Identification, synthesis and quantification of eutylone consumption markers in a chemsex context.

Eutylone is a cathinone-derived synthetic amphetamine scheduled by the World Health Organization and European Monitoring Centre for Drugs and Drug Addiction since 2022 due to its growing consumption. We report here an eutylone intoxication involving a 38-year-old man and a 29-year-old woman in a chemsex context. A bag containing a white crystalline powder labelled as a research product was found in their vehicle. Nuclear magnetic resonance and liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analyses identified the powder as eutylone and confirmed purity superior to 99%. LC-HRMS data analysis using molecular networking allowed to propose new eutylone metabolites in blood samples in a graphical manner. We described 16 phase I (e.g. hydroxylated or demethylated) and phase II metabolites (glucuroconjugates and sulfoconjugates). The same metabolites were found both in male and female blood samples. Toxicological analyses measured eutylone concentration in blood samples at 1374 ng/mL and 1536 ng/mL for the man and the woman, respectively. A keto-reduced metabolite (m/z 238.144) was synthesized to permit its quantification at 67 ng/mL and 54 ng/mL in male and female blood samples, respectively. Overall, the identification of these metabolites will increase the knowledge of potential drug consumption markers and allow to implement mass spectrometry databases to better monitor future drug abuse or consumption.

In silico and in vitro metabolism studies of the new synthetic opiate AP-237 (bucinnazine) using bioinformatics tools.

The recent emergence of new synthetic opioids (NSOs) compounds in the illicit market is increasingly related to fatal cases. Identification and medical care of NSO intoxication cases are challenging, particularly due to high frequency of new products and extensive metabolism. As the study of NSO metabolism is crucial for the identification of these drugs in cases of intoxication, we aimed to investigate the metabolism of the piperazine NSO AP-237 (= bucinnazine). Two complementary approaches (in silico and in vitro) were used to identify putative AP-237 metabolites which could be used as consumption markers. In silico metabolism studies were realized by combining four open access softwares (MetaTrans, SyGMa, Glory X, Biotransformer 3.0). In vitro experiments were performed by incubating AP-237 (20 µM) in differentiated HepaRG cells during 0 h, 8 h, 24 h or 48 h. Cell supernatant were extracted and analyzed by liquid chromatography coupled to high-resolution mass spectrometry and data were reprocessed using three strategies (MetGem, GNPS or Compound Discoverer®). A total of 28 phase I and six phase II metabolites was predicted in silico. Molecular networking identified seven putative phase I metabolites (m/z 203.154, m/z 247.180, m/z 271.180, two m/z 289.191 isomers, m/z 305.186, m/z 329.222), including four previously unknown metabolites. Overall, this cross-disciplinary approach with molecular networking on data acquired in vitro and in silico prediction enabled to propose relevant candidate as AP-237 consumption markers that could be added to mass spectrometry libraries to help diagnose intoxication.