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

Abstract

e Detection of testosterone and/or its pro-drugs in the gelding is currently regulated by the application of an international threshold for urinary testosterone of 20 ng/mL. The use of steroid ratios may provide a useful supplementary approach to aid in differentiating between the administration of these steroids and unusual physiological conditions that may result in atypically high testosterone concentrations. In the current study, an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify testosterone (T) and epitestosterone (E). The method was used to analyze 200 post-race urine samples from geldings in order to generate the ratios for the reference population. Following statistical analysis of the data, an upper limit of 5 for T:E ratio in geldings is proposed. Samples collected from 15 geldings with atypical urinary testosterone concentrations (>15 ng/mL) but otherwise normal steroid profile, had T:E ratios within those observed for the reference population. The applicability of an upper T:E ratio to detect an administration was demonstrated by the analysis of a selection of incurred samples from testosterone propionate, dehydroepiandrosterone (DHEA), and a mixture of DHEA and pregnenolone (Equi-Bolic®) administrations. These produced testosterone concentrations above the threshold of 20 ng/mL, but also T:E ratios above the proposed limit of 5. In conclusion, consideration of the T:E ratio appears to be a valuable complementary aid to evaluate whether an atypical testosterone concentration could be caused by a natural biological outlier as opposed to the administration of these steroids.

Monitoring dehydroepiandrosterone (DHEA) in the urine of Thoroughbred geldings for doping control purposes

“…Analysis was performed as previously described for testosterone and epitestosterone, but modified to include DHEA, using an LC–MS/MS system consisting of a Waters Acquity I‐Class UPLC coupled with a Waters Xevo TQ‐S triple quadrupole mass spectrometer in positive ESI mode at a capillary voltage of 2.0 kV, a source temperature of 150°C and a desolvation gas temperature at 550°C. Collision gas was argon at a flow rate of 0.15 mL/min.…”

Important considerations for the utilisation of methanolysis in steroid analysis

“…15 Derivatisation was performed by addition of 30 μL of MSTFA:ammonium iodide: ethanethiol and incubation at 80°C for an hour. 15 Derivatisation was performed by addition of 30 μL of MSTFA:ammonium iodide: ethanethiol and incubation at 80°C for an hour.…”

“…15 Eluents were dried and then reconstituted with 50 μL of methanol and 50 μL of purified water prior to transferring to LC vials for analysis. Either 0.5 mL of methanolysis solvent or methanol (n = 2 each) was added prior to incubation and solid-phase extraction (SPE) as previously described.…”

“…[14][15][16][17] This paper describes deuterium-hydrogen exchange observed for a deuterium-labelled internal marker, D 9 -progesterone, when using methanolysis for deconjugation of sulphate and glucuronide moieties in a screening method for a number of AAS, oestrogens and progestogens in equine urine. Even so, due to good recoveries and ease of use, methanolysis is widely used in equine doping-control laboratories as a convenient alternative to the combination of solvolysis and enzyme hydrolysis.…”

Intelligence‐based anti‐doping from an equine biological passport

“…one‐quarter of the conventional dose), it is important for racing laboratories to detect T misuse at sub‐threshold levels using an EBP approach. Furthermore, recent cases of elevated urinary T levels in geldings determined by racing laboratories in Australia and the UK to be of endogenous origin have been assisted by assessment of T/E values . It is envisaged that longitudinal T/E profiling, along with other endogenous steroids such as dehydroepiandrosterone will form part of a steroidomic component of an EBP.…”