissociatives (2F-deschloroketamine, 2-oxo-PCE). The method was validated in terms of linearity, range, precision (interday and intraday), limit of detection and limit of quantification, while filtration losses and matrix effects were also examined. The method was applied to wastewater samples collected from New South Wales and Queensland over the 2021-22 New Year period, when recreational drug use was expected to increase. Three NPS were found: eutylone, clonazolam and etizolam, with eutylone having the highest mass loads.

Identification of the New Psychoactive Substance Eutylone.

OBJECTIVES: To establish a method to identify unknown sample based on the combined use of Fourier transform infrared spectroscopy (FTIR), gas chromatography-quadrupole time-of-flight mass spectrometry (GC-QTOF-MS), ultra-high performance liquid chromatography-linear ion trap quadrupole-orbitrap mass spectrometry (UPLC-LTQ-Orbitrap MS) and 1H-nuclear magnetic resonance spectroscopy (1H-NMR) technique. METHODS: The unknown sample was directly analyzed by FTIR. The unknown sample was dissolved in methanol solution containing internal standard SKF525A and the supernatant was detected by GC-QTOF-MS and UPLC-LTQ-Orbitrap MS. The unknown sample was dissolved in methanol-d4 solution for structural analysis of 1H-NMR. RESULTS: The characteristic absorption peaks of FTIR spectra obtained from unknown sample were 1 682 (C=O bond), 1 503, 1 488, 1 436, 1 363, 1 256, 1 092, 1 035, 935, 840 and 800 cm-1, the characteristic fragment ions (m/z) of GC-QTOF-MS were 86.096 4 (base peak), 58.065 1, 149.023 5, 121.028 6 and 65.038 6, the accurate mass [M+H]+ detected by UPLC-LTQ-Orbitrap MS was 236.127 7. The sample was identified as synthetic cathinone new psychoactive substance Eutylone by 1H-NMR. CONCLUSIONS: The method established in this study can be used for structural confirmation of Eutylone.

A fatal case of intoxication from a single use of eutylone: Clinical symptoms and quantitative analysis results.

Eutylone is a synthetic cathinone that is becoming an increasingly popular drug in the US and Europe. This report describes a fatal case of eutylone intoxication. A 32-year-old man went into cardiac arrest after several minutes of abnormal behavior. Rectal temperature was 37.0 °C at 5 h after death. Autopsy revealed no remarkable injuries apart from several small abrasions and no signs of rhabdomyolysis. Toxicological examination revealed only aripiprazole in the therapeutic range and eutylone. The eutylone concentration in cardiac blood was 4290 ng/g. This case is valuable because it involved fatal intoxication from a single use of eutylone and quantitative analysis, whereas most previous reports of eutylone intoxication have involved a mixture of drugs with limited quantitative analysis.

Quantitative determination and metabolic profiling of synthetic cathinone eutylone in vitro and in urine samples by liquid chromatography tandem quadrupole time-of-flight mass spectrometry.

In Taiwan, synthetic cathinones are the most prevalent new psychoactive substances, and their use is growing continuously. Urine samples are currently analysed to determine drug abuse, but the metabolic profiles and metabolites of these compounds are not widely reported. Given that cases of eutylone abuse have been growing since 2020, this study established a method employing supported liquid extraction combined with liquid chromatography tandem quadrupole time-of-flight mass spectrometry to identify and quantify eutylone and its metabolites in urine samples. Method validation was performed, and eight authentic samples were analysed. Moreover, in vitro metabolism experiments were conducted, and metabolites were generated by incubating eutylone with human liver microsomes and cytosol. Metabolite characterisation was achieved by confirming the accurate mass and product ions in full MS/MS spectra. Five metabolites were identified in in vitro experiments; they resulted from eutylone N-dealkylation, ß-ketone reduction, demethylenation, aliphatic hydroxylation and sequential demethylenation and O-methylation. The metabolic profile was obtained evaluating the metabolites at different incubation times: Demethylenation occurred first, followed by N-dealkylation, ß-ketone reduction and aliphatic hydroxylation. Three additional metabolites were identified in authentic samples. Based on in vitro and in vivo evidence, we propose that the demethylenation and O-methylation metabolite, the ß-ketone reduction metabolite, and the ß-ketone reduction, demethylenation and O-methylation metabolite are the most appropriate biomarkers of eutylone consumption. Using these markers can help expand the eutylone detection window and provide information for toxicology research.

