Suzetrigine in Equestrian Sports: Optimized Extraction and LC-HRMS Detection Strategies.

Rationale: Suzetrigine, a recently approved Na1.8 sodium channel blocker, shows strong potential in the treatment of neurological, psychiatric, and pain-related conditions. Its peripheral selectivity enables effective pain management while avoiding central nervous system complications and addiction risks linked to opioid use. Following FDA approval in January 2025, concerns have emerged regarding its possible misuse for performance enhancement in sports, highlighting the need for reliable detection tools in doping control.

Methods: An analytical procedure was designed and validated to detect suzetrigine in equine urine and plasma. Different chromatographic columns, mobile phase compositions, and ionization modes were systematically tested. Extraction efficiency was evaluated using solid-phase extraction (SPE), liquid-liquid extraction (LLE), and dilute-and-inject techniques to identify the most suitable approach for sensitivity and recovery.

Results: Of the various chromatographic columns evaluated, the AQUA C18 column (3.0 μm, 4.6 × 150 mm) exhibited the best separation performance. Among the extraction techniques tested, LLE optimized at specific pH levels and solvent conditions consistently achieved superior recovery rates and lower limits of detection. The fully validated procedure, utilizing high-resolution mass spectrometry, demonstrated excellent sensitivity, reproducibility, and robustness, making it suitable for routine detection of suzetrigine in biological matrices.

Conclusions: The validated approach offers a reliable tool for doping control laboratories to detect suzetrigine in equestrian samples. Beyond equine testing, this protocol provides a methodological framework that can be extended to broader anti-doping programs, supporting the monitoring of emerging substances with misuse potential in sports.