Testosterone Administration to Mares: Criteria for Detection of Testosterone Abuse by Analysis of Metabolites in Plasma and Urine

Abstract

A pharmacological dose of a long-acting testosterone ester, testosterone hexahydrobenzoate, was administered intramuscularly to two mares. The time course for some characteristic metabolites in blood and urine was then studied using an analytical method based on gas chromatography-mass spectrometry associated with stable isotope dilution. Among the plasma analytes, testosterone glucuronide was found to be the most adequate indicator for the monitoring of exogenous testosterone up to 2 weeks postadministration if a threshold value of 200 ng/L was applied. In urine, the simultaneous measurement of the concentrations of testosterone sulfate (TS) and epitestosterone sulfate (ES) allowed the calculation of the concentration ratio, TS/ES, which was independent of urine flow and which offered the possibility of detecting testosterone misuse 20 to 30 days after dosing if a tentative threshold value of 8 was adopted. In addition to this ratio, particularly when the TS/ES ratio was close to the cutoff point, it seemed advisable to take into account the concentrations of 5 alpha-androstane-3 beta, 17 alpha-diol (glucuronide) and its 17 beta-isomer (sulfate), which should not exceed 50 micrograms/L.

Application of testosterone to epitestosterone ratio to horse urine – a complementary approach to detect the administrations of testosterone and its pro‐drugs in Thoroughbred geldings

“…Product ion spectrum of testosterone‐MO (17β‐OH) and epitestosterone‐MO (17α‐OH) at collision energy of 30 V. Structures of the fragment ions consistent with those previously suggested for oxime derivatives elsewhere …”

“…Epitestosterone concentrations were not increased following the administration of testosterone propionate, although a small increase in epitestosterone concentrations was observed following dosing with DHEA and Equi‐Bolic®. This suggests that the exogenous testosterone is not converted to epitestosterone, which is supported by the previous studies following the administrations of 14 C‐testosterone and testosterone esters . Exogenous DHEA and androstenedione have been previously shown to convert to epitestosterone but this occurs to a lesser extent than conversion to testosterone.…”

“…Furthermore, a T:E ratio of 12 was adopted as a threshold for the detection of testosterone in mares/fillies . This threshold was based on data collected from the analysis of 294 post‐race urine samples and also following the IM dose of 1000 mg of testosterone hexahydrobenzoate . The current international concentration based threshold of 55 ng/mL in fillies replaced the T:E ratio of 12 in 2000 following a study of 1531 animals in six international laboratories…”

First mass spectrometric detection of boldenone in horse mane samples

“…Nowadays, abuse of endogenous or exogenous anabolic steroids for doping purposes is one of the most serious issues. Major steroids can be detected and quantified in horse urine [6]. However, due to the large detection time window generally available from hair analysis, it is necessary to evaluate the interest of this medium for their detection.…”

Important considerations for the utilisation of methanolysis in steroid analysis

“…9 The details of steroid analysis methodologies used by individual laboratories are not regulated in equine sports and veterinary drug residue analysis in the same way as for human sports by WADA. [10][11][12] Enzyme hydrolysis is generally performed using β-glucuronidase extracted from Escherichia coli or Helix pomatia. In the horse, the phase II metabolism of AAS is strongly affected by the stereochemistry of the steroids.…”