# In vitro toxicokinetics and metabolic profiling of methoxycathinones and methylthiocathinones using human liver systems and hyphenated mass spectrometry

## TL;DR

This study examines how certain synthetic cathinones are processed in the human liver and identifies potential metabolites for drug screening.

## Contribution

The study provides new insights into the toxicokinetics and metabolism of methoxycathinones and methylthiocathinones using human liver systems.

## Key findings

- Methoxycathinones showed lower plasma protein binding compared to methylthiocathinones.
- Phase I and II metabolic reactions were identified, including demethylation, hydroxylation, and conjugation.
- A total of 42 and 45 metabolites were identified in pooled human liver S9 and HepaRG systems, respectively.

## Abstract

Ring-substituted synthetic cathinones represent a major subgroup within new psychoactive substances. This study investigated the in vitro toxicokinetics of the three 4-methoxy-substituted representatives 4MeO-NE-BP (4’-methoxy-N-ethylbutyrophenone), 4MeO-αP-BP (4’-methoxy-α-pyrrolidinobutyrophenone), and 4MeO-αP-VP (4’-methoxy-α-pyrrolidinovalerophenone) and the three related novel 4-methylthio analogs 4MeS-NE-BP (4’-methylthio-N-ethylbutyrophenone), 4MeS-αP-BP (4’-methylthio-α-pyrrolidinobutyrophenone), and 4MeS-αMor-PrP (4’-methylthio-2-morpholinopropiophenone). This included plasma protein binding (PPB), phase I and phase II metabolism in pooled human liver S9 fraction (pHLS9) and HepaRG cells, and monooxygenases activity. Methoxycathinones exhibited lower PPB (~ 40–60%) compared to methylthiocathinones (~ 85%). Predominant phase I metabolic reactions included O-/S-demethylation and hydroxylation, with additional transformations such as N-dealkylation, N-oxidation, and oxo reduction. Phase II conjugation reactions, such as glucuronidation and sulfation, were observed post-demethylation. Overall, 42 and 45 metabolites were identified in pHLS9 and HepaRG systems, respectively, with metabolite number increasing alongside alkyl chain length and heterocyclic substitution. All compounds were substrates for multiple monooxygenases, suggesting a low risk for drug–drug interactions. Based on metabolic stability and abundance, parent compounds and O-/S-desmethyl and hydroxylated metabolites might be proposed as urinary screening targets in clinical and forensic toxicology, as well as doping control settings.

The online version contains supplementary material available at 10.1007/s00204-025-04205-x.