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Methylxanthines positives in competition samples have challenged doping control laboratories and racing jurisdictions since methylxanthines are naturally occurring prohibited substances and often constituents of feed. For theobromine, an international threshold (renamed in International Residue Limit, IRL) of 2 µg/mL in urine has been established. On the basis of the data presented herein, a threshold or rather an IRL for theobromine in plasma of 0.3 µg/mL was proposed and was thereupon approved by the International Federation of Horseracing Authorities (IFHA). Official recommendations for reporting caffeine and theophylline are still lacking. The aim of the study was to investigate IRLs for theobromine in blood and for caffeine and theophylline in blood and urine. Therefore, a set of six administrations were carried out including both single i.v. and single oral administrations of caffeine, theobromine and theophylline. Plasma and urine concentrations were determined using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS). Applying the Toutain model approach an effective plasma concentration (EPC) of caffeine was estimated at 3.05 µg/mL, irrelevant concentrations in blood (IPC) and urine (IUC) approached 6 and 12 ng/mL, respectively. EPC of theobromine was calculated with 3.80 µg/mL, and irrelevant concentrations of theobromine were determined at 8 ng/mL in plasma and at 142 ng/mL in urine. Toutain modelling of the theophylline data produced an EPC, IPC, and IUC of 3.20 µg/mL, 6 ng/mL, and 75 ng/mL, respectively. The obtained irrelevant concentrations were used to postulate IRLs for theobromine in plasma and for caffeine and theophylline in plasma and urine. Copyright © 2016 John Wiley & Sons, Ltd.

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In horses, nickel is considered a micronutrient and feed supplements containing nickel are available; hence, follow-up studies are deemed warranted to consolidate potential future threshold levels concerning urine and blood nickel concentrations in horses using larger sets of samples for both matrices and to provide in-depth insights by conducting elimination studies with soluble Ni(2) (+) -salt species. Copyright © 2016 John Wiley & Sons, Ltd.

OBJECTIVE To evaluate the influence of respiratory tract disease (ie, recurrent airway obstruction [RAO]) and mode of inhalation on detectability of inhaled budesonide in equine plasma and urine samples. ANIMALS 16 horses (8 healthy control horses and 8 horses affected by RAO, as determined by results of clinical examination, blood gas analysis, bronchoscopy, and cytologic examination of bronchoalveolar lavage fluid). PROCEDURES 4 horses of each group inhaled budesonide (3 μg/kg) twice daily for 10 days while at rest, and the remaining 4 horses of each group inhaled budesonide during lunging exercise. Plasma and urine samples were obtained 4 to 96 hours after inhalation and evaluated for budesonide and, in urine samples, the metabolites 6β-hydroxybudesonide and 16α-hydroxyprednisolone. RESULTS Detected concentrations of budesonide were significantly higher at all time points for RAO-affected horses, compared with concentrations for the control horses. All samples of RAO-affected horses contained budesonide concentrations above the limit of detection at 96 hours after inhalation, whereas this was found for only 2 control horses. Detected concentrations of budesonide were higher, but not significantly so, at all time points in horses that inhaled budesonide during exercise, compared with concentrations for inhalation at rest. CONCLUSIONS AND CLINICAL RELEVANCE Results of this study indicated that the time interval between inhalation of a glucocorticoid and participation in sporting events should be increased when inhalation treatment is administered during exercise to horses affected by respiratory tract disease.

The anti-doping rules of national and international sport federations ban any use of tetrahydrogestrinone (THG) in human as well as in horse sports. Initiated by the THG doping scandals in human sports a method for the detection of 3-keto-4,9,11-triene steroids in horse blood and urine was developed. The method comprises the isolation of the analytes by a combination of solid phase and liquid-liquid extraction after hydrolysis and solvolysis of the steroid conjugates. The concentrations of THG in blood and urine samples were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A THG excretion study on horses was conducted to verify the method capability for the analysis of postadministration urine samples. In addition, blood samples were collected to allow for determination of the pharmacokinetics of THG in horses. Following the administration of a single oral dose of 25 microg THG per kg bodyweight to 10 horses, samples were collected at appropriate intervals. The plasma levels of THG reached maximal concentrations of 1.5-4.8 ng/mL. Twenty-four hours after the administration plasma levels returned to baseline. In urine, THG was detectable for 36 h. Urinary peak concentrations of total THG ranged from 16 to 206 ng/mL. For the 10 horses tested, the mean plasma clearance of THG was 2250 mL/h/kg and the plasma elimination half-life was 1.9 h.

