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Drug testing and analysis2023; doi: 10.1002/dta.3521

Detection of boldenone in the urine of female horses-ex vivo formation versus administration.

Abstract: Boldenone is an anabolic-androgenic steroid (AAS) that is prohibited in equine sports. However, in certain situations, it is endogenous, potentially formed by the microbes in urine. An approach to the differentiation based on the detection of the biomarkers Δ1-progesterone, 20(S)-hydroxy-Δ1-progesterone and 20(S)-hydroxyprogesterone was assessed, and their concentrations were monitored in the urine of untreated female horses (n = 291) alongside boldenone, boldienone, testosterone and androstenedione. Using an ultra-sensitive analytical method, boldenone (256 ± 236 pg/mL, n = 290) and the biomarkers (Δ1-progesterone up to 57.6 pg/mL, n = 8; 20(S)-hydroxy-Δ1-progesterone 85.3 ± 181 pg/mL, n = 130; 20(S)-hydroxyprogesterone 43.5 ± 92.1 pg/mL, n = 158) were detected at low concentrations. The ex vivo production of Δ1-steroids was artificially induced following the storage of urine samples at room temperature for 7 days in order to assess the concentrations and ratios of the monitored steroids. The administration of inappropriately stored feed source also resulted in an increase in 20(S)-hydroxy-Δ1-progesterone concentrations and the biomarker ratios. Using the results from different datasets, an approach to differentiation was developed. In situations where the presence of boldenone exceeds a proposed action limit of 5 ng/mL, the presence of the biomarkers would be investigated. If Δ1-progesterone is above 50 pg/mL or if 20(S)-hydroxy-Δ1-progesterone is above 100 pg/mL with the ratio of 20(S)-hydroxy-Δ1-progesterone:20(S)-hydroxyprogesterone greater than 5:1, then this would indicate ex vivo transformation or consumption of altered feed rather than steroid administration. There remains a (small) possibility of a false negative result, but the model increases confidence that adverse analytical findings reported in female horses are caused by AAS administrations.
© 2023 John Wiley & Sons, Ltd.
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Publication Date: 2023-06-01 PubMed ID: 37264746DOI: 10.1002/dta.3521Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article examines if the detection of a prohibited anabolic-androgenic steroid (Boldenone) in female horse urine is linked to external administration or is an outcome of natural formation within the body. The study determines markers for differentiation and establishes a testing model.

Study Rationale and Overview

  • The research is premised on the controversy surrounding the source of Boldenone, an anabolic-androgenic steroid banned from use in horse racing. The study is driven by challenges where the steroid can be endogenously created as a natural byproduct of biological processes in the animal’s system, bringing doping accusations under scrutiny.

Research Methodology

  • The study monitored the concentrations of Boldenone, Boldienone, testosterone, androstenedione as well as three markers (Δ1-progesterone, 20(S)-hydroxy-Δ1-progesterone, and 20(S)-hydroxyprogesterone) in the urine of untreated female horses.
  • It utilised an ultra-sensitive analytical method to detect these substances in low concentrations in the samples. The process also tested the possibility of artificial manufacturing of Δ1-steroids from urine samples stored at everyday room temperatures for a week.

Outcomes and Observations

  • The study found an increase in biomarker concentrations when horse feed sources were not properly stored. The detection of elevated 20(S)-hydroxy-Δ1-progesterone concentrations and biomarker ratios suggested ex vivo transformation or consumption of altered feed, as opposed to intentional steroid administration.
  • The research crew developed an approach to differentiate between endogenous formation and external administration. It proposed a model that signaled for investigation into biomarker presence if Boldenone exceeded a certain action limit.

Conclusion and Implications

  • The study observed that detection of Δ1-progesterone above 50 pg/mL, or elevated levels of 20(S)-hydroxy-Δ1-progesterone (above 100 pg/mL) combined with a specific ratio of biomarkers indicated natural formation or altered feed consumption rather than steroid administration.
  • While a small possibility of a false negative was admitted, the overall confidence in adverse analytical findings indicating administration of anabolic-androgenic steroids in female horses increased significantly.

