LC-HRMS-Based Metabolic Profiling of the REV-ERB Agonist SR9009 in Camel Liver Homogenate and Cunninghamella elegans
Ansar Babu Palathinkal1,2, S Farook Basha2, Saraswathy Laya3
1Camel Forensic Laboratory, Central Veterinary Research Laboratory, Dubai, UAE.
Rationale:
SR9009 is a synthetic REV-ERB agonist with potential performance-enhancing properties and is included on the World Anti-Doping Agency (WADA) Prohibited List for human sport. Its potential misuse has also raised concerns in animal sports, particularly equine and camel racing. Although SR9009 metabolism has been investigated in human and equine models, its metabolic fate in racing camels (Camelus dromedarius) remains unclear. Characterizing its biotransformation pathways and identifying suitable biomarkers are essential for developing reliable anti-doping screening strategies for camel racing.
Methods:
An in vitro metabolic study was conducted using camel liver homogenate and the fungal model Cunninghamella elegans. Metabolites were characterized using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Putative structural elucidation was achieved through accurate mass measurements of precursor and product ions. Diagnostic fragmentation pathways were established using collision-induced dissociation (CID) tandem mass spectrometry (MS/MS), enabling the systematic identification of metabolic modifications based on characteristic fragment ions and neutral losses.
Results:
Seventeen Phase I metabolites of SR9009 were identified. Major biotransformation pathways included N-dealkylation, mono-hydroxylation, di-hydroxylation, and combinations thereof. The N-dealkylated metabolite was the predominant metabolite detected in both camel liver homogenate and Cunninghamella elegans, indicating its suitability as a primary analytical target for anti-doping screening. No Phase II metabolites were detected under the experimental conditions employed. Putative structural assignments were supported by characteristic high-resolution tandem mass spectral fragmentation patterns.
Conclusions:
This study provides the first comprehensive metabolic profile of SR9009 in a camel-specific in vitro model. The identification of 17 Phase I metabolites, particularly the predominant N-dealkylated derivative, provides valuable analytical targets for doping control. These findings establish a metabolic foundation for future in vivo studies to determine pharmacokinetics and detection windows in racing camels.
