A validated UHPLC-MS/MS method to quantify low levels of anabolic-androgenic steroids naturally present in urine of untreated horses.

This study presents an improved method for detecting low levels of anabolic-androgenic steroids (AASs) in horse urine, which aids in anti-doping control in equestrian sports. This new method uses ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and has been validated against industry standards.

Objective and Methodology of the Study

• This study was aimed at increasing the understanding of the naturally occurring AAS in horse urine. This was motivated by the suspicion that some AASs, especially β-boldenone, may originate from within the horse’s body.
• To this end, the researchers developed and validated a new method for detecting these steroids, using ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).
• This method was validated based on guidelines provided by the Association of Official Racing Chemists (AORC) and the European Commission (EC), thus demonstrating its effectiveness.

Performance of the New Method

• The new method developed was able to detect very low levels of AASs (0.2 ng/mL). It exhibited good reproducibility (with the standard deviation percentage below 10%), high recovery (between 94.6 and 117.1%).
• The method also showed high selectivity and specificity, and a linear response was observed (confirmed with an R-squared value above 0.99 and lack-of-fit analysis) for all included AAS.

Testing the New Method on Samples

• The researchers used the method to screen the urine samples of 105 guaranteed untreated horses, including 47 geldings, 53 mares, and 5 stallions, which served as controls.
• They looked for β-boldenone and five other related natural steroids: androstadienedione (ADD), androstenedione (AED), alpha-testosterone (αT), beta-testosterone (βT), and progesterone (P).
• Progesterone, β-testosterone, and α-testosterone were found in over half the horses at low concentrations. Occasionally, low concentrations of AED, ADD, and boldenone were also detected.

Conclusion

• In conclusion, the researchers developed and validated a sensitive UHPLC-MS/MS method to detect and quantify low levels of anabolic-androgenic steroids naturally present in urine of untreated horses.
• This new method strengthens the toolset for anti-doping control in the horse racing and equestrian sports industry, helping to ensure fair competition and the welfare of the animals.