Cite This Article

APA
Viljanto M, Kaabia Z, Taylor P, Hincks P, Muir T, Habershon-Butcher J, Bailly-Chouriberry L, Scarth J. (2023). Detection of boldenone in the urine of female horses-ex vivo formation versus administration. Drug Test Anal. https://doi.org/10.1002/dta.3521

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English

Researcher Affiliations

Viljanto, Marjaana
  • LGC, Fordham, Cambridgeshire, UK.
Kaabia, Zied
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.
Taylor, Polly
  • LGC, Fordham, Cambridgeshire, UK.
Hincks, Pamela
  • LGC, Fordham, Cambridgeshire, UK.
Muir, Tessa
  • British Horseracing Authority, London, UK.
Habershon-Butcher, Jocelyn
  • British Horseracing Authority, London, UK.
Bailly-Chouriberry, Ludovic
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.
Scarth, James
  • LGC, Fordham, Cambridgeshire, UK.

References

This article includes 40 references
  1. International Federation of horseracing Authorities. 2022 International Agreement on breeding, racing and wagering. .
  2. Viljanto M, Scarth J, Hincks P. Application of testosterone to epitestosterone ratio to horse urine - a complementary approach to detect the administration of testosterone and its pro-drugs in thoroughbred geldings. Drug Test Anal 2017;9(9):1328-1336.
    doi: 10.1002/dta.2109google scholar: lookup
  3. Houghton E, Ginn A. Nandrolone in the colt: a review of the science relating to the threshold value. In: Auer DE, Houghton E, eds. Proceedings of the 13th international conference of racing analysts and veterinarians. R&W Publications; 2000:29-33.
  4. Gray BP, Teale P, Pearce CM. Analysis of methyloxime derivatives of intact esters of testosterone and boldenone in equine plasma using ultra high performance liquid chromatography tandem mass spectrometry. Drug Test Anal 2011;3(4):206-213.
    doi: 10.1002/dta.237google scholar: lookup
  5. Kaabia Z, Dervilly-Pinel G, Hanganu F. Ultra high performance liquid chromatography/tandem mass spectrometry based identification of steroid esters in serum and plasma: an efficient strategy to detect natural steroids abuse in breeding and racing animals. J Chromatogr a 2013;1284:126-140.
    doi: 10.1016/j.chroma.2013.02.010google scholar: lookup
  6. Gray B, Viljanto M, Menzies E, Vanhaecke L. Detection of prohibited substances in equine hair by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry - application to doping control samples. Drug Test Anal 2018;10(7):1050-1060.
    doi: 10.1002/dta.2367google scholar: lookup
  7. Kwok KY, Choi TLS, Kwok WH, Wong JKY, Wan TSM. Detection of anabolic and androgenic steroids and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry. J Chromatogr 2017;1493:76-86.
    doi: 10.1016/j.chroma.2017.03.007google scholar: lookup
  8. Piper T, Geyer H, Gougoulidis V, Flenker U, Schanzer W. Determination of 13C/12C ratios of urinary excreted boldenone and its main metabolite 5β-androst-1-en-17β-ol-3-one. Drug Test Anal 2010;2(5):217-224.
    doi: 10.1002/dta.124google scholar: lookup
  9. . WADA Technical Document - TD2022IRMS. .
  10. Kaabia Z. Investigation of novel analytical strategies to detect natural steroid hormones misuse in bovine and equine. 2013. PhD thesis, L'Université Nantes Angers Le Mans.
  11. Viljanto MJ, Kicman AT, Walker CJ. Elucidation of the biosynthetic pathways of boldenone in the equine testis. Steroids 2019;146:79-91.
    doi: 10.1016/j.steroids.2019.03.011google scholar: lookup
