Doping control in horses: housing conditions and oral recycling of flunixin by ingestion of contaminated straw

Nonsteroid anti-inflammatory drugs (NSAID) such as flunixin, phenylbutazone and naproxen are prohibited substances in competition horses, and they are subjected to regular doping controls. The aforementioned NSAIDs are also reported to be major contaminants present in the horse environment (Barker, 2008). Norgren et al. (2000) and Wennerlund et al. (2000) reported that untreated horses were excreting flunixin or naproxen for several days when housed in boxes previously used for horses treated with these drugs. Russell and Maynard (2000) have pointed out that contamination of the horses environment with isoxsuprine can lead to the presence of the drug up to 10 weeks after the end of treatment. An experimental study with dipyrone and chlorpheniramine (Duluard et al., 2006) has also drawn attention to the need to control the bedding conditions for horses, to avoid selfor cross-contamination. Spurious urinary excretion profiles in horses, owing to bedding contamination and drug recycling, were also observed with meclofenamic acid administered intravenously (IV) (Popot et al., 2007). We report here on the flunixin excretion profiles in urine collected from 17 horses subjected to different housing conditions with respect to bedding and management. Flunixin is a NSAID marketed for equine use, either as an injectable at the concentration of 50 mg ⁄ mL or as an oral preparation at the concentration of 0.5 g ⁄ 10 g (Finadyne ; Intervet ⁄ Schering-Plough Animal Health, Beaucouze, France), and for which the pharmacokinetics and metabolism have been intensively studied (Jaussaud et al., 1987; Johansson & Anler, 1988; Soma et al., 1988; Toutain et al., 1994; Lee et al., 2004; Pellegrini-Masini et al., 2004). Flunixin is extensively eliminated by urinary clearance (Soma et al., 1988) and as such, flunixin is a drug for which recycling from bedding ingestion could be expected. In this observational survey, flunixin was administered either by the IV route at 1 mg ⁄ kg in one single administration or by the oral route at a dosing regimen of 0.5 mg ⁄ kg twice a day for 3 days. For the oral route, the paste was given outside the box. For the flunixin IV administration (Finadyne ), six thoroughbreds (two females, two geldings and two stallions) weighing from 450 to 600 kg and aged 2–16 years were used in the experiment. The study on oral flunixin involved six standard-bred horses and three thoroughbreds (four females, three geldings, two stallions) weighing from 450 to 600 kg and aged 2–17 years. A washout period of at least 1 month separated the two administration protocols for the horses selected more than once. The horses were accommodated in individual boxes and received a regular diet composed of manufactured feed and hay. Unlimited water was available. The horses were allowed either a moderate exercise daily (20 min of walking, 35 min of trotting, 5 min of galloping) or one hour per day in a paddock. Bedding conditions were the investigated factor and differed by the amount of straw in the box (about 20 vs. 45 kg), the daily cleaning (straw completely vs. not completely removed every day). Daily cleaning means the complete removal of the straw every day. In case of no daily cleaning, a complete cleaning was done once every week, and the messy straw was taken away the other days. For a group of three horses, the subjects were moved to another clean box 24 h after the single IV administration, while for the other horses, they stayed in the same box for the entire observational period. Table 1 details the bedding conditions for the different groups of observed horses. Urine was sampled from spontaneously urinating horses. Based upon results obtained by Sams et al., 1999; urine samples were collected before administration and after from at least 24 h up to 15 days. Urine samples were kept at )20 C and thawed just before analysis. For quantification, flunixin in plasma and urine was extracted by solid-phase extraction according to the method previously described (Popot et al., 2007) in which an in-house preparation of clonixin was used as internal standard. Flunixin was quantified using a gas chromatography ⁄ mass spectrometry in the electronic impact ionization mode (GC ⁄ EI-MS) method employed for the screening of NSAID in urine, which was slightly modified for quantification purposes. Linearity was observed from 10 to 300 ng ⁄ mL; (when necessary, urine is diluted with blank urine negative for flunixin) the limit of detection was 2 ng ⁄ mL and the limit of quantification was 10 ng ⁄ mL. Following a previous study (Jaussaud et al., 1987), urine concentrations of total flunixin (i.e. the sum of flunixin and flunixin glucuronide) were measured. Thus, it is the total J. vet. Pharmacol. Therap. 34, 612–614. doi: 10.1111/j.1365-2885.2011.01276.x. SHORT COMMUNICATION