Application of testosterone to epitestosterone ratio to horse urine – a complementary approach to detect the administrations of testosterone and its pro-drugs in Thoroughbred geldings.

The researchers developed a technique to detect unnatural levels of testosterone and its pro-drugs in racing horses by using a ratio of testosterone to epitestosterone, facilitating differentiation between natural biological outliers and the doping of horses.

Technique Development

• The study first developed a method to quantify testosterone (T) and epitestosterone (E) in horse urine samples, using ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS).
• This technique was tested on 200 post-race urine samples from geldings to create reference T:E ratios.
• Statistical analysis of this data generated an upper limit of 5 for the T:E ratio in geldings.

Application Test

• The team then applied their method to urine samples collected from 15 geldings with higher than typical levels of urinary testosterone (>15 ng/mL) but otherwise typical steroid profiles.
• These yielded T:E ratios within the range observed for the control population, suggesting that the elevated testosterone levels were within normal physiological bounds rather than a result of testosterone administration.

Test of Additional Steroids

• Finally, the applicability of the method to detect steroid administration was tested using urine samples from horses given testosterone propionate, dehydroepiandrosterone (DHEA), and a mixture of DHEA and pregnenolone (Equi-Bolic®).
• Urine from these horses showed both testosterone concentrations above the existing threshold (20 ng/mL) and T:E ratios above the newly proposed limit of 5, suggesting steroid administration.

Conclusion

• The researchers conclude that examining the T:E ratio in horse urine samples could be an effective supplementary technique to determine whether elevated testosterone levels are due to natural variability or the administration of testosterone or its pro-drugs.