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Many new psychoactive substances (NPS) are stimulants, and information about their potency and abuse potential is often lacking. To start addressing this need, a method measuring the inhibition of the dopamine, serotonin, and norepinephrine transporters (DAT, SERT, and NET) by stimulant drugs was developed. The use of a proprietary fluorescent dye mixture and three cell lines (CHO-K1, HEK 293, and MDCK), each expressing a single transporter, allowed for a semiautomated, one-pot determination of inhibition in a 384-well format. The method was validated using well characterized stimulants, including cocaine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), & alpha;-PVP, and fluoxetine and performed similarly to other methods. Seven synthetic cathinones all showed highest potency for DAT inhibition, followed by NET and SERT. The rank potency for DAT inhibition IC50 (nM) was MPHP (4.53) > 4Cl-& alpha;-PVP (8.05) > 3F-& alpha;-PVP (12.7) > & alpha;-PiHP (13.4) > N-ethylpentylone (16.9) > N-ethylhexedrone (44.5) > 4-methylpentedrone (261). All but 4-methylpentedrone were more potent than amphetamine (257) and cocaine (111). The DAT/SERT inhibition ratio for the cathinones was in the range from 5.02 for 4-methylpentedrone to >3730 for & alpha;-PiHP, compared to 1.64 for cocaine and >4030 for & alpha;-PVP. All seven substances had inhibition profiles similar to those of potent stimulants with high abuse potential., Funding Agencies|Public Health Agency of Sweden; National Board of Forensic Medicine

Acetylbenzylfentanyl, benzoylbenzylfentanyl, 3-fluoro-methoxyacetylfentanyl, and 3-phenylpropanoylfentanyl are fentanyl analogs that have been reported to the European Monitoring Centre for Drugs and Drug Addiction in recent years. The aim of this study was to identify metabolic pathways and potential biomarker metabolites of these fentanyl analogs. The compounds were incubated (5 mu M) with cryopreserved hepatocytes for up to 5 h in vitro. Metabolites were analyzed with liquid chromatography-quadrupole time of flight-high-resolution mass spectrometry (LC-QTOF-HRMS). The experiments showed that acetylbenzylfentanyl, benzoylbenzylfentanyl, and 3-phenylpropanoylfentanyl were mainly metabolized through N-dealkylation (forming nor-metabolites) and 3-fluoro-methoxyacetylfentanyl mainly through demethylation. Other observed metabolites were formed by mono-/dihydroxylation, dihydrodiol formation, demethylation, dehydrogenation, amide hydrolysis, and/or glucuronidation. The experiments showed that a large number of metabolites of 3-phenylpropanoylfentanyl were formed. The exact position of hydroxy groups in formed monohydroxy metabolites could not be established solely based upon recorded MSMS spectra of hepatocyte samples. Therefore, potential monohydroxy metabolites of 3-phenylpropanoylfentanyl, with the hydroxy group in different positions, were synthesized and analyzed together with the hepatocyte samples. This approach could reveal that the beta position of the phenylpropanoyl moiety was highly favored; beta-OH-phenylpropanoylfentanyl was the most abundant metabolite after the nor-metabolite. Both metabolites have the potential to serve as biomarkers for 3-phenylpropanoylfentanyl. The nor-metabolites of acetylbenzylfentanyl, benzoylbenzylfentanyl, and 3-fluoro-methoxyacetylfentanyl do also seem to be suitable biomarker metabolites, as do the demethylated metabolite of 3-fluoro-methoxyacetylfentanyl. Identified metabolic pathways and formed metabolites were in agreement, Funding Agencies|Eurostars-2 Joint Programme (European Commission) [E! 113377]; European Unions Horizon 2020 research and innovation program; Swedens Innovation Agency VINNOVA [2019-03566]; Strategic Research Area in Forensic Sciences (Strategiomradet forensiska vetenskaper) at Linkoeping University

4 Cl-cumyl-PINACA (SGT-157), 4 F-cumyl-5F-PINACA (4F-cumyl-5F-PINACA, SGT-65) and 4 F-cumyl-5F-PICA (4F-cumyl-5F-PICA, SGT-64) are a series of new halogenated cumyl synthetic cannabinoid receptor agonists (SCRAs). Due to rapid metabolism, monitoring and screening for SCRAs in biological matrices requires identification of their metabolites. It is an essential tool for estimating their spread and fluctuations in the global illicit market. The purpose of this study was to identify human biotransformations of 4 Cl-cumyl-PINACA, 4 F-cumyl-5F-PINACA and 4 F-cumyl-5F-PICA in vitro and characterize for the first time the metabolic pathways of halogenated cumyl SCRAs. 4 Cl-cumyl-PINACA, 4 F-cumyl-5F-PINACA and 4 F-cumyl-5F-PICA were incubated with human hepatocytes in duplicates for 0, 1, 3 and 5 h. The supernatants were analysed in data-dependent acquisition on a UHPLC-QToF-MS, and the potential metabolites were tentatively identified. A total of 11 metabolites were detected for 4 Cl-cumyl-PINACA, 21 for 4 F-cumyl-5F-PINACA and 10 for 4 F-cumyl-5F-PICA. The main biotransformations were oxidative defluorination, followed by hydroxylation with dehydrogenation, N-dealkylation, dihydrodiol formation and glucuronidation. Hydroxylations were most common at the tail moieties with higher abundancy for indole than indazole compounds. N-dealkylations were more common for fluorinated tail chain compounds than the non-fluorinated 4 Cl-cumyl-PINACA. In conclusion, many metabolites retained halogen groups at the cumyl moieties which, in various combinations, may be suitable as analytical biomarkers., Funding Agencies|VINNOVA [2019-03566]; Eurostars-2 Joint Programme [E! 113377]; European Union; Swedens Innovation Agency Vinnova; Strategic Research Area in Forensic Sciences [2016:7]

