Performance assessment of an equine metabolomics model for screening a range of anabolic agents.

INTRODUCTION: Despite their ban, Anabolic Androgenic Steroids (AAS) are considered as the most important threat for equine doping purposes. In the context of controlling such practices in horse racing, metabolomics has emerged as a promising alternative strategy to study the effect of a substance on metabolism and to discover new relevant biomarkers of effect. Based on the monitoring of 4 metabolomics derived candidate biomarkers in urine, a prediction model to screen for testosterone esters abuse was previously developed. The present work focuses on assessing the robustness of the associated method and define its scope of application. MATERIALS AND METHODS: Several hundred urine samples were selected from 14 different horses of ethically approved administration studies involving various doping agents' (AAS, SARMS, ß-agonists, SAID, NSAID) (328 urine samples). In addition, 553 urine samples from untreated horses of doping control population were included in the study. Samples were characterized with the previously described LC-HRMS/MS method, with the objective of assessing both its biological and analytical robustness. RESULTS: The study concluded that the measurement of the 4 biomarkers involved in the model was fit for purpose. Further, the classification model confirmed its effectiveness in screening for testosterone esters use; and it demonstrated its ability to screen for the misuse of other anabolic agents, allowing the development of a global screening tool dedicated to this class of substances. Finally, the results were compared to a direct screening method targeting anabolic agents demonstrating complementary performances of traditional and omics approaches in the screening of anabolic agents in horses.

Pharmacokinetics of tiludronate in horses: A field population study.

BACKGROUND: Tiludronate is a bisphosphonate drug marketed to treat different bone conditions in horses. OBJECTIVES: The goal of this study was to measure the plasma concentrations of tiludronate in a population of race and sport horses under field conditions, and using pharmacokinetic population modelling, to estimate detection times for doping control. STUDY DESIGN: Prospective cohort. METHODS: This study was conducted under field conditions on 39 race or sport horses diagnosed with bone conditions based on a lameness examination and treated with tiludronate. Each horse received 1 mg/kg of tiludronate (Tildren® ) intravenously (i.v.). Blood samples (from 1 to 4 per horse with a total of 93 samples) were collected around 10, 20, 30, 40 and 50 days after tiludronate administration. Tiludronate was quantified by HPLC/ESI-MSn . Tiludronate concentrations were analysed using nonlinear mixed-effects modelling (population approach). Monte Carlo simulations were then used to compute a prediction interval to estimate the corresponding quantile of horses predicted to have concentrations below some potential screening limits. RESULTS: This study highlighted pharmacokinetic differences between healthy experimental horses and the population of horses being treated in the field as well as the effect of level of training on plasma tiludronate. Different detection times were computed corresponding to different possible screening limits. MAIN LIMITATIONS: The number of horses in each group was limited, and the specific disease being treated with tiludronate is unknown. CONCLUSIONS: This population pharmacokinetic study on tiludronate will enable racing and other sports authorities to provide a detection time reflecting field conditions for the medication control of tiludronate. More generally, our study design and the data modelling serve as an example of how to generate detection times directly from the target horse population rather than from experimental horses.

Mouldy feed: A possible explanation for the excretion of anabolic-androgenic steroids in horses.

To ensure fair competition and to protect the horse's welfare, horses have to compete on their own merits, without any unfair advantage that might follow the use of drugs. Therefore, regulatory authorities list all substances that are not allowed in competition, including most anabolic-androgenic steroids. As zero-tolerance is retained, the question arose whether the consumption of mouldy feed could lead to the excretion of steroids, due to the biotransformation of plant phytosterols to steroids. A rapid ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analytical method, previously validated according to AORC (Association of Official Racing Chemists) and EC (European Commission) guidelines, was used to measure steroids in different sample types. Multiple mouldy feed samples were tested for the presence of steroids. The effect of digestion was tested by in vitro simulation of the horse's hindgut in batch incubations. In most feed samples no steroids were detected, even when the products were mouldy. Mouldy corn however showed to contain up to 3.0 ± 0.4 µg/kg AED (4-androstenedione), the main testosterone precursor. This concentration increased when mouldy corn (with added phytosterols) was digested in vitro. An herbal phytosupplement also showed to contain α-testosterone. These results demonstrate that it is important to caution against the consumption of any feed or (herbal) supplement of which the detailed ingredients and quantitative analysis are unknown. The consumption of mouldy corn should especially be avoided, not only from a horse health and welfare point of view, but also to avoid possible inadvertent positive doping results. Copyright © 2016 John Wiley & Sons, Ltd.

In vitro simulation of the equine hindgut as a tool to study the influence of phytosterol consumption on the excretion of anabolic-androgenic steroids in horses.

