Hydroxy levamisole and its phase II conjugates as potential indicators of levamisole doping in thoroughbred horses

Rationale According to previous research, aminorex is the major metabolite of levamisole; however, in the screening of levamisole‐positive racehorse urine and plasma samples, aminorex could only be detected in trace amounts or not at all. In forensic laboratories, hydroxy levamisole and its phase II conjugates make it easier to confirm levamisole misuse and to differentiate between the abuse of levamisole and aminorex. This study aimed to identify the major levamisole metabolites that can be detected along with the parent drug. Methods The study describes levamisole and its metabolites in thoroughbred horses following oral administration and in vitro with equine liver microsomes. The plausible structures of the detected metabolites were postulated using liquid chromatography combined with high‐resolution mass spectrometry. Results Under the experimental conditions 26 metabolites (17 phase I, 2 phase II, and 7 conjugates of phase I metabolites) were detected (M1–M26). The major phase I metabolites identified were formed by hydroxylation. In phase II, the glucuronic acid conjugates of levamisole and hydroxy levamisole were detected as the major metabolites. In plasma, the parent drug and major metabolites are detectable for up to eight days, while in urine, they are detectable for up to twenty days. Levamisole levels rapidly increased at 45 min following administration, then declined gradually until detectable levels were reached approximately 8 days after administration, according to a pharmacokinetics study. Conclusions A prolonged elimination profile and relatively high concentration of hydroxy metabolites suggest that the detection of hydroxy metabolites is imperative for investigating levamisole doping in horses.