Metabolite profile of synthetic cannabinoids seized in Portugal

Synthetic cannabinoids represent the most extensive group of new psychoactive substances (NPS) that are monitored by the EMCDDA. Comprehensive knowledge of the metabolic profile of cannabinoids is crucial for confirming their consumption. Due to their potentially fast metabolic degradation, the only way to attest their intake in clinical and forensic contexts, even shortly after its intake, often relies on monitoring their metabolites in biofluids. However, the rapid emergence of new cannabinoids in the market makes it difficult for competent authorities to do this type of research work, as a result, most of new cannabinoids remain undetected in biological samples. Therefore, in a cooperative effort between forensic and research institutions, we decided to determine the metabolite profiles of the recently seized cannabinoids in Portugal: ADB-5Br-INACA, ADB-5Br-BUTINACA, and ADB-5Br-PINACA.

The selected cannabinoids were incubated with pooled human S9 fractions in the presence of Phase I and II cofactors. Liquid chromatography coupled with tandem High-Resolution Mass Spectrometry (LC-HRMS/MS) was used to monitor the consumption of the parent cannabinoid over the incubation period for half-life calculations and to characterize the generated metabolites.

Short half-lives of 12 min and 21 min were obtained for ADB-5Br-INACA and ADB-5Br-BUTINACA, respectively. Within a few hours, the concentrations of these cannabinoids are expected to be lower more than those of their metabolites, suggesting their potential use as biomarkers. Surprisingly, ADB-5Br-PINACA was stable under the incubation conditions, suggesting that this cannabinoid is a suitable biomarker for its intake. The main metabolic pathways identified for the three cannabinoids are hydroxylation, amide hydrolysis, and glucuronidation. 

These results can be crucial in forensic and clinical contexts for validating the consumption of these substances using the detected metabolites. Based on the synthetic ability of the team, we plan to synthesize standards of the main metabolites for their unequivocal detection in urine and/or blood samples.

Fundação para a Ciência e Tecnologia (FCT) is acknowledged for funding the projects: UIDB/00100/2020 (DOI: 10.54499/UIDB/00100/2020) and UIDP/00100/2020 (DOI:10.54499/UIDP/00100/2020) to Centro de Química Estrutural, LA/P/0056/2020 (DOI 10.54499/LA/P/0056/2020) to the Associate Laboratory Institute of Molecular Sciences; UIDB/04046/2020 (DOI:10.54499/UIDB/04046/2020) and UIDP/04046/2020 (DOI: 10.54499/UIDP/04046/2020) to BioISI-Biosystems & Integrative Sciences Institute; UIDB/04565/2020 and UIDP/04565/2020 to Institute for Bioengineering and Biosciences;  LA/P/0140/2020 to the Associated laboratory Institute for Health and Bioeconomy and through Investimento RE-C05-i02 – Missão Interface N.o01/C05-i02/22. Joint funding from FCT and the COMPETE Program through grant RNEM-LISBOA-01-0145-FEDER-022125. FCT is also acknowledged for the PhD grant 2022.11339.BD to RPL.