Comparison of authentic urine N-ethylpentedrone metabolites to predicted in silico and in vitro human hepatocyte metabolism

Gloria Daziani, Omayema Taoussi, Diletta Berardinelli, Giulia Bambagiotti, Marilyn A. Huestis, Francesco Paolo Busardò et al. · 2025 · 8 citationsRead the paper

Synthetic cathinones are the second most common class of substances belonging to the category of new psychoactive substances (NPS). N-ethylpentedrone (NEPD) self-administration was reported, making it important to elucidate its metabolic pathway to improve its detection. NEPD is often taken together with other substances; confidently identifying its intake is important for accurate toxicological analysis and understanding its toxicity. NEPD metabolism was studied following incubation with human hepatocytes and analysis by liquid chromatography-high-resolution mass spectrometry paired with Compound Discoverer software (Thermo Fisher Scientific). Prediction of possible metabolites was performed with the online software GLORYx. In vivo NEPD metabolism was evaluated by the analysis of two authentic human urine samples collected after NEPD ingestion. Twenty metabolites were identified by in vitro hepatocyte incubation, with eighteen identified in the human urine samples. The most abundant reaction was N-deethylation (in positive ionization mode), followed by carbonyl reduction, and a combination of these processes. A pathway previously identified for other synthetic cathinone metabolism, 3-CMC, 4-CMC, and 4-BMC, consisting of the combination of carbonyl reduction, oxidative deamination, and glucuronidation was identified, with the important addition of N-deethylation. These metabolites were identified in urine through additional analyses conducted using negative ionization and retrospectively also in hepatocytes, as they would not have been detectable when analysing the sample with positive ionization alone. It was therefore possible to identify probable markers of NEPD intake and confirm the relevance of the newly proposed metabolic pathway.

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