  12. Ho ENM, Yiu KCH, Tang FPW. Detection of endogenous boldenone in the entire male horses. J Chromatogr 2004;B808:287-294.
  13. Viljanto MJ, Kaabia Z, Taylor P. Differentiation of boldenone administration from ex vivo transformation in the urine of castrated male horses. Drug Test Anal 2022;14(5):887-901.
    doi: 10.1002/dta.3240google scholar: lookup
  14. Wong J, Leung D, Curl P, Schiff P, Lam K, Wan T. Evidence of boldenone, nandrolone, 5(10)-estrene-3β-17β-diol and 4-estrene-3,17-dione as minor metabolites of testosterone in equine. Drug Test Anal 2017;9(9):1337-1348.
    doi: 10.1002/dta.2192google scholar: lookup
  15. Schänzer W, Geyer H, Gotzmann A. Endogenous production and excretion of boldenone (17beta-hydroxyandrosta-1,4-dien-3-one), an androgenic anabolic steroid. In: Donike M, Geyer H, Gotzmann U, Mareck-Engelke U, eds. Proceedings of the Manfred Donike workshop 12th Cologne workshop on dope analysis. Recent advances in doping analysis 2. Sport and Buch Strauß; 1994:211.
  16. Levy HR, Talalay P. Bacterial oxidation of steroids. II studies on the enzymatic mechanism of ring a dehydrogenation. J Biol Chem 1959;234(8):2014-2021.
    doi: 10.1016/s0021-9258(18)69859-xgoogle scholar: lookup
  17. Levy HR, Talalay P. Bacterial oxidation of steroids. I. Ring A dehydrogenations by intact cells. J Biol Chem 1959;234(8):2009-2013.
    doi: 10.1016/s0021-9258(18)69858-8google scholar: lookup
  18. Donova M, Egorova O. Microbial steroid transformations: current state and prospects. Appl Microbiol Biotechnol 2012;94(6):1423-1447.
    doi: 10.1007/s00253-012-4078-0google scholar: lookup
  19. Fidani M, Gamberini MC, Pasello F. Evaluation of equine urine reactivity towards phase II metabolites of 17-hydroxy steroids by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 2009;23(1):65-76.
    doi: 10.1002/rcm.3858google scholar: lookup
  20. Viljanto M, Kicman AT, Walker CJ. Bioformation of boldenone and related precursors/metabolites in equine feces and urine, with relevance to doping control. Drug Test Anal 2019;12(2):215-229.
    doi: 10.1002/dta.2706google scholar: lookup
  21. van de Kerkhof DH, van der Voort PM, de Boer D, Maes RAA. Confirmation of endogenous boldenone production: a procedure for an in vitro experiment. Proceedings of the Manfred Donike Workshop 17th Cologne Workshop on Dope Analysis, 1999; 161-168.
  22. Pompa G, Arioli F, Fracchiolla ML. Neoformation of boldenone and related steroids in faeces of veal calves. Food Addit Contam 2006;23(2):126-132.
    doi: 10.1080/02652030500442508google scholar: lookup
  23. Bailly-Chouriberry L, Thiebaud G, Garcia P. Boldenone positive cases associated to altered black oats. In: Houghton E, Symons P, Knight P, Sams R, Wynne P, Gordon GC, eds. Proceedings of the 21st international conference of racing analysts and veterinarians. Regent Press Co Ltd; 2016:75-81.
  24. Bonnaire Y, Dehennin L, Popot MA, Plou P. Testosterone in mares and fillies: a new threshold. In: Auer DE, Houghton E, eds. Proceedings of the 13th international conference of racing analysts and veterinarians. R&W Publications; 2000:75-81.
  25. Strompen O, Opfermann G, Enbergs H, Schänzer W. Determination of endogenous steroid profiles in equine urine by gas chromatography/mass spectrometry. In: Hill DW, Hill WT, eds. Proceedings of 14th international conference of racing analysts and veterinarians. R&W Publications; 2002:188-194.
