Detection of efaproxiral (RSR13) and its metabolites in equine by liquid chromatography tandem mass spectrometry.

The study examines how efaproxiral (an experimental compound known for enhancing muscle oxygen uptake) is detected and eliminated in horses, using a technique called liquid chromatography tandem mass spectrometry. The researchers also identified the metabolites of efaproxiral in the horses’ urine after administration of the compound.

Methodology of the Research

• The research team used a 2.5g efaproxiral dose, administering it intravenously to the equine subjects.
• Following administration, both urine and plasma samples were collected over a 120-hour period. Efaproxiral was traceable up to 120 hours in urine and up to 78 hours in plasma samples.
• The highest concentration noted in the plasma was 42µg/ml at 5 minutes post-administration. Meanwhile, in the urine, the peak concentration was 2.8mg/ml and detected between 0-1 hour post-administration.

Identification and Detection Technique

• A validated liquid chromatography tandem mass spectrometry method was employed for the quantification of efaproxiral in equine plasma and urine. This technique pairs two different types of mass spectrometry to increase accuracy in separation, identification, and quantification of the chemical compounds.
• The limit of detection using this method is exceptionally low: 0.05 ng/ml in plasma and 0.1 ng/ml in urine.
• The researchers noted that the method was highly accurate, precise, and capable of good recovery, demonstrating its suitability for this type of research.

Discovery of Metabolites

• The manuscript also encompasses the systematic identification of the efaproxiral metabolites detected in post-administration equine urine samples.
• Mass spectra and enhanced product ion scans were used for identifying metabolites. The researchers utilized both positive and negative mode ionizations to achieve precise results.
• In addition to free efaproxiral, one phase 1 metabolite (a compound resulting from the body processing efaproxiral) and two phase 2 metabolites were also identified in the equine urine post drug-administration.

Significance of Findings

• These findings contribute to anti-doping efforts in sports involving equines. Efaproxiral’s potential as a performance-enhancing compound has led to prohibition by the World Anti-Doping Agency. Thus, effective detection techniques are of paramount importance in maintaining the integrity of competitive sport.