Cannabidiol (CBD) products gain increasing popularity amongst animal owners and veterinarians as an alternative remedy for treatment of stress, inflammation or pain in horses. Whilst the use of cannabinoids is banned in equine sports, there is limited information available concerning CBD detection times in blood or urine. The aim of this study was to determine the pharmacokinetic properties of CBD following oral administration in the horse to assist doping control laboratories with interpreting CBD analytical results. Part 1: dose escalation study: Single oral administration of three escalating doses of CBD paste (0.2 mg/kg, n = 3 horses; 1 mg/kg, n = 3; 3 mg/kg, n = 5) with >7 days wash-out periods in between. Part 2: multiple dose study: oral administration of CBD paste (3 mg/kg, n = 6) twice daily for 15 days. Multiple blood and urine samples were collected daily throughout both studies. Following study part 2, blood and urine samples were collected for 2 weeks to observe the elimination phase. Concentrations of CBD, its metabolites and further cannabinoids were evaluated using gas-chromatography/tandem-mass-spectrometry. Pharmacokinetic parameters were assessed via two approaches: population pharmacokinetic analysis using a nonlinear mixed-effects model and non-compartmental analysis. AUC0–12 h and Cmax were tested for dose proportionality. During the elimination phase, the CBD steady-state urine to serum concentration ratio (Rss) was calculated. Oral CBD medication was well-tolerated in horses. Based on population pharmacokinetics, a three-compartment model with zero-order absorption most accurately described the pharmacokinetic properties of CBD. High volumes of distribution into peripheral compartments and high concentrations of 7-carboxy-CBD were observed in serum. Non-compartmental analysis identified a Cmax of 12.17 ± 2.08 ng/mL after single administration of CBD (dose: 3 mg/kg). AUC0–12 h showed dose proportionality, increase for Cmax leveled off at higher doses. Following multiple doses, the CBD terminal half-life was 161.29 ± 43.65 h in serum. Rss was 4.45 ± 1.04. CBD is extensively metabolized and shows high volumes of tissue distribution with a resulting extended elimination phase. Further investigation of the potential calming and anti-inflammatory effects of CBD are required to determine cut-off values for medication control using the calculated Rss.

In horses, the benzodiazepine diazepam (DIA) is used as sedative for pre‐medication or as an anxiolytic to facilitate horse examinations. As the sedative effects can also be abused for doping purposes, DIA is prohibited in equine sports. DIA is extensively metabolized to several active metabolites such as nordazepam, temazepam and oxazepam (OXA). For veterinarians, taking into account the detection times of DIA and its active metabolites is needed for minimizing the risk of an anti‐doping rule violation. Therefore, a pharmacokinetic study on 6 horses was conducted using a single intravenous (IV) dose of 0.2 mg/kg DIA Plasma and urine samples were collected at specified intervals until 16 and 26 days post‐administration, respectively. Samples were analysed by a sensitive liquid chromatography–electrospray ionization/tandem mass spectrometry method. DIA showed a triphasic elimination pattern in the horse. The mean plasma clearance of DIA was 5.9 ml/min/kg, and the plasma elimination half‐life in the terminal phase was 19.9 h. Applying the Toutain model approach, an effective plasma concentration of DIA was estimated at 24 ng/ml, and irrelevant plasma concentration (IPC) and irrelevant urine concentration (IUC) were computed to 0.047 and 0.1 ng/ml, respectively. The detection time according to the European Horserace Scientific Liaison Committee (EHSLC), that is the time for which observed DIA plasma concentrations of all investigated horses were below the IPC was 10 days. Using Monte Carlo Simulations, it was estimated that concentrations of DIA in plasma would fall below the IPC 18 days after the DIA administration for 90% of horses. However, in the present study, a single administration of DIA could be detected for 24 days in urine via the presence of OXA, its dominant metabolite.