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

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 / Ezra Mikhail, Erik Siccardi, Ali Bawazir, Ambika Rajesh, Seetharani Prathyush, Duaa Mohammad Kamal Al Wazani, Muhammed Sabeek, Thomas John. - (Drug Testing and Analysis (2021) 2 February)

  • PMID: 33533201
  • DOI: 10.1002/dta.3010

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.

Keywords: IOX-2; PHD inhibitor; equine doping; plasma; urine.

Parameters

Science
Study
Date
2 February 2021
People
Al Wazani, Duaa Mohammad Kamal
Bawazir, Ali
John, Thomas
Mikhail, Ezra
Prathyush, Seetharani
Rajesh, Ambika
Sabeek, Muhammed
Siccardi, Erik
Country
United Arab Emirates
Language
English
Sport/IFs
Equestrian (FEI) - International Equestrian Federation
Other organisations
United Arab Emirates Al Nasr-2 Equine Clinic
United Arab Emirates Veterinary Sports and Research Centre (VSRC)
Analytical aspects
Animal doping
Mass spectrometry analysis
New substance, not on prohibited list
Testing method development
Doping classes
M1. Manipulation Of Blood And Blood Components
Substances
Hypoxia-inducible factor (HIF) activating agents
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Abstract
Date generated
18 March 2021
Date of last modification
20 March 2021
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  • ADRV
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  • Sport/IFs
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  • Laboratories
  • Analytical aspects
  • Doping classes
  • Substances
  • Medical terms
  • Various
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