Development of a gas chromatographic-mass spectrometric method using multiple analytes for the confirmatory analysis of anabolic steroid residues in horse urine. II. Detection of administration of 19-nortestosterone phenylpropionate to equine male castrates and fillies.

A study has developed a method to confirm the use of anabolic steroids, particularly 19-nortestosterone, in horses. Specific metabolites are identified by a process of extraction, separation, hydrolysis, purification, and gas chromatographic-mass spectrometric analysis.

Research Objective and Importance

• The aim of this research was to create a process that could confirm the administration of anabolic drugs to horse athletes, specifically the esters of 19-nortestosterone.
• This is crucial as these performance-enhancing drugs can give an unfair advantage in competitions and may have adverse effects on the horse’s health. The reliable detection of such substances is therefore essential for ensuring equity and animal welfare.

Method Development

• The method was based on the use of multiple analytes, chemicals used to analyze other substances.
• Having administered the anabolic preparations, urinary conjugates were extracted using a solid-phase extraction technique.
• The conjugate groups were separated before a process called hydrolysis was used to identify specific metabolites, compounds involved in metabolism, associated with either glucuronic acid or sulfate.

Chromatographic and Spectrometric Analysis

• After the hydrolysis process, the free neutral steroids were purified on thin-layer chromatography, a technique used to separate non-volatile mixtures.
• The method then utilized derivatization, a technique that chemically modifies a compound, and a gas chromatographic-mass spectrometric analysis for further examination.
• The primary metabolites, including 19-nortestosterone and two isomers of estrane-3,17-diol, were found in the sulfate fraction. Estrane-3,17 alpha-diol was discovered in the glucuronic acid fraction.

Detectable Period

• An essential finding of this study is that these metabolites could be confirmed for 17-18 days after the administration of Nandrolin, a particular type of 19-nortestosterone phenylpropionate.
• This gives regulators and stakeholders a detection window for ensuring fairness in competitions and the health of the horse athletes.