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The metabolism of the masking agent tolcapone in the horse has been investigated. This substance was found to have undergone various chemical transformations that produced a large variety of phase I metabolites, as well as glucuronide and sulfate conjugation. Confirmation of the presence of tolcapone and the 3‐O‐methylated metabolite in the blood samples collected up to 240 minutes and in urine obtained up to 24 hours, was successfully conducted using both gas chromatography– and liquid chromatography–tandem mass spectrometry techniques. The 3‐O‐methyl tolcapone is the better marker to use in a screening method because, in comparison to tolcapone, we have found that this substance offers superior chromatographic performance that should potentially give a lower limit of detection.

The abuse of performance-enhancing catecholamine-based stimulants, such as levodopa, is controlled in horse racing through the application of a regulatory threshold for the common major metabolite. However, catechol-O-methyltransferase (COMT) enzyme inhibitors can be used to restrict the catalysis of the stimulant, and so the concurrent administration of both substances would be a viable strategy to enhance racing performance while removing the risk of exceeding the threshold. A 200 mg dose of nitecapone, a COMT inhibitor, was administered to a Thoroughbred horse, and we have analysed the blood (≤24 h) and urine (≤48 h) samples that were collected. The extracts, analysed by UHPLC coupled to a high-resolution accurate mass spectrometer, were consistent with the presence of nitecapone glucuronide in all the samples collected. An in-depth examination of the samples was then carried out using targeted accurate mass extracted ion chromatograms to identify whether the metabolites that have been found in other species were also present in the extracts.Once these were tentatively identified, MS/MS experiments were conducted on some of the metabolites (M1-M5), as well as decomposition products (DP1 and DP2), to verify that spectrum included MS fragments were consistent with their proposed structures. The accumulated data provided evidence that is consistent with this drug having been converted into many metabolites and a few decomposition products. An unexpected finding was that O-methylation was a very minor pathway until after the reduction of the 2,4-pentanedione side chain had occurred.