Metabolic profile of 25E-NBOH in human liver microsomes, rat urine, and fungus Cunninghamella elegans.
Magdaléna Vágnerová, Petr Palivec, Monika Mrňavá, Eliška Mizerová, Bronislav Jurásek, Silvie Rimpelová, Jan Sácký, Klára Šíchová, Čestmír Vejmola, Tomáš Páleníček, David Sýkora, Martin Kuchař
Journal of pharmaceutical and biomedical analysis August 1, 2026 Peer reviewed DOI: 10.1016/j.jpba.2026.117417 via PubMed
Summary
The metabolism of the recreational drug 25E-NBOH was investigated using three systems: human liver microsomes, rat urine, and the fungus Cunninghamella elegans. A total of 56 metabolites were detected, primarily through hydroxylation, O-demethylation, and N-debenzylation, followed by conjugation. Ten synthetic reference standards were used to confirm metabolite identities, and the known psychoactive substance 2C-E was confirmed as a metabolite. Three main biomarkers were identified. This work provides a comprehensive metabolic profile to aid future pharmacological and toxicological studies and clinical diagnosis of intoxication.
Study at a glance
| Design | metabolite profiling study |
|---|---|
| Population | human liver microsomes, Wistar rat urine, and Cunninghamella elegans fungus |
| Key finding | The metabolism of 25E-NBOH produces 56 metabolites across three systems, with primary pathways including hydroxylation, O-demethylation, and N-debenzylation, and three main biomarkers were identified. |
Abstract
N-benzylphenethylamines are a class of new psychoactive substances (NPS) that are increasingly being used as recreational drugs with a wide range of adverse effects, possibly even death. Currently, the appearance of new N-benzylphenethylamines far outweighs the studies of their metabolism. One of such compounds, 25E-NBOH, has been on the market for the last seven years but its pharmacological and toxicological effects have not yet been thoroughly reported. To provide a basis for such studies, we investigated 25E-NBOH metabolism in three different systems: human liver microsomes, Wistar rat urine, and Cunninghamella elegans fungus, which contains enzymes similar to those found in mammals and serves as an environmentally sustainable and ethically favourable alternative to animal-based metabolic models. Untargeted LC-HRMS/MS was used to detect phase I and phase II 25E-NBOH metabolites in all three systems. A total of 56 metabolites were annotated, many of which occurred in multiple isomeric forms. Despite metabolic differences between the systems, several abundant metabolites were found in all of them. The primary metabolic pathways detected were hydroxylation at various positions, O-demethylation, and N-debenzylation, followed by conjugation with glucuronic acid, sulphate, or glucose. To confirm the presence of metabolites, we synthesised and measured ten substances under the same LC-HRMS/MS conditions as the real samples. Seven of these were successfully matched to detected metabolites based on retention time and MS/MS spectra, enabling structural assignment and isomer distinction. Additionally, the identity of 2C-E, a known psychoactive substance, was confirmed as one of the metabolites using a commercial reference standard. Lastly, we report the structures of three main biomarkers, suggested by both this study and prior literature. This study provides the first comprehensive in vitro and in vivo metabolic profile of 25E-NBOH, identifying the structures of specific metabolites using in-house synthesised reference standards and proposing structures for the main biomarkers. These findings establish a solid foundation for future pharmacological and toxicological studies, supporting clinicians in the accurate diagnosis of intoxication cases.