Results of this study support once-daily administration of meloxicam regardless of the feeding status of a horse and suggest a period of at least 3 days before urine concentrations of meloxicam reach concentrations that could be used in drug control programs.

Doping control is a main priority for regulatory bodies of both the horse racing industry and the equestrian sports. Urine and blood samples are screened for the presence of hundreds of forbidden substances including anabolic-androgenic steroids (AASs). Based on the suspected endogenous origin of some AASs, with β-boldenone as the most illicit candidate, this study aimed to improve the knowledge of the naturally present AAS in horse urine. To this extent, a novel ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated according to the Association of Official Racing Chemists (AORC) and European Commission (EC) guidelines, proving the power of this new method. Low limits of detection (0.2 ng/mL), good reproducibility (percentage of standard deviation (%RSD) < 10%), high recovery (94.6 to 117.1%), selectivity and specificity, and a linear response (confirmed with R(2) > 0.99 and lack-of-fit analysis) were obtained for all included AASs. With this method, urine samples of 105 guaranteed untreated horses (47 geldings, 53 mares, and 5 stallions serving as a control) were screened for β-boldenone and five related natural steroids: androstadienedione (ADD), androstenedione (AED), alpha-testosterone (αT), beta-testosterone (βT), and progesterone (P). Progesterone, β-testosterone, and α-testosterone were detected in more than half of the horses at low concentrations (<2 ng/mL). Occasionally, not only testosterone and progesterone but also low concentrations of AED, ADD, and boldenone (Bol) were found (0.5-5 ng/mL). Graphical Abstract A sensitive, new and fully validated UHPLC-MS/MS method has been developed that is able to quantify low levels of anabolic-androgenic steroids naturally present in urine of untreated horses (mares and geldings).

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Recombinant human erythropoietin (rHuEPO) is a 30-34 kDa glycoprotein banned by the racing authorities. For some years this molecule has been detected in race horses in USA and in Europe, and even in racing camels. Although direct methods to differentiate horse endogenous EPO and rHuEPO have been developed either by LC-MS/MS or by isoelectric focusing (IEF) with double-blotting, the short confirmation time of such prohibited hormone in plasma remains a problem for horseracing doping control laboratories. In order to improve the rHuEPOs confirmation process in horse plasma or urine in terms of reliability and delay, a small anti-EPO monolith membrane contained in a disposable column (anti-EPO monolith column) has been successfully used and validated (n = 10). This new sample preparation, combined with LC-FAIMS-MS/MS, has been performed on plasma and urine samples collected from one horse which received an Eprex® treatment during six consecutive days and a second one with a single injection of Aranesp®. This inventive technology allowed the possibility to confirm the presence of rHuEPO within one day with a limit of detection validated for both urine and plasma at 250 pg mL(-1) by means of a disposable, ready to use immunoaffinity column. The lower limit of detection (LLOD) obtained for each matrix was 100 pg mL(-1). These results provide an important improvement for rHuEPO doping control in horseracing especially the possibility to confirm these banned molecules in both matrices, urine and plasma, with a confidence of two specific target peptides.

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Growth hormones are proteins produced by the anterior pituitary gland responsible for bone and tissue growth through their effects on carbohydrates, lipids and proteins metabolisms. Despite strict regulations banning the use of recombinant equine growth hormone, this substance is suspected to be misused to improve the horse physical performances. In order to check whether the regulation is fulfilled or not, controls are organized and a new analytical screening tool potentially able to detect such abuse was investigated in this paper. An untargeted metabolomics approach, based on liquid chromatography coupled to high resolution mass spectrometry, was developed and applied to characterize and compare horse urinary and plasmatic metabolic profiles upon reGH administrations. After minimal sample preparation, biological fluids were analyzed by LC-ESI(±)-Q-TOF. Data processing was performed by XCMS software and multivariate data analysis applied to the generated data set allowed building OPLS models to discriminate control versus treated populations. Results demonstrated significant metabolic modifications consecutively to the reGH treatment. A comparative study between urinary and plasmatic signatures was performed to evaluate the resulting metabolomic models and to asses their respective interests in the scope of real application for screening reGH administration.