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.
IOX4 is a hypoxia‐inducible factor prolyl hydroxylase (HIF‐PHD) inhibitor, which was developed for the treatment of anemia by exerting hematopoietic effects. The administration of HIF‐PHD inhibitors such as IOX4 to horses is strictly prohibited by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale. To the best of our knowledge, this is the first comprehensive metabolic study of IOX4 in horse plasma and urine after a nasoesophageal administration of IOX4 (500 mg/day, 3 days). A total of four metabolites (three mono‐hydroxylated IOX4 and one IOX4 glucuronide) were detected from the in vitro study using homogenized horse liver. As for the in vivo study, post‐administration plasma and urine samples were comprehensively analyzed with liquid chromatography/electrospray ionization high‐resolution mass spectrometry to identify potential metabolites and determine their corresponding detection times. A total of 10 metabolites (including IOX4 glucuronide, IOX4 glucoside, O‐desbutyl IOX4, O‐desbutyl IOX4 glucuronide, four mono‐hydroxylated IOX4, N‐oxidized IOX4, and N‐oxidized IOX4 glucoside) were found in urine and three metabolites (glucuronide, glucoside, and O‐desbutyl) in plasma. Thus, the respective quantification methods for the detection of free and conjugated IOX4 metabolites in urine and plasma with a biphase enzymatic hydrolysis were developed and applied to post‐administration samples for the establishment of elimination profiles of IOX4. The detection times of total IOX4 in urine and plasma could be successfully prolonged to at least 312 h.
Rationale
The use of GW1516, a peroxisome proliferator‐activated receptor δ (PPAR δ) agonist, is strictly prohibited in both horseracing and equestrian competitions. However, little is known about its metabolic fate in horses. To the best of our knowledge, this is the first reported metabolic study of GW1516 in equine urine.
Methods
Urine samples obtained from a thoroughbred after nasoesophageal administration with GW1516 were protein‐precipitated and the supernatants were subsequently analyzed by liquid chromatography/electrospray ionization high‐resolution mass spectrometry (LC/ESI‐HRMS) with a Q‐Exactive mass spectrometer. Monoisotopic ions of GW1516 and its metabolites were monitored from the full‐scan mass spectral data of pre‐ and post‐administration samples. A quantification method was developed and validated to establish the excretion profiles of GW1516, its sulfoxide, and its sulfone in equine urine.
Results
GW1516 and its nine metabolites [including GW1516 sulfoxide, GW1516 sulfone, 5‐(hydroxymethyl)‐4‐methyl‐2‐(4‐trifluoromethylphenyl)thiazole (HMTT), methyl 4‐methyl‐2‐[4‐(trifluoromethyl)phenyl]‐1,3‐thiazole‐5‐carboxylate (MMTC), 4‐methyl‐2‐[4‐(trifluoromethyl)phenyl]‐1,3‐thiazole‐5‐carboxylic acid (MTTC), and M1 to M4] were detected in post‐administration urine samples. GW1516 sulfoxide and GW1516 sulfone showed the longest detection times in post‐administration urine samples and were therefore recommended as potential screening targets for doping control purposes. Quantitative analysis was also conducted to establish the excretion profiles of GW1516 sulfoxide and GW1516 sulfone in urine.
Conclusions
For the purposes of doping control of GW1516, the GW1516 sulfoxide and GW1516 sulfone metabolites are recommended as the target analytes to be monitored in equine urine due to their high specificities, long detection times (1 and 4 weeks, respectively), and the ready availability of their reference materials.
IOX4, a hypoxia-inducible factor stabilizer, is classified as a banned substance for horses in both horse racing and equestrian sports. We recently reported the pharmacokinetic profiles of IOX4 in horse plasma and urine and also identified potential monitoring targets for the doping control purpose. In this study, a long-term longitudinal analysis of IOX4 in horse hair after a nasoesophageal administration of IOX4 (500 mg/day for 3 days) to three thoroughbred mares is presented for the first time for controlling the abuse/misuse of IOX4. Six bunches of mane hair were collected at 0 (pre), 1, 2, 3, and 6 month(s) postadministration. Our results showed that the presence of IOX4 was identified in all postadministration horse hair samples, but no metabolite could be detected. The detection window for IOX4 could achieve up to 6-month postadministration (last sampling point) by monitoring IOX4 in hair. In order to evaluate the longitudinal distribution of IOX4 over 6 months, a validated quantification method of IOX4 in hair was developed for the analysis of the postadministration samples. Segmental analysis of 2-cm cut hair across the entire length of postadministration hair showed that IOX4 could be quantified up to the level of 1.84 pg/mg. In addition, it was found that the movement of the incorporated IOX4 band in the hair shaft over 6 months varied among the three horses due to individual variation and a significant diffusion of IOX4 band up to 10 cm width was also observed in the 6-month postadministration hair samples.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.