Traditionally, steroids other than testosterone are considered to be synthetic, anabolic steroids. Nevertheless, in stallions, it has been shown that ß-Bol can originate from naturally present testosterone. Other precursors, including phytosterols from feed, have been put forward to explain the prevalence of low levels of steroids (including ß-Bol and ADD) in urine of mares and geldings. However, the possible biotransformation and identification of the precursors has thus far not been investigated in horses. To study the possible endogenous digestive transformation, in vitro simulations of the horse hindgut were set up, using fecal inocula obtained from eight different horses. The functionality of the in vitro model was confirmed by monitoring the formation of short-chain fatty acids and the consumption of amino acids and carbohydrates throughout the digestion process. In vitro digestion samples were analyzed with a validated UHPLC-MS/MS method. The addition of ß-Bol gave rise to the formation of ADD (androsta-1,4-diene-3,17-dione) or αT. Upon addition of ADD to the in vitro digestions, the transformation of ADD to ß-Bol was observed and this for all eight horses' inocula, in line with previously obtained in vivo results, again confirming the functionality of the in vitro model. The transformation ratio proved to be inoculum and thus horse dependent. The addition of pure phytosterols (50% ß-sitosterol) or phytosterol-rich herbal supplements on the other hand, did not induce the detection of ß-Bol, only low concentrations of AED, a testosterone precursor, could be found (0.1 ng/mL). As such, the digestive transformation of ADD could be linked to the detection of ß-Bol, and the consumption of phytosterols to low concentrations of AED, but there is no direct link between phytosterols and ß-Bol.

HPLC/ESI-MS(n) method for non-amino bisphosphonates: application to the detection of tiludronate in equine plasma.

Tiludronate is a non-nitrogen-containing biphosphonate drug approved in equine veterinary medicine for the treatment of navicular disease and bone sparvin in horse. Its hydrophilic properties and its strong affinity for the bone have made the control of its use quite difficult. After an initial step of method development in plasma and urine, due to a strong matrix effect and erratic detection in urine, the final method development was conducted in plasma. After addition of (3-trifluoromethylphenyl) thiomethylene biphosphonic acid as internal standard, automated sample preparation consisted of a filtration on a Nexus cartridge followed by a Solid Phase Extraction on an Oasis WAX cartridge with weak anion exchange properties. After methylation of the residue with trimethyl orthoacetate (TMOA), analysis was conducted by HPLC/ESI-MS(n) on a LTQ mass spectrometer. The method has been validated with a LOD and LOQ of respectively 1 and 2.5ng/mL. Using a weighting factor of 1/concentration(2), a linear model was suitable in the range of 2.5 up to 500ng/mL. Precision and accuracy data determined at two concentrations were satisfactory (i.e. less than 15%). Carryover would have been a problem but this has finally been fixed using the additional steps of washing during robotised SPE extraction and analysis on both the autosampler and the analytical column. The method was successfully employed for the first time to the quantification of tiludronate in plasma samples collected from horses treated with Tildren™ (Intravenous administration at the dose of 0.1mg/kg/day for 10 days).

Monitoring the endogenous steroid profile disruption in urine and blood upon nandrolone administration: An efficient and innovative strategy to screen for nandrolone abuse in entire male horses.

Nandrolone (17ß-hydroxy-4-estren-3-one) is amongst the most misused endogenous steroid hormones in entire male horses. The detection of such a substance is challenging with regard to its endogenous presence. The current international threshold level for nandrolone misuse is based on the urinary concentration ratio of 5α-estrane-3ß,17α-diol (EAD) to 5(10)-estrene-3ß,17α-diol (EED). This ratio, however, can be influenced by a number of factors due to existing intra- and inter-variability standing, respectively, for the variation occurring in endogenous steroids concentration levels in a single subject and the variation in those same concentration levels observed between different subjects. Targeting an efficient detection of nandrolone misuse in entire male horses, an analytical strategy was set up in order to profile a group of endogenous steroids in nandrolone-treated and non-treated equines. Experiment plasma and urine samples were steadily collected over more than three months from a stallion administered with nandrolone laurate (1 mg/kg). Control plasma and urine samples were collected monthly from seven non-treated stallions over a one-year period. A large panel of steroids of interest (n = 23) were extracted from equine urine and plasma samples using a C18 cartridge. Following a methanolysis step, liquid-liquid and solid-phase extractions purifications were performed before derivatization and analysis on gas chromatography-tandem mass spectrometry (GC-MS/MS) for quantification. Statistical processing of the collected data permitted to establish statistical models capable of discriminating control samples from those collected during the three months following administration. Furthermore, these statistical models succeeded in predicting the compliance status of additional samples collected from racing horses.