Comprehensive metabolic study of IOX4 in equine urine and plasma using liquid chromatography/electrospray ionization Q Exactive high‐resolution mass spectrometer for the purpose of doping control

Abstract

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.

Long‐term monitoring of IOX4 in horse hair and its longitudinal distribution with segmental analysis using liquid chromatography/electrospray ionization Q Exactive high‐resolution mass spectrometry for the purpose of doping control

“…For the purification of IOX4 from crude hair extract, two major problems of matrix effect and absorption were encountered. In the early stage of method development, the liquid–liquid extraction with chloroform used for the analysis of IOX4 in horse plasma and urine previously developed was attempted; 16 however, the extraction efficiency was not sufficient for hair analysis, most likely due to relatively high ionization suppression (around 75%). Mazzarino reported that analytical method for IOX4 in human urine equipped with strong cation exchange (SCX) solid‐phase extraction (SPE) showed high extraction recovery (88%) and acceptable matrix effect (33%) 57 .…”

“…All experimental procedures were approved by the Animal Welfare and Ethics Committee of the Laboratory of Racing Chemistry (LRC) (authorization number 20-06) as reported earlier. 16 After a nasoesophageal administration of IOX4 at a dose of 500 mg to three thoroughbred mares (with chestnut or bay hair) for three consecutive days, six bunches of mane hair (approximately 5 mm in diameter and the hair length ranging from approximately 10 to 16 cm) were collected from each horse by cutting at 0 (pre), 1, 2, 3, and 6 month(s) postadministration at different locations of the horse's mane area. The cuts were made as close to the skin as possible.…”

“…For the optimization of liquid chromatography electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) conditions, the LC condition used was very similar to our previously reported one with minor modifications. 16 In the horse racing industry, guidelines for the minimum criteria for the identification of a prohibited substance have been established by the AORC. 58 According to the criteria, for the identification (confirmation) of a prohibited substance based on the relevant hyphenated mass spectrometric techniques, the relative abundance of at least three product ions must match within a predefined allowance between the test sample and the reference standard.…”

“…When stepped CEs of 20, 30, and 40 eV were set, several product ions were observed as shown in Figure 1 and the proposed mass fragmentations have been reported earlier. 16 Among those ions, three characteristic product ions of m/z 165, m/z 204, and m/z 273 were selected for confirmation due to their acceptable ion abundances, high sensitivity, and specificity.…”

“…Philip et al reported the in vitro metabolic study of IOX4 using equine liver microsomes 15 . In addition, the authors recently reported the comprehensive in vitro and in vivo metabolic studies of IOX4 in horses 16 . The elimination profiles of IOX4 were established, and potential monitoring targets including IOX4 glucuronide, IOX4 glucoside, and N ‐oxidized IOX4 were identified in both plasma and urine after a nasoesophageal administration of IOX4 (500 mg/day for 3 days) to three thoroughbred mares.…”