Hexahydrocannabinol (HHC), hexahydrocannabiphorol (HHCP) and their acetates, HHC‐O and HHCP‐O, respectively, are emerging in Europe as alternatives to tetrahydrocannabinol (THC). This study aimed to elucidate the metabolic pathways of the semi‐synthetic cannabinoids HHC, HHCP, HHC‐O and HHCP‐O from incubation with human hepatocytes. The metabolites of HHC were also identified in authentic urine samples. HHC, HHCP, HHC‐O and HHCP‐O were incubated with primary human hepatocytes for 1, 3 and 5 h. Authentic urine samples from cases screened positive for cannabis in blood using ELISA but confirmed negative were analysed both non‐hydrolysed and hydrolysed for HHC metabolites. Potential metabolites were identified using ultra‐high performance liquid chromatography (UHPLC) coupled to a quadrupole time‐of‐flight mass spectrometer (QToF‐MS). HHC and HHCP were primarily metabolised through monohydroxylation (monoOH), followed by oxidation to a carboxylic acid metabolite. HHC‐O and HHCP‐O were rapidly metabolised to HHC and HHCP, respectively. In authentic urine samples, 18 different metabolites were identified, and 99.3% of hydroxylated metabolites were glucuronidated. 11‐OH‐HHC, 5′OH‐HHC and another metabolite with a monoOH on the side chain were the only metabolites present in all 16 urine samples. The metabolism of HHC and HHCP were similar, although the longer alkyl side chain of HHCP (heptyl) led to greater hydroxylation on the side chain than HHC (pentyl). The use of HHC and HHCP can be differentiated from the use of THC and other phytocannabinoids, but the use of the acetate analogues may not be differentiable from their non‐acetate analogues.

Synthetic cannabinoid receptor agonists (SCRAs) remain a major public health concern, with their use implicated in intoxications and drug-related deaths worldwide. Increasing our systematic understanding of SCRA metabolism supports clinical and forensic toxicology casework, facilitating the timely identification of analytical targets for toxicological screening procedures and confirmatory analysis. This is particularly important as new SCRAs continue to emerge on the illicit drug market. In this work, the metabolism of ADB-HEXINACA (ADB-HINACA, N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-hexyl-1H-indazole-3-carboxamide), which has increased in prevalence in the United Kingdom and other jurisdictions, was investigated using in vitro techniques. The (S)-enantiomer of ADB-HEXINACA was incubated with pooled human hepatocytes over 3 hours to identify unique and abundant metabolites using liquid chromatography–quadrupole time-of-flight mass spectrometry. In total, 16 metabolites were identified, resulting from mono-hydroxylation, di-hydroxylation, ketone formation (mono-hydroxylation then dehydrogenation), carboxylic acid formation, terminal amide hydrolysis, dihydrodiol formation, glucuronidation and combinations thereof. The majority of metabolism took place on the hexyl tail, forming ketone and mono-hydroxylated products. The major metabolite was the 5-oxo-hexyl product (M9), while the most significant mono-hydroxylation product was the 4-hydroxy-hexyl product (M8), both of which were confirmed by comparison to in-house synthesized reference standards. The 5-hydroxy-hexyl (M6) and 6-hydroxy-hexyl (M7) metabolites were not chromatographically resolved, and the 5-hydroxy-hexyl product was the second largest mono-hydroxylated metabolite. The structures of the terminal amide hydrolysis products without (M16, third largest metabolite) and with the 5-positioned ketone (M13) were also confirmed by comparison to synthesized reference standards, along with the 4-oxo-hexyl metabolite (M11). The 5-oxo-hexyl and 4-hydroxy-hexyl metabolites are suggested as biomarkers for ADB-HEXINACA consumption.

An emerging new trend reveals that potent synthetic cannabinoid receptor agonists (SCRAs) that were prevalent prior to the Chinese class-wide ban in July 2021 have remained on the market or re-emerged. This is likely because of a new production route where unscheduled tail-less precursors are converted into the desired controlled SCRA via a one-step synthesis. This study reports the discovery of a clandestine production site in Switzerland for this synthesis. Controlled recreations of the synthesis of ADB-BUTINACA (also known as ADB-BINACA or ADMB-BUTINACA) and MDMB-4en-PINACA based on a recipe recovered from the Swiss production site were undertaken. The recreations yielded oils or waxy products with chromatographic purities ranging from 39.3 to 93.4% with the precursors (ADB-INACA or MDMB-INACA) remaining in all products. Mixtures of a precursor (e.g., MDMB-INACA, ADB-INACA, AB-INACA, MDMB-ICA, ADB-IATA, MDMB-5’Me-INACA) with corresponding final SCRA(s) (e.g., MDMB-4en-PINACA, 4F-MDMB-BUTINACA, MDMB-BUTINACA, MDMB-FUBINACA, ADB-BUTINACA, ADB-4en-PINACA, AB-CHMINACA, 4F-MDMB-BUTICA, ADB-FUBIATA) were found in 28 forensic casework samples in the US received between February 2023 and February 2024 and 87 samples seized from the Scottish prisons between February 2023 and July 2024. In vitro CB₁ receptor activity of the precursors, synthesized products, and mixtures of precursors and final SCRAs were examined using β-arrestin 2 recruitment and intracellular calcium release assays. The precursors were much less potent than the final SCRAs and the concentration of the final SCRA dictated the activity of mixtures. This study raises awareness of the emergence of a new production method for SCRAs as observed in multiple countries.

