Doping control analysis of four JWH-250 metabolites in equine urine by liquid chromatography-tandem mass spectrometry.

This journal article presents a validated method for analysing the presence of a synthetic cannabinoid, JWH-250, in equine urine to enforce doping control in horse racing. The process involves treating urine samples and using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for detection.

Objective of the Research

• This study focuses on developing and validating a method for doping control analysis that can identify four metabolites of JWH-250 in equine urine. JWH-250 is a synthetic cannabinoid that is prohibited in horse racing due to its performance-enhancing properties.

Methodology

• Equine urine samples were treated with acetonitrile and the analytes were concentrated by evaporation. This was done so that the LC-MS/MS (liquid chromatography-tandem mass spectrometry) analysis could be more precise and effective.
• The researchers used a specific chromatographic column (Phenomenex Lux 3 µm AMP column) for separating the substances in the test samples.
• A triple quadrupole mass spectrometer was used in positive electrospray ionization mode, together with multiple reaction monitoring (MRM), for detecting the analytes.

Results

• The study established the limits of the detection, quantification and confirmation for the four JWH-250 metabolites at 25 pg/mL, 50 pg/mL, and 50 pg/mL respectively with a linear dynamic range from 50-10000 pg/mL.
• Through enzymatic hydrolysis, it was indicated that three of the metabolites (JWH-250 4-OH-pentyl, JWH-250 5-OH-pentyl, and JWH-250 5-OH indole) are highly conjugated while JWH-250 N-pentanoic acid is not conjugated.
• The presence of the metabolites in the samples was confirmed using relative retention time and product ion intensity ratios as the criteria.

Conclusion

• The method was applied to post-race urine samples from horses suspected to be exposed to JWH-250, and it successfully confirmed the presence of all four JWH-250 metabolites.
• This validation and successful application demonstrate that this LC-MS/MS method can be used for doping control analysis in equine sports involving JWH-250.