A validated method for the quantification of IOX‐2, a potent prolyl hydroxylase inhibitor in equine urine and plasma using liquid chromatography–high‐resolution mass spectrometry

Abstract

IOX-2 is a potent inhibitor of enzyme prolyl hydroxylases-2 (PHD) that plays a critical role in regulating hypoxia inducible factor (HIF) abundance and oxygen homeostasis. Federation for Equestrian Sports has listed HIF activators as prohibited substances to prevent their usage in doping. Consequently, it became essential to develop adequate knowledge and testing methods to detect it in equine sports drug testing samples. The validated method utilizes ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry in order to detect extremely low concentration of the analyte present in both matrices. Confirmation for the presence of the analyte was achieved by comparing ion ratios, retention time, and accurate mass. Method linear range for plasma was between 0.25 to 100 ng/ml, and limit of detection (LOD) was 0.075 ng/ml. The linear range for urine was 0.125 to 100 ng/ml, and LOD was 0.025 ng/ml. Intraday precision at 0.5, 10, and 50 ng/ml was between 4.0% and 9.7% for plasma and 4.2% and 10.4% for urine. Accuracy at 0.5, 10, and 50 ng/ml was between 91% and 94% for plasma and 99% and 103% for urine. Elimination profile of IOX-2 in equine plasma and urine was carried out using the developed method in which two horses were intravenously administered IOX-2 and samples were collected. Metabolic profile in plasma and urine was investigated. IOX-2 was detected for a minimum of 54 and 151 h of post administration in plasma and urine, respectively, thereby providing a valuable tool for evaluating its misuse in equine racing.