The metabolic profile of the synthetic cannabinoid receptor agonist ADB-HEXINACA using human hepatocytes, LC–QTOF-MS and synthesized reference standards

Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances (NPS) and new structural scaffolds have emerged on the recreational drug market since the enactment of Chinese SCRA analog controls in 2021. This study reports the first SCRAs to be detected with a bromide at the 5 position (5′Br) on the phenyl ring of the indazole core and without a tail moiety. ADB‐5′Br‐INACA (ADMB‐5′Br‐INACA) and MDMB‐5′Br‐INACA were detected in seized samples from Scottish prisons, Belgian customs, and US forensic casework. The brominated analog with a tail moiety, ADB‐5′Br‐BUTINACA (ADMB‐5′Br‐BUTINACA), was also detected in Scottish prisons and US forensic casework. The metabolites of these compounds and the predicted compound MDMB‐5′Br‐BUTINACA were identified through incubation with primary human hepatocytes to aid in their toxicological identification. The bromide on the indazole remains intact on metabolites, allowing these compounds to be easily distinguished in toxicological samples from their non‐brominated analogs. Glucuronidation was more common for tail‐less analogs than their butyl tail‐containing counterparts. Forensic toxicologists are advised to update their analytical methods with the characteristic ions for these compounds, as well as their anticipated urinary markers: amide hydrolysis and monoOH at tert‐butyl metabolites (after β‐glucuronidase treatment) for ADB‐5′Br‐INACA; monoOH at tert‐butyl and amide hydrolysis metabolites for ADB‐5′Br‐BUTINACA; and ester hydrolysis metabolites with additional metabolites for MDMB‐5′Br‐INACA and MDMB‐5′Br‐BUTINACA. Toxicologists should remain vigilant to the emergence of new SCRAs with halogenation of the indazole core and tail‐less analogs, which have already started to emerge.

Detection in seized samples, analytical characterization, and in vitro metabolism of the newly emerged 5‐bromo‐indazole‐3‐carboxamide synthetic cannabinoid receptor agonists

Norman,

Webling,

Kyslychenko

et al. 2023

ADB‐HEXINACA—a novel synthetic cannabinoid with a hexyl substituent: phase I metabolism in authentic urine samples, a case report and prevalence on the German market

Giorgetti,

Zschiesche,

Groth

et al. 2024

Synthetic cannabinoid receptor agonists (SCRAs) are one of the largest groups of new psychoactive substances (NPS). Yet, another novel analog started spreading on the NPS market around 2021. Soon after, the substance could be analytically characterized in herbal material as ADB‐HEXINACA, an SCRA containing a hexyl‐substituted tail on the indazole core. Here, we present suitable urinary markers to prove the consumption of this analog, a case report of acute polydrug intoxication and data on its prevalence in Germany. Anticipated phase I metabolites were detected in 12 authentic urine samples that were collected for abstinence control and analyzed by ultra‐performance liquid chromatography coupled to a time‐of‐flight mass spectrometer (UPLC‐qToF‐MS). The results of in vivo samples were confirmed by analysis of in vitro incubates with pooled human liver microsomes (pHLMs). Forensic samples were used to assess the prevalence of ADB‐HEXINACA. Thirty‐two phase I metabolites were detected in the authentic urine samples. The main metabolites resulted from amide hydrolysis in combination with either monohydroxylation or ketone formation at the hexyl moiety (M15 and M26), the monitoring of which is recommended as a proof of consumption. ADB‐HEXINACA was detected in 3.5% of SCRA positive urine samples collected for abstinence control in Freiburg up to December 2022 and in 5.5% of the SCRA positive blood/serum samples. The hexyl substituent of ADB‐HEXINACA allows for the detection of specific urinary biomarkers suggested as analytical targets to confirm its prior intake. ADB‐HEXINACA had a rather low prevalence in Germany, alternating months of higher prevalence with periods of total absence.

Human metabolism of the semi‐synthetic cannabinoids hexahydrocannabinol, hexahydrocannabiphorol and their acetates using hepatocytes and urine samples

Lindbom,

Norman,

Baginski

et al. 2024

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.