Synthetic cannabinoid receptor agonists (SCRAs) are a group of new psychoactive substances (NPS) that bind to and activate the cannabinoid 1 and 2 (CB₁ and CB₂) receptors. Following the introduction of SCRA analog controls in China in July 2021, new SCRAs with a bromine at the 5 position on the indazole core emerged on the recreational drug market. In this study, the in vitro CB₁ receptor activity of 19 different SCRAs with a halogenated indazole core was systematically examined using a AequoScreen^® CB₁ assay alongside four available non-halogenated analogs, ADB-BUTINACA, MDMB-BUTINACA, MDMB-4en-PINACA, and MDMB-INACA, and the newly emerged MDMB-5′Me-INACA (MDMB-5-methyl-INACA). Of SCRAs with a substitution at the 5 position on the indazole core, analogs with a fluorine had the lowest EC₅₀ values. For SCRAs with a methyl ester head moiety (MMB or MDMB), chlorinated analogs had the next lowest EC₅₀ values followed by brominated analogs; however, the opposite relationship was observed for SCRAs with an amide head moiety (AB or ADB). All halogenated compounds had similar potency to their non-halogenated analog, except the brominated tert-leucine methyl ester SCRAs, which had significantly reduced potency. Of the different head moieties, tert-leucine methyl ester SCRAs were the most potent, followed by tert-leucinamide, valinamide, and valine methyl ester. The potencies of the SCRAs with pentyl (ADB-5′Br-PINACA/ADMB-5′Br-PINACA/ADB-P-5′Br-INACA), pent-4-enyl (ADB-4en-5′Br-PINACA/ADMB-4en-5′Br-PINACA/ADB-4en-P-5′Br-INACA and MDMB-4en-5′Br-PINACA/MDMB-4en-P-5′Br-INACA), and butyl tails (ADB-5′Br-BUTINACA/ADMB-5′Br-BUTINACA/ADB-B-5′Br-INACA) were not significantly different, whereas fluorobenzyl (ADB-5′Br-FUBINACA), tail-less (ADB-5′Br-INACA), and decyl tail (ADB-5′Br-DECINACA/ADMB-5′Br-DECINACA/ADB-D-5′Br-INACA) analogs were significantly less potent.

Synthetic cannabinoids (SCs) remain a major public health concern, as they continuously are linked to severe intoxications and drug‐related deaths worldwide. As new SCs continue to emerge on the illicit drug market, an understanding of SC metabolism is needed to identify formed metabolites that may serve as biomarkers in forensic toxicology screening and for understanding the pharmacokinetics of the drugs. In this work, the metabolism of ADB‐4en‐P‐5Br‐INACA and ADB‐P‐5Br‐INACA ((S)‐N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐5‐bromo‐1‐(pent‐4‐en‐1‐yl)‐1H‐indazole‐3‐carboxamide, (S)‐N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐5‐bromo‐1‐pentyl‐1H‐indazole‐3‐carboxamide respectively) were investigated using human hepatocytes in vitro and in‐house synthesized references. Both SCs were incubated with pooled human hepatocytes over 3 h, with the aim to identify unique and abundant metabolites using liquid chromatography–quadrupole time‐of‐flight mass spectrometry (LC‐QTOF‐MS). In total nine metabolites were identified for ADB‐4en‐P‐5Br‐INACA and 10 metabolites for ADB‐P‐5Br‐INACA. The observed biotransformations included dihydrodiol formation, terminal amide hydrolysis, hydroxylation, dehydrogenation, carbonyl formation, glucuronidation, and combinations thereof. The major metabolites were confirmed by in‐house synthesized references. Recommended biomarkers for ADB‐P‐5Br‐INACA and ADB‐4en‐P‐5Br‐INACA are the terminal hydroxy and dihydrodiol metabolite respectively.

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 μM) 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 β position of the phenylpropanoyl moiety was highly favored; β‐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 with findings in previous studies of similar fentanyl analogs.