A new class of synthetic cannabinoids termed OXIZIDs has recently emerged on the recreational drug market. In order to continue the detection of new drugs in biological specimens, the identification of metabolites is essential. The aim of this study was to elucidate the metabolites of BZO-4en-POXIZID produced in human liver microsomes (HLMs) and human hepatocyte incubations and to compare the results with closely related analogs using the same experimental setup. Each drug was incubated for 1 h in HLM and BZO-4en-POXIZID was also incubated in human hepatocytes for up to 3 h. Subsequently, the incubates were analyzed by liquid chromatography-high-resolution mass spectrometry. BZO-4en-POXIZID metabolites were obtained in the incubation with HLMs and human hepatocytes, via the metabolic pathways of dihydrodiol formation, hydroxylation, reduction of the alkene bond and glucuronidation. The major metabolic pathway was found to be dihydrodiol formation at the pentenyl tail moiety. BZO-POXIZID, 5 F-BZO-POXIZID, BZO-HEXOXIZID and BZO-CHMOXIZID underwent similar metabolism to those reported in the literature, via the metabolic pathways of N-dealkylation, hydroxylation, ketone formation and oxidative defluorination (to alcohol or carboxylic acid). The results suggest that OXIZIDs are mainly metabolized at the N-alkyl moiety and the major metabolic pathways are hydroxylation when the N-alkyl moiety is a simple hydrocarbon, whereas functional-group-specific pathways (dihydrodiol formation and oxidative defluorination) are preferred when the moiety contains specific functional groups (alkene or fluoro), as has been observed for other synthetic cannabinoids. The major metabolites generated via these major metabolic pathways should serve as useful analytical targets for urine analysis. Furthermore, the higher abundance of glucuronidated metabolite suggests that enzymatic hydrolysis of glucuronides may be necessary for urine analysis to increase phase I metabolite concentration and, Funding Agencies|Japan Society for the Promotion of Science (KAKENHI) [21K17329]; Eurostars-2 joint programme [E! 113377]; European Union [2019-03566]; Strategic Research Area in Forensic Sciences (Styrkeomradet forensiska vetenskaper) at Linkoping University; Leverhulme Trust [RC-2015-01]

Supercritical fluid chromatography (SFC) is a technique that analyzes compounds that are temperature-labile, have moderately low weight, or are chiral compounds. Methylphenidate (MPH) is a chiral compound with two chiral centers. MPH has two chiral metabolites, ethylphenidate (EPH) and ritalinic acid (RA). MPH is sold as a racemic mixture. The d-enantiomer of threo-MPH is responsible for medicinal effects. Due to the differing effects of the enantiomers, it is important to analyze the enantiomers individually to better understand their effects. This method utilizes SFCand solid-phase extraction (SPE) to separate and analyze the enantiomers of MPH, EPH, and RA in postmortem blood. The objective of this method was to assess a unique approach with SFC for enantiomeric separation of MPH, EPH, and RA. A SPE method was developed and optimized to isolate the analytes in blood and validated as fit-for-purpose following international guidelines. The linear range for MPH and EPH was 0.25-25 and 10-1000 ng/mL for RA in blood. Bias was -8.6% to 0.8%, and precision was within 15.4% for all analytes. Following method validation, this technique was applied to the analysis of 49 authentic samples previously analyzed with an achiral method. Quantitative results for RA were comparable to achiral technique, whereas there was loss of MPH and EPH over time. The l:d enantiomer ratio was calculated, and MPH demonstrated greater abundance of the d-enantiomer. This is the first known method to separate and quantify the enantiomers of all three analytes utilizing SFC and SPE.

Early warning systems detect new psychoactive substances (NPS), while dedicated monitoring programs and routine drug and toxicology testing identify fluctuations in prevalence. We report the increasing prevalence of the synthetic cannabinoid receptor agonist (SCRA) ADB-BUTINACA (N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-butyl-1H-indazole-3-carbox-amide). ADB-BUTINACA was first detected in a seizure in Sweden in 2019 and we report its detection in thirteen routine Swedish forensic toxicology cases soon after. In January 2021, ADB-BUTINACA was detected in SCRA-infused papers seized in Scottish prisons and has rapidly increased in prevalence, being detected in 60.4% of the SCRA-infused papers tested between January and July 2021. In this work, ADB-BUTINACA was incubated with human hepatocytes (HHeps) and 21 metabolites were identified in vitro, 14 being detected in authentic case samples. The parent drug and metabolites B9 (mono-hydroxylation on the n-butyl tail) and B16 (mono-hydroxylation on the indazole ring) are recommended biomarkers in blood, whilst metabolites B4 (dihydrodiol formation on the indazole core), B9 and B16 are suitable biomarkers in urine. ADB-4en-PINACA (N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(pent-4-en-1-yl)-1H-indazole-3-carboxamide) was detected in Scottish prisons in December 2020 but, unlike ADB-BUTINACA, prevalence has remained low. ADB-4en-PINACA was incubated with HHeps and 11 metabolites were identified. Metabolites E3 (dihydrodiol formed in the tail moiety) and E7 (hydroxylation on the linked/head group) are the most abundant metabolites in vitro and are suggested as urinary biomarkers. The in vitro potencies of ADB-BUTINACA (EC50, 11.5 nM and ADB-4en-PINACA (EC50, 11.6 nM) are similar to that of MDMB-4en-PINACA (EC50, 4.3 nM). A third tert-leucinamide SCRA, ADB-HEXINACA was also detected in prison samples and warrants further investigation.