  26. Tang PW, Ip WC, Law WC, Wan TSM, Crone DL. Further studies on the testosterone threshold for geldings. In: Laviolette B, Koupai-Abyazani MR, eds. Proceedings of the 12th international conference of racing analysts and veterinarians. R&W Publications; 1998:107-112.
  27. Clarke A, Scarth JP, Hands J. Detection of nandrolone and boldenone abuse in the ovine by GC-MS-MS. Chromatographia 2011;73(11-12):1155-1169.
    doi: 10.1007/s10337-011-2026-2google scholar: lookup
  28. Dumasia MC, Houghton E. Studies related to the metabolism of anabolic steroids in the horse: the identification of some 16-oxygenated metabolites of testosterone and a study of the phase II metabolism. Xenobiotica 1981;11(5):323-331.
    doi: 10.3109/00498258109045311google scholar: lookup
  29. Holtan DW, Houghton E, Silver M, Fowden AL, Ousey J, Rossdale PD. Plasma progestogens in the mare, fetus and newborn foal. J Reprod Fertil Suppl 1991;44:517-528.
  30. Kuhl H. Pharmacology of estrogens and progestogens: influence of different routes of administration. Climacteric 2005;8(sup1):3-63.
    doi: 10.1080/13697130500148875google scholar: lookup
  31. Decloedt A, Bailly-Chouriberry L, Vanden Bussche J. A validated UHPLC-MS/MS method to quantify low levels of anabolic-androgenic steroids naturally present in urine of untreated horses. Anal Bioanal Chem 2015;407(15):4385-4396.
    doi: 10.1007/s00216-014-8428-xgoogle scholar: lookup
  32. Liu S, Sjövall J, Griffiths WJ. Analysis of oxosteroids by nano-electrospray mass spectrometry of their oximes. Rapid Commun Mass Spectrom 2000;14(6):390-400.
    doi: 10.1002/(sici)1097-0231(20000331)14:6003c390::aid-rcm882003e3.0.co;2-7google scholar: lookup
  33. Bonnaire Y, Dehennin L, Plou P, Toutain PL. Testosterone administration to mares: criteria for detection of testosterone abuse by analysis of metabolites in plasma and urine. J Anal Toxicol 1995;19(3):175-181.
    doi: 10.1093/jat/19.3.175google scholar: lookup
  34. Soma LR, Uboh CE, Guan F, McDonnell S. Plasma concentrations of testosterone and 19-nortestosterone (nandrolone) in the nonracing intact male horse by liquid chromatography-mass spectrometry. J Vet Pharmacol Ther 2008;31(6):587-590.
    doi: 10.1111/j.1365-2885.2008.00997.xgoogle scholar: lookup
  35. Donadeu FX, Watson ED. Seasonal changes in ovarian activity: lessons learnt from the horse. Anim Reprod Sci 2007;100(3-4):225-242.
    doi: 10.1016/j.anireprosci.2006.12.001google scholar: lookup
  36. Soma LR, Uboh CE, Guan F, McDonnell S, Pack J. Pharmacokinetics of boldenone and stanozolol and the results of quantification of anabolic and androgenic steroids in racehorses and nonrace horses. J Vet Pharmacol Ther 2007;30(2):101-108.
    doi: 10.1111/j.1365-2885.2007.00824.xgoogle scholar: lookup
  37. Pu F, McKinney AR, Stenhouse AM, Suann CJ, McLeod MD. Direct detection of boldenone sulfate and glucuronide conjugates in horse urine by ion trap liquid chromatography-mass spectrometry. J Chromatogr B 2004;813(1-2):241-246.
    doi: 10.1016/j.jchromb.2004.09.052google scholar: lookup
  38. Dumasia MC, Houghton E. Biotransformation of 1-dehydrotestosterone in the equine male castrate: identification of the neutral unconjugated and glucuronic acid conjugated metabolites in horse urine. Biomed Environ Mass Spectrom 1988;17(5):383-392.
    doi: 10.1002/bms.1200170507google scholar: lookup
  39. Dehennin L, Bonnaire Y, Plou P. Human nutritional supplements in the horse. Dehydroepiandrosterone versus androstenedione: comparative effects on the androgen profile and consequences for doping analysis. J Anal Toxicol 