Detection of methandienone (methandrostenolone) and metabolites in horse urine by gas chromatography-mass spectrometry.

The research investigated how the drug methandienone transforms in horses. The main discovery was three unique metabolites of the drug, and a new method was proposed to detect methandienone administration in horses by tracking these metabolites.

Objective of the Research

The objective of this study was to understand how methandienone, a popular performance-enhancing drug, is metabolized in horses. This insight could provide an effective way to detect the use of this drug in racehorses, ensuring fairness and integrity in competitive events.

Methodology

• Methandienone was administered to a female horse, after which urine samples were collected for up to 96 hours.
• Rather than using enzymic hydrolysis, the urine samples were processed by a series of solid-liquid and liquid-liquid extractions eliminating the need for complex purification techniques.
• For analysis, the extracts were treated to undergo trimethylsilylation – a process that prepares the samples for gas chromatography-mass spectrometry.

Findings

• The researchers found not only the parent compound of methandienone and its C-17 epimer (a single compound that exists in two forms), but also three monohydroxylated metabolites which were named as: 6 beta-hydroxymethandienone (III), its C-17 epimer (IV) and 16 beta-hydroxymethandienone (V).
• Besides these findings, three isomers (molecules with the same chemical formula but different arrangement of atoms) of 6 beta,16-dihydroxymethandienone (VIa-c) were identified.
• Additional analysis suggested that the breakdown of 16 beta-hydroxymethandienone (V) in the horse produces 16 beta,17 beta-dihydroxy-17 alpha-methyl-5 beta-androst-1-en-3-one (VII), which on reduction of the isomers results in the corresponding 6 beta,16,17-trihydroxy-17-methyl-5 beta-androst-1-en-3-ones (VIIIa-c).

Implications

• These findings suggest a new methodology to detect the administration of methandienone in horses: by screening for the isomers of VI and VIII.
• This approach uses the selected-ion monitoring technique, which has shown to be a promising method to track the metabolites produced by the drug breakdown and, consequently, its use.