A Comparative In Vitro Metabolic Study of Butonitazene Using Camel Liver and Fungus Cunninghamella elegans for Doping Control Applications

Rationale: The growing emergence of synthetic opioids in the nitazene subclass such as butonitazene poses a significant challenge for doping control and toxicological monitoring. Butonitazene is a highly potent synthetic opioid, structurally distinct from classical opioids; its potential misuse in competitive events such as horse and camel racing cannot be overlooked. The in vitro metabolism of butonitazene was studied using camel liver, and findings were compared with metabolic profiles obtained from Cunninghamella elegans. Methods: In vitro metabolic studies of butonitazene were conducted using homogenized camel liver and the fungus Cunninghamella elegans. A liquid chromatography–high resolution mass spectrometry method was used to identify and characterize the metabolites of butonitazene. Extracted metabolites were analyzed on a Thermo Fisher Orbitrap Exploris LC–MS system, renowned for its high resolution and accurate mass detection. Results: A total of 17 Phase I and one Phase II metabolites were identified. The Phase I metabolism primarily involved N-dealkylation, O-dealkylation, and hydroxylation, whereas the Phase II metabolite was formed through the sulfonation of a hydroxylated Phase I metabolite. These metabolites demonstrate substantial potential as biomarkers for the long-term detection of butonitazene in doping control analysis. Conclusion: The study utilized advanced high-resolution LC–MS techniques in identifying and characterizing the in vitro metabolites of butonitazene in camels. Given its high potency and its potential for misuse in competitive events, the identified metabolites provide a foundation for effective doping control measures, further enhancing regulatory frameworks designed to protect animal welfare and uphold the integrity of the sport.