In vitro metabolism of two synthetic cannabinoids and using the obtained metabolites in routine drug screening methods.

The present study aims to generate glucuronic acid-conjugated metabolites of the carboxylic acid metabolites of two of the most prevalent synthetic cannabinoids in humans, MDMB-4en-PINACA and ADBBUTINACA, using in vitro pHLM and pS9 systems. Additionally, the study seeks to optimize and integrate the resulting metabolites into routine drug screening methods. Another objective was to enhance our understanding of phase 2 metabolic transformations of next-gene ation synthetic cannabinoids by simultaneously detecting both conjugated and free forms. Furthermore, by monitoring the decomposition of glucuronides, we evaluated the efficiency of our routine hydrolysis method—used for confirmatory measurements of synthetic cannabinoids—by comparing it to two alternative hydrolysis procedures. For in vitro experiments, an NADP regeneration system and a UGT system were utilized. Sample acquisitions were performed using a Shimadzu LC-MS/MS-8040 system. A total of 14 urine samples were analyzed, both with and without hydrolysis, followed by acetonitrile precipitation. The hydrolysis efficiency of Escherichia coli β-D-glucuronidase-IX-A was compared with that of alkaline hydrolysis and Abalonase. The in vitro-produced ADB-BUTINACA-carboxylic acid glucuronide and MDMB-4en-PINACA-carboxylic acid glucuronide were MRM-optimized and incorporated into the screening method, supported by chromatographic separation. The glucuronide-bound form of MDMB-4en-PINACA was present in 70% of the samples, while that of ADB-BUTINACA was detected in 93%. The average concentration of ADB-BUTINACA-carboxylic acid was 181 ng/mL, whereas that of MDMB-4en-PINACA-carboxylic acid was 4.17 ng/mL. Alkaline hydrolysis demonstrated the highest efficiency, completely degrading glucuronide-bound metabolites in all samples and yielding the highest concentrations of free metabolites. On average, the concentrations increased by more than 700% following alkaline hydrolysis, compared to increases of approximately 300% with β-D-glucuronidase and 190% with Abalonase. Our findings indicate that the preparation and monitoring of phase 2 metabolites of synthetic cannabinoids enhance drug detection and reduce the likelihood of false-negative results. However, further studies are required to fully assess the reliability of hydrolysis methods.