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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.
© 2021 John Wiley & Sons, Ltd.
Publication Date: 2021-02-06 PubMed ID: 33533201DOI: 10.1002/dta.3010Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Validation Study
Summary
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This research developed and validated a method to detect a prohibited doping substance, a prolyl hydroxylases-2 inhibitor (IOX-2), in horse urine and blood. The method employs ultra-high-performance liquid chromatography and high-resolution mass spectrometry for the detection, measurement and the determination of elimination profile of IOX-2 in equine urine and plasma.
Methodology
In this study, the researchers used:
- Ultra-high-performance liquid chromatography, a type of liquid chromatography that improves separation of components in a mixture,
- Combined with high-resolution mass spectrometry, which separates ions based on their mass-to-charge ratio, for the detection of extremely low concentration of IOX-2 in equine urine and plasma.
Confirmation of IOX-2 Presence
The presence of IOX-2 was confirmed by:
- Comparing ion ratios,
- Assessing retention time, the time a compound takes to travel through the system, and
- Accurate mass analysis, which helps to obtain precise molecular weight for the target compound.
Linear Range and Limit of Detection
The method’s limit of detection, which is the lowest amount of a substance that can be detected, and its linear range, which specifies the minimum and maximum concentration over which the method has a clear relationship between detector response and analyte concentration, were established:
- For plasma, the linear range was established to be between 0.25 to 100 ng/ml, and the limit of detection was 0.075 ng/ml.
- For urine, the linear range was between 0.125 to 100 ng/ml, and the limit of detection was 0.025 ng/ml.
Precision and Accuracy Assessment
Precision (the closeness of two or more measurements to each other) and accuracy (how close a measured value is to the actual value) were evaluated at three concentration levels (0.5, 10, and 50 ng/ml):
- Between 4.0% and 9.7% for plasma, and 4.2% and 10.4% for urine for precision,
- Between 91% and 94% for plasma and 99% and 103% for urine for accuracy.
IOX-2 Elimination Profile
The researchers also elucidated the elimination profile of IOX-2, meaning how the body gets rid of the drug, in equine plasma and urine:
- Two horses were intravenously administered IOX-2, and samples were collected and tested.
- IOX-2 was detected for a minimum of 54 hours post-administration in plasma and 151 hours in urine.
- This provided a basis for evaluating the possible misuse of IOX-2 in horse racing.
Cite This Article
APA
Mikhail E, Siccardi E, Bawazir A, Rajesh A, Prathyush S, Al Wazani DMK, Sabeek M, John T.
(2021).
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.
Drug Test Anal, 13(6), 1178-1190.
https://doi.org/10.1002/dta.3010 Publication
Researcher Affiliations
- Veterinary Sports and Research Centre, Wadi Al Safa, Dubai, UAE.
- Al Nasr-2 Equine Clinic, Dubai, UAE.
- Veterinary Sports and Research Centre, Wadi Al Safa, Dubai, UAE.
- Veterinary Sports and Research Centre, Wadi Al Safa, Dubai, UAE.
- Veterinary Sports and Research Centre, Wadi Al Safa, Dubai, UAE.
- Veterinary Sports and Research Centre, Wadi Al Safa, Dubai, UAE.
- Veterinary Sports and Research Centre, Wadi Al Safa, Dubai, UAE.
- Veterinary Sports and Research Centre, Wadi Al Safa, Dubai, UAE.
MeSH Terms
- Animals
- Chromatography, High Pressure Liquid / methods
- Doping in Sports / prevention & control
- Horses
- Limit of Detection
- Mass Spectrometry / methods
- Prolyl-Hydroxylase Inhibitors / administration & dosage
- Prolyl-Hydroxylase Inhibitors / analysis
- Prolyl-Hydroxylase Inhibitors / pharmacokinetics
- Substance Abuse Detection / methods
Grant Funding
- Zabeel Equestrian office
References
This article includes 18 references
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Citations
This article has been cited 2 times.- Ishii H, Shibuya M, Kusano K, Sone Y, Kamiya T, Wakuno A, Ito H, Miyata K, Sato F, Kuroda T, Yamada M, Leung GN. Pharmacokinetic Study of Vadadustat and High-Resolution Mass Spectrometric Characterization of its Novel Metabolites in Equines for the Purpose of Doping Control. Curr Drug Metab 2022;23(10):850-865.
- Prajapati P, Singh P, Doshi G. Emerging Therapeutic Targets in Rheumatoid Arthritis: Focusing on HIF-1α, Nrf2, STATs, and RORγt. Curr Drug Targets 2025;26(8):507-533.
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