In 2021, almost one new psychoactive substance per week entered the European market according to the European Monitoring Centre for Drugs and Drug Addiction. Among the new psychoactive compounds are a subclass of synthetic cannabinoids which mimics the effects of Δ9-tetrahydrocannabinol (THC), the major psychoactive component in cannabis. Synthetic cannabinoids are full cannabinoid receptor agonists and are more potent than THC, leading to intoxications that results of seizures, psychoses, acute kidney injury, tachycardia, stroke, agitation, and there are even reports of synthetic cannabinoid associated deaths. Among the newly introduced new synthetic cannabinoids seized in the European Union are Benzyl-4CN-BUTINACA (N-benzyl-1-(4-cyanobutyl)-1H-indazole-3-carboxamide) and 4CN-MDMB-BUTINACA (methyl 2-{[1-(4-cyanobutyl)-1H-indazole-3-carbonyl]amino}-3,3-dimethylbutanoate). Synthetic cannabinoids are known to be extensively metabolized in the body, resulting in very low concentrations of the parent compound. Therefore, the metabolites found in biological samples can become of great importance to prove the use of new synthetic cannabinoids.

The aim of this study was to characterize metabolites after incubation with human hepatocytes from these two drugs, namely Benzyl-4CN-BUTINACA and 4CN-MDMB-BUTINACA, analysed by liquid chromatography-quadrupole time-of-flight mass spectrometry, LC-QTOF-MS and to generate two reference spectra of the in vitro phase I and phase II metabolites.

After five hours of incubation with human hepatocytes were ten metabolites detected for Benzyl-4CN-BUTINACA. The major metabolites were hydroxylation, dihydrodiol, dehydration, dealkylation, decyanation and glucuronidation. Three metabolites were also the result of cyanide release. The most abundant metabolite was a dihydrodiol followed by a metabolite resulting from decyanation, dehydration and dihydroxylation. After five hours of incubation with human hepatocytes for 4CN-MDMB-BUTINACA were twelve metabolites detected. The major metabolites were–ester hydrolysis, ester hydrolysis+hydroxylation and ester hydrolysis+dehydrogenation. Two phase II metabolites were also detected, and both were glucuronides–hydroxylation+glucuronidation and ester hydrolysis+glucuronidation. Three metabolites also resulted from cyanide release.

Interestingly, for both 4CN-MDMB-BUTINACA and Benzyl-4CN-BUTINACA were several biotransformations indicted the release of cyanide, a proposed toxic metabolic pathway.