Recently, the illegal use of novel technologies, such as gene and cell therapies, has become a great concern for the horseracing industry. As a potential way to control this, metabolomics approaches that comprehensively analyze metabolites in biological samples have been gaining attention. However, it may be difficult to identify metabolic biomarkers for doping because physiological conditions generally differ between resting and exercise states in horses. To understand the metabolic differences in horse plasma between the resting state at training centres and the sample collection stage after racing for doping test (SAD), we took plasma samples from these two stages (n=30 for each stage) and compared the metabolites present in these samples by liquid chromatography-high resolution mass spectrometry. This analysis identified 5,010 peaks, of which 1,256 peaks (approximately 25%) were annotated using KEGG analysis. Principal component analysis showed that the resting state and SAD groups had entirely different metabolite compositions. In particular, the levels of inosine, xanthosine, uric acid, and allantoin, which are induced by extensive exercise, were significantly increased in the SAD group. In addition, many metabolites not affected by extensive exercise were also identified. These results will contribute to the discovery of biomarkers for detecting doping substances that cannot be detected by conventional methods.

Rationale GW1516 is a peroxisome proliferator‐activated receptor‐δ (PPAR‐δ) agonist that is banned in horseracing and equestrian sports. Long‐term detection and longitudinal distribution of GW1516 in the mane of a horse are reported for the first time and this hair analysis could prolong the detection window of GW1516 for doping control. Methods Mane hairs were divided into three segments (0–7, 7–15, and >15 cm from the cut end) and completely pulverized and homogenized for analysis. The pulverized hair samples were extracted with methanol followed by further purification and the extracts were analyzed by liquid chromatography/electrospray ionization high‐resolution mass spectrometry (LC/ESI‐HRMS) using a Q‐Exactive instrument. This method was successfully validated and applied to post‐administration samples to confirm the presence of GW1516 and its metabolites and estimate the uptake amounts of GW1516. Results After administration of 150 mg of GW1516 to a thoroughbred mare, GW1516 was detected in one of two segments of all mane hairs, and four metabolites, namely GW1516 sulfoxide, GW1516 sulfone, 5‐(hydroxymethyl)‐4‐methyl‐2‐(4‐trifluoromethylphenyl)thiazole (HMTT), and 4‐methyl‐2‐[4‐(trifluoromethyl)phenyl]‐1,3‐thiazole‐5‐carboxylic acid (MTTC), were also identified. The longitudinal distribution analysis results showed that the maximum uptake of GW1516 into hair (approximately 0.05 pg/mg) was observed at around 13 weeks post‐administration and GW1516 could be detected and confirmed up to 6 months post‐administration. Conclusions The parent drug GW1516 was identified as the most appropriate monitoring target in equine hair for controlling its misuse in horses. The use of hair analysis could extend the detection time of GW1516 to at least 6 months after the administration of 150 mg of GW1516 to a thoroughbred mare.