Investigating 3-CMC metabolism: Insights from liver microsomes and postmortem biological matrix

3-Chloromethcathinone (3-CMC) is a synthetic cathinone that has been identified as a new psychoactive substance (NPS) by the European Monitoring Centre for Drugs and Drug Addiction. Despite its increasing prevalence in the recreational drug market since 2014, scientific literature on 3-CMC remains limited. This study employed a multi-step approach to investigate 3-CMC metabolism. First, an in-silico prediction was conducted to compile a list of potential metabolites. Then, in vitro assays were performed using human liver microsomes at two concentrations of 3-CMC. Samples were analyzed using an ultra-performance liquid chromatography system coupled with a high-resolution mass spectrometer. Chromatographic separation was obtained with an Acquity UPLC HSS C18 1.8 µm, 2.1 × 150 mm column on an Ultimate 3000 system chromatography coupled with a QExactivePlus mass spectrometer). Finally, data mining for metabolite identification was conducted using Compound Discoverer software. The combined in silico and in vitro approaches identified four primary metabolites of 3-CMC in HLM assays:1) hydroxylation of the aliphatic group to give M1 2) followed by reduction of the β-keto group, yielding M4; 3) N-demethylation, affording M2; and 4) Reduction of the β-keto group, yielding M3. Subsequent analysis of biological samples from two postmortem cases revealed that urine was the most informative matrix for detecting 3-CMC and its metabolites. The M3 metabolite, was identified as the third abundant metabolite in human liver microsome but was identified as the predominant metabolite in human postmortem samples. Identifying these key metabolites is crucial for improving the accuracy of forensic investigations and extending the detection window beyond the parent compound.