Quantitative analysis of paracetamol, metacetamol, and orthocetamol in equine urine from racehorses in Japan using liquid chromatography–electrospray ionization–tandem mass spectrometry

Paracetamol is commonly used as an over-the-counter analgesic and antipyretic medication for humans, but not sold as a legitimate therapeutic medication for horses in Japan. However, paracetamol is commonly found in horses together with its two isomers, metacetamol and orthocetamol. We previously reported that paracetamol and orthocetamol were both present in selected feed consumed by Japanese racehorses. For the purpose of the doping control of paracetamol in local Japanese horses, we proposed establishing residue limits (Japanese residue limits, JRLs) to minimize the risk of reporting paracetamol from environmental contributions and to differentiate its presence from active administration. Recently, we proposed a preliminary JRL for paracetamol in equine plasma based on a population study of more than 300 Japanese racehorses. In this paper, we will present our studies on the urinary concentrations of paracetamol, metacetamol, and orthocetamol in postrace samples collected from 403 Japanese racehorses over a 1 year period, detected using liquid chromatography–electrospray ionization–tandem mass spectrometry. Our results revealed that the hydrolyzed urinary concentrations of paracetamol, metacetamol, and orthocetamol were in the range 15.7–2,360 ng/mL (median 363 ng/mL), 8.07–382 ng/mL (84.5 ng/mL), and 919–74,418 ng/mL (13,475 ng/mL), respectively. Based on our statistical model, the preliminary JRL of hydrolyzed paracetamol in equine urine was determined to be 7,400 ng/mL, at a risk factor of 1 in 10,000. Further investigations will be required to demonstrate the applicability and validity of our preliminary plasma and urine JRLs to local Japanese racehorses.