Analysis of exogenous nandrolone metabolite in horse urine by gas chromatography/combustion/carbon isotope ratio mass spectrometry.

This study focuses on developing a method to differentiate steroid hormones that naturally occur in racehorses from those introduced externally, particularly nandrolone (NAD), which has been involved in horse doping practices. The technique applied is gas chromatography/combustion/carbon isotope ratio mass spectrometry (GC/C/IRMS). The results of the study reveal the ability of GC/C/IRMS for detecting exogenous nandrolone metabolite in horse urine, which can act as a supplement to the existing doping control procedures.

The Issue with Nandrolone Doping in Racehorses

• A major challenge lies in the doping control of nandrolone (NAD), an endogenous steroid hormone, in male horses. Detecting its presence is not adequate proof of doping as it is already naturally present in horses.
• The existing control is based on the concentration threshold of NAD metabolites, specifically the ratio of 5alpha-estran-3beta,17alpha-diol (ETA) to 5(10)-estren-3beta,17alpha-diol (ETE). The ETA/ETE ratio of 1/1 as per the International Federation of Horseracing Authorities guidelines signals possible doping.
• However, this threshold might not be applicable for horses with complex metabolic disorders.

The Proposed Solution and Methodology

• To address the issue, this study set out to create an analytical method that would distinguish between naturally occurring steroids and externally introduced ones after administering NAD, using gas chromatography/combustion/carbon isotope ratio mass spectrometry (GC/C/IRMS).
• Urine was sourced from both NAD-administered horses and horses without NAD administration. A 10 mL urine sample underwent processes like hydrolyzation, liquid-liquid extraction, and solid-phase extraction.
• Following this, the residue from the purified extracts was then derivatized by acetylation.
• The (13)C/(12)C ratio (delta(13)C) was measured via GC/C/IRMS. The delta(13)C values of ETA were determined for both categories of horses.

Findings and Conclusion

• The outcomes showed significantly different delta(13)C values of ETA for NAD-administered horses -32.20+/-0.35 per thousand, and authentic (non-NAD administered) horses -27.85+/-0.75 per thousand.
• The detection limit for ETA in this GC/C/IRMS analysis was approximately 25 ng/mL.
• This suggests that the (13)C/(12)C ratio measurement through GCC/IRMS can discern exogenous ETA (arising from NAD administration) from endogenous ETA, making GC/C/IRMS a valuable tool for enhancing the ETA/ETE ratio method.