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

Abstract

The use of testosterone and its pro-drugs, such as dehydroepiandrosterone (DHEA), is currently regulated in horseracing by the application of international testosterone thresholds. However, additional steroidomic approaches, such as steroid ratios, to distinguish overall adrenal stimulation from drug administrations and an equine biological passport for longitudinal steroid profiling of individual animals could be advantageous in equine doping testing. Thus, DHEA concentrations and related ratios (testosterone [T] to DHEA and DHEA to epitestosterone [E]) were assessed in the reference population by quantitative analysis of 200 post-race gelding urine samples using liquid chromatography-tandem mass spectrometry. DHEA concentrations ranged between 0.9 and 136.6 ng/mL (mean 12.8 ng/mL), T:DHEA ratios between 0.06 and 1.85 (mean 0.43), and DHEA:E ratios between 0.21 and 13.56 (mean 2.20). Based on the reference population statistical upper limits of 5.4 for T:DHEA ratio and 48.1 for DHEA:E ratio are proposed with a risk of 1 in 10 000 for a normal outlier exceeding the value. Analysis of post-administration urine samples collected following administrations of DHEA, Equi-Bolic® (a mix of DHEA and pregnenolone) and testosterone propionate to geldings showed that the upper limit for T:DHEA ratio was exceeded following testosterone propionate administration and DHEA:E ratio following DHEA administrations and thus these ratios could be used as additional biomarkers when determining the cause of an atypical testosterone concentration. Additionally, DHEA concentrations and ratios can be used as a starting point to establish reference ranges for an equine biological passport.

In vivo Administration of Testosterone Propionate in Cattle Analyzed by High Performance Liquid Chromatography-Tandem Mass Spectrometry: An Enzymatic Hydrolysis Study and Drug Abuse Issues

“…However, these ratios were calculated when high βTE concentrations were obtained (around 15 ppb), which did not occur in any of the samples in this work. 14,16 As an outcome of this study, it can be seen that the T/E ratio could be useful for sample treatment classification in castrate cattle, since high values are suggestive of exogenous treatment, whereas low or non-existent values of βTE were detected in the untreated castrated animals. The decision limit for this analyte was equal to 0.23 μg mL -1 , which indicated that the endogenous concentrations should be lower than this value.…”

“…Urine samples of 200 geldings were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) in order to evaluate the T/E ratio in a random population not subject to drug treatment. 14 For 15 of the 200 analyzed samples the concentration of βTE exceeded the limit of 15 ng mL -1 (LOQ = 1.6 µg L -1 ), and the T/E ratios were lower than the proposed limit of 5 ng mL -1 . In the same study, two geldings (one 7-year-old and one 10-year-old) were also intramuscularly injected with testosterone propionate (TEP), and the T/E ratio exceeded the proposed limit for all of the collected samples.…”

“…The alpha epimer αTE was observed at greater intensities than βTE for all of the collected urine samples, which corroborates the literature, 15 although βTE was not detected in the vast majority of urine samples collected from the untreated steers, probably because of their castrate condition. The methods reported in the literature describe endogenous testosterone as dependent on the animal age and gender, 14,15,24 and an aim of this work was to establish that animal castration should also be evaluated when dealing with testosterone monitoring. Here, the calculation of the T/E ratio to assess a cut-off limit for a control group of animals was not possible, and most of the concentrations obtained for both isomers were lower than the validated LOQ (0.50 µg mL -1 ), which is equal or inferior to other reported limits found in the literature.…”

“…Here, the calculation of the T/E ratio to assess a cut-off limit for a control group of animals was not possible, and most of the concentrations obtained for both isomers were lower than the validated LOQ (0.50 µg mL -1 ), which is equal or inferior to other reported limits found in the literature. 14,15,24 However, it should be noted that the method validation and applicability were developed in the frame of the European Decision 657/2002/EC, 1 which recommends that any result above the CCα of the method should be reported for prohibited drugs. According to this regulation, the CCα is used to define the limit above which it can be concluded that a sample contains the analyte, with an error probability of α equal to 1% (probability of false noncompliant decision) for prohibited drugs.…”

“…The steroid 17α-testosterone (αTE) is the main metabolite of 17β-testosterone (βTE) in human and bovine urine and the βTE/αTE ratio (T/E ratio), is widely used in human doping analysis. 13,14 Exogenous administration of commercial testosterone formulations causes an increased excretion rate of urinary βTE and decreased excretion rate of αTE resulting in an overall increase in the T/E ratio. However, false-negative results should be carefully considered, since individual metabolic patterns may be correlated with abnormal T/E ratios.…”