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. 2025 May;17(5):647-654.
doi: 10.1002/dta.3771. Epub 2024 Jul 11.

Simultaneous detection of three hypoxia-inducible factor stabilizers-molidustat, roxadustat, and vadadustat-in multiple keratinized matrices and its application in a doping context

Anne Checkouri  1 , Laurie Gheddar  1 , Nadia Arbouche  1 , Jean-Sébastien Raul  1 , Pascal Kintz  1   2
Affiliations

Simultaneous detection of three hypoxia-inducible factor stabilizers-molidustat, roxadustat, and vadadustat-in multiple keratinized matrices and its application in a doping context

Anne Checkouri et al. Drug Test Anal. 2025 May.

Abstract

In a doping case, a top athlete challenged an anti-doping rule violation, involving molidustat. Molidustat is a stabilizing agent of the hypoxia-inducible factor (HIF) recently developed. It is currently undergoing clinical trials for anemia associated with chronic kidney disease. HIF stabilizers are banned at all times by the World Anti-Doping Agency (class S2). Because of their pharmacological proprieties, these new drugs can enhance athletic performance. The athlete's defense wanted to analyze multiple keratinized matrices as they allow long-term investigations. Requests concerning HIF stabilizers are constantly growing. We have therefore developed a liquid chromatography coupled with tandem mass spectrometry method to identify and quantify three molecules of this class: molidustat, vadadustat, and roxadustat. Thirty milligrams of keratinized matrices were incubated in 1 mL of pH 8.4 diammonium hydrogen phosphate buffer for 16 h at 40°C with 1 ng of testosterone-D3, used as internal standard. After extraction with ethyl acetate/diethyl ether (80/20), the organic phase was evaporated, and the dry residue was reconstituted in 30 μL of initial phase. The method was linear from 5 to 1000 pg/mg for the three analytes. Limits of quantification were 2, 0.5, and 5 pg/mg for molidustat, roxadustat, and vadadustat, respectively. The analysis of the athlete's head hair (collected 1 month after the urine test) showed a concentration of molidustat of 135 pg/mg, and his beard hair and his fingernails clippings contained 55 and 40 pg/mg, respectively.

Keywords: HIF; body hair; hair; molidustat; nails.

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Figures

FIGURE 1
FIGURE 1
Action of HIF in chronic kidney disease. EPO, erythropoietin; HIF, hypoxia‐inducible factor; HSC, hematopoietic stem cell; O2, oxygen; VEGF, vascular endothelial growth factor. Created with BioRender.com.
FIGURE 2
FIGURE 2
Chromatogram of a 10 pg/mg point for the three HIF stabilizers. From bottom to top: Transitions of testosterone‐d3 (292.24 > 96.99), vadadustat (307.03 > 204.06), molidustat (315.10 > 207.00), and roxadustat (353.01 > 278.09).
FIGURE 3
FIGURE 3
(a) Fingernail clippings chromatogram. Molidustat concentration is 40 pg/mg. From bottom to top: Transition for internal standard testosterone‐d3 (292.24 > 96.99) and two transitions of molidustat (315.0 > 137.10 and 315.10 > 207.00). (b) Beard hair chromatogram. Molidustat concentration is 55 pg/mg. From bottom to top: Transition for internal standard testosterone‐d3 (292.24 > 96.99) and two transitions of molidustat (315.0 > 137.10 and 315.10 > 207.00). (c) Head hair chromatogram. Molidustat concentration is 135 pg/mg. From bottom to top: Transition for internal standard testosterone‐d3 (292.24 > 96.99) and two transitions of molidustat (315.0 > 137.10 and 315.10 > 207.00).
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References

    1. Akizawa T, Nobori K, Matsuda Y, et al. Molidustat for the treatment of anemia in Japanese patients undergoing peritoneal dialysis: a single‐arm, open‐label, phase 3 study. Ther Apher Dial. 2022;26(2):368‐377. doi:10.1111/1744-9987.13713 - DOI - PMC - PubMed
    1. Lentini S, van der Mey D, Kern A, et al. Absorption, distribution, metabolism and excretion of molidustat in healthy participants. Basic Clin Pharmacol Toxicol. 2020;127(3):221‐233. doi:10.1111/bcpt.13409 - DOI - PMC - PubMed
    1. Flamme I, Oehme F, Ellinghaus P, Jeske M, Keldenich J, Thuss U. Mimicking hypoxia to treat anemia: HIF‐stabilizer BAY 85‐3934 (molidustat) stimulates erythropoietin production without hypertensive effects. PLoS ONE. 2014;9(11):e111838. doi:10.1371/journal.pone.0111838 - DOI - PMC - PubMed
    1. World Anti‐Doping Agency . 2017 anti‐doping testing figures by laboratory [Internet]. Available from: https://www.wada-ama.org/sites/default/files/resources/files/2017_anti-d.... Accessed May 5, 2024.
    1. ITIA [Internet] . ITIA – independent tribunal sanctions Simona Halep for anti‐doping rule violations. Available from: https://www.itia.tennis/news/sanctions/independent-tribunal-sanctions-si.... Accessed February 26, 2024.

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