In vivo biotransformation of 17 alpha-methyltestosterone in the horse revisited: identification of 17-hydroxymethyl metabolites in equine urine by capillary gas chromatography/mass spectrometry.

The research article investigates the in vivo biotransformation of 17 alpha-methyltestosterone in horses, leading to the identification of new metabolites in equine urine using capillary gas chromatography/mass spectrometry technique.

Understanding the Metabolism of 17 alpha-methyltestosterone

• According to the study, the in vivo phase I biotransformation of 17 alpha-methyltestosterone in horses results in a complex mixture of C(20)O(2), C(20)O(3), and C(20)O(4) metabolites which are excreted out through the urine as phase II conjugates of glucuronide and sulphates.
• The primary metabolic pathways involve the reduction of the A-ring, epimerisation at C-17, and predominant oxidations at C-6 and C-16. These metabolic processes reflect how the pathway of 17 alpha-methyltestosterone is altered and metabolised in the horse’s body.

Identification of phase I Metabolites

• Earlier phase I metabolites were identified by GC/EI + MS mainly from the fragmentation patterns of their MO-TMS, enol-TMS, or TMS ether derivatives. This system yields data on the structure and type of the metabolites produced.
• The study administered 17 alpha-methyltestosterone orally to two castrated thoroughbred male horses and selectively hydrolysed the glucuronic acid conjugates excreted in post-administration urine samples using E. coli beta-glucuronidase enzymes.
• Post the enzymatic deconjugation, the metabolites and steroid aglycones were isolated from the urine and analysed by GC/EI + MS.

Identification of New Metabolites

• The research discovered new metabolites, presenting a loss of 103 mass units from the molecular ions with losses of trimethylsilanol or methoxy groups and an absence of the classical D-ring fragment ion.
• The mass spectral data identified these compounds as 17-hydroxymethyl metabolites. These were formed in vivo in horses by the oxidation of the 17-methyl moiety of 17 alpha-methyltestosterone.
• This significant finding underscores the viability of using GC/EI + MS to identify these new C(20)O(3) and C(20)O(4) 17-hydroxymethyl metabolites of 17 alpha-methyltestosterone in thoroughbred horse urine.