Characterization of urinary metabolites of testosterone, methyltestosterone, mibolerone and boldenone in greyhound dogs

Abstract

Androgenic steroids are used in female greyhound dogs to prevent the onset of estrus; moreover, these steroids also have potent anabolic activity. As anabolic steroids increase muscle mass and aggression in animals, the excessive use of these agents in racing greyhounds gives an unfair performance advantage to treated dogs. The biotransformation of most anabolic steroids has not been determined in greyhound dogs. The objective of the present study was to identify the urinary metabolites of testosterone, methyltestosterone, mibolerone, and boldenone in greyhound dogs. These steroids were administered orally (1 mg/kg) to either male or female greyhound dogs and urine samples were collected pre-administration and at 2, 4, 8, 12, 24, 72, and 96 h post-administration. Urine extracts were analyzed by high-performance liquid chromatography/mass spectrometry (HPLC/MS) to identify major metabolites and to determine their urinary excretion profiles. Major urinary metabolites, primarily glucuronide, conjugated and free, were detected for the selected steroids. Sulfate conjugation did not appear to be a major pathway for steroid metabolism and excretion in the greyhound dog. Phase I biotransformation was also evaluated using greyhound dog liver microsomes from untreated dogs. The identification of several in vivo steroid metabolites generated in this study will be useful in detecting these steroids in urine samples submitted for drug screening.

Metabolism of stanozolol: Chemical synthesis and identification of a major canine urinary metabolite by liquid chromatography–electrospray ionisation ion trap mass spectrometry

“…In addition to the evidence derived from MS 2 fragmentation patterns, hydroxylation of the steroidal C6 position has previously been confirmed by comparison with reference materials for the canine in vitro biotransformation of both testosterone and 17␣-methyltestosterone [5,6]. Furthermore 6-hydroxystanozolol has been proposed but not confirmed as a minor human metabolite of stanozolol on the basis of triple quadrupole MS 2 fragmentation patterns [15].…”

“…In the context of sporting pursuits, the metabolism of anabolic-androgenic steroids in humans [1,2] and horses [3] has been the subject of numerous detailed studies, leading to the development of a range of reference materials and robust drug screens. However, much less is known about the canine metabolism of this class of drugs [4][5][6][7][8][9].…”

Metabolism of methyltestosterone in the greyhound

“…Williams et al . studied both the in vivo 13 and the in vitro 14 metabolism of methyltestosterone in the dog using liquid chromatography/mass spectrometry (LC/MS). However, due to the limitations of this analytical technique for steroid metabolite detection, only metabolites containing a keto function were observed, since fully reduced metabolites tend to show a poor response under electrospray ionisation conditions 15, 16.…”

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

“…The in vivo biotransformation and detection of anabolic steroids in the horse have been the subject of a number of studies from this laboratory1–11and from other laboratories 12–15. Following enzymatic hydrolysis of the glucuronides and chemical solvolysis of the sulfoconjugates,1,4,8 the phase I aglycones are isolated from urine by liquid‐liquid or solid‐phase extraction (SPE).…”