Identification of the New Psychoactive Substance Eutylone / 法医学杂志

OBJECTIVES@#To establish a method to identify unknown sample based on the combined use of Fourier transform infrared spectroscopy (FTIR), gas chromatography-quadrupole time-of-flight mass spectrometry (GC-QTOF-MS), ultra-high performance liquid chromatography-linear ion trap quadrupole-orbitrap mass spectrometry (UPLC-LTQ-Orbitrap MS) and 1H-nuclear magnetic resonance spectroscopy (1H-NMR) technique.@*METHODS@#The unknown sample was directly analyzed by FTIR. The unknown sample was dissolved in methanol solution containing internal standard SKF525A and the supernatant was detected by GC-QTOF-MS and UPLC-LTQ-Orbitrap MS. The unknown sample was dissolved in methanol-d4 solution for structural analysis of 1H-NMR.@*RESULTS@#The characteristic absorption peaks of FTIR spectra obtained from unknown sample were 1 682 (C=O bond), 1 503, 1 488, 1 436, 1 363, 1 256, 1 092, 1 035, 935, 840 and 800 cm-1, the characteristic fragment ions (m/z) of GC-QTOF-MS were 86.096 4 (base peak), 58.065 1, 149.023 5, 121.028 6 and 65.038 6, the accurate mass [M+H]+ detected by UPLC-LTQ-Orbitrap MS was 236.127 7. The sample was identified as synthetic cathinone new psychoactive substance Eutylone by 1H-NMR.@*CONCLUSIONS@#The method established in this study can be used for structural confirmation of Eutylone.

Eutylone and Its Structural Isomers Interact with Monoamine Transporters and Induce Locomotor Stimulation.

Synthetic cathinones are a class of new psychoactive substances that induce psychostimulant effects and pose risk for hospitalizations, overdose, and death. At the present time, derivatives of the synthetic cathinone, methylone, are being confiscated in nonmedical (i.e., recreational) drug markets worldwide. In particular, eutylone is a newly emerging methylone analog that possesses ethyl groups at the α-carbon and amine positions. Little information is available about the pharmacological effects of eutylone, but based on its structure, we surmised that the compound interacts with monoamine transporters in the brain. To test this hypothesis, we compared the effects of eutylone and its structural isomers, dibutylone and pentylone, using in vitro transporter assays in rat brain synaptosomes and in vivo locomotor activity assessments in mice. All drugs displayed dose-related inhibition of [3H]neurotransmitter uptake at dopamine transporters (DAT) and norepinephrine transporters (NET), but effects at DAT were 10-fold more potent (IC50 = 120 nM). Eutylone and pentylone inhibited uptake at serotonin transporters (SERT), while dibutylone did not. Additionally, eutylone and pentylone displayed weak partial releasing actions at SERT which achieved 50% of maximal response. All drugs stimulated dose-related locomotion in mice, and eutylone was most potent and efficacious in this regard (ED50 = 2 mg/kg, sc). Our results demonstrate that eutylone is a hybrid transporter compound with uptake inhibition properties at DAT and NET but substrate activity at SERT. The effects of eutylone are similar to those produced by pentylone, which suggests that eutylone will exhibit abuse liability and pose risks for psychostimulant side-effects in human users.

Sample Mining and Data Mining: Combined Real-Time and Retrospective Approaches for the Identification of Emerging Novel Psychoactive Substances.

Novel psychoactive substances (NPS) are synthetic drugs that pose serious public health and safety concerns. A multitude of NPS have been identified in the United States, often implicated in forensic investigations. The most common and effective manner for identifying NPS is by use of mass spectrometry and the true utility lies within nontargeted acquisition techniques. During this study, a liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) assay was developed, validated, and implemented for forensic toxicology testing. A SCIEX TripleTOF™ 5600 + with SWATH® acquisition was used. Resulting data were compared against an extensive library database containing more than 800 compounds. The LC-QTOF-MS assay was applied to the reanalysis of biological sample extracts to discover emergent NPS. More than 3,000 sample extracts were analyzed, and more than 20 emerging NPS were detected for the first time. Among these were isopropyl-U-47700, 3,4-methylenedioxy-U-47700, fluorofuranylfentanyl, N-methyl norfentanyl, 2F-deschloroketamine, 3,4-methylenedioxy-alpha-PHP, eutylone, and N-ethyl hexedrone.