Liquid chromatography electrospray ionization tandem mass spectrometry for the detection of mesocarb abuse in horse doping.

This research article describes a new method for detecting mesocarb abuse in horse racing by using a combination of liquid chromatography and mass spectrometry. The method detected mesocarb and seven of its metabolites in the urine of horses.

Methodology

• The researchers used gradient liquid chromatography and electrospray ionization tandem mass spectrometry in order to determine mesocarb abuse in the sport of horse racing.
• Mesocarb was administered orally to two horses at a dose of 50 µg/kg. As part of the research, they collected and analyzed urine samples up to 120 hours post administration.
• They executed the handling of hydrolyzed and conjugated urine fractions using liquid-liquid extraction, a common method in chemical analysis.

Identification and Detection

• They confirmed the identity of the parent drug and its metabolites using liquid chromatography, a technique for separating mixtures, combined with tandem mass spectrometry (MS/MS), a method used for identifying specific substances within a test sample.
• In the urine samples, they detected mesocarb and seven of its metabolites. The main metabolites they found were mono- and di-hydroxylated ones.

Formation Process of Metabolites

• From the differences they observed in the MS/MS spectra, they supposed that the hydroxylation of the phenylisopropyl moiety of mesocarb produced these metabolites. They also noted that the main process of its hydroxylation in humans occurred in the phenylcarbamoyl moiety, implying a notable difference between species.

Observation of Metabolites

• They found that the major metabolites were almost completely glucuroconjugated. This is a process that helps make substances more water-soluble for easier excretion.
• They also detected minor metabolites such as p-hydroxymesocarb and three di-hydroxylated metabolites, along with the parent mesocarb, in the urine’s free fraction.

Conclusion

• The research concluded that the two mono-and two di-hydroxylated metabolites, discovered through the employed method, can effectively control mesocarb abuse in horses. This shows the value of the technique for ensuring the integrity of competitive equestrian sports.