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

Overview

• This study developed and validated a sensitive and reliable method to detect the drug suzetrigine in horse urine and plasma, addressing concerns about its potential misuse as a performance-enhancing substance in equestrian sports.

Introduction to Suzetrigine

• Suzetrigine is a sodium channel blocker (Na1.8) recently approved by the FDA in January 2025.
• It has therapeutic potential for neurological, psychiatric, and pain-related conditions due to its peripheral selectivity.
• This selectivity allows effective pain management without the central nervous system side effects or addiction risks typical of opioids.
• Given these benefits, concerns arose about its potential misuse as a doping agent to enhance athletic performance in equestrian sports.

Research Objectives

• To create a robust analytical method capable of sensitively detecting suzetrigine in equine biological samples (urine and plasma).
• To optimize extraction and detection strategies to maximize recovery and sensitivity.
• To validate the developed method for potential routine use in doping control laboratories.

Methodology

• Sample Matrices: Equine urine and plasma.
• Chromatographic Testing:
  • Multiple chromatographic columns were compared for suzetrigine separation performance.
  • The AQUA C18 column (3.0 μm particle size; 4.6 × 150 mm dimension) was identified as optimal.
• Extraction Techniques:
  • Solid-Phase Extraction (SPE)
  • Liquid-Liquid Extraction (LLE)
  • Dilute-and-inject technique
  • Systematic comparisons were performed to evaluate recovery efficiency and sensitivity.
  • LLE was optimized by adjusting pH and solvent conditions to maximize extraction efficiency.
• Detection:
  • High-Resolution Mass Spectrometry (HRMS) was used for detection.
  • Both ionization modes and mobile phase compositions were tested for optimal sensitivity.

Results

• The AQUA C18 chromatographic column demonstrated superior separation quality compared to other columns tested.
• Liquid-liquid extraction, when optimized for specific pH and solvent conditions, consistently produced the highest recovery rates.
• The optimized LLE method provided the lowest limits of detection, enabling very sensitive detection of suzetrigine.
• The finalized analytical method showed excellent reproducibility and robustness upon validation.
• This method is suitable for routine implementation in equine doping control laboratories.

Conclusions and Implications

• This validated method offers doping control authorities a reliable tool for detecting suzetrigine misuse in equestrian sports.
• The approach provides a scalable framework that can be adapted to monitor other emerging substances with abuse potential in various sports disciplines.
• The study highlights the importance of method optimization, especially in extraction and chromatography, for developing sensitive and reproducible anti-doping assays.
• Overall, this work supports broader anti-doping efforts by improving detection capabilities for newly approved therapeutic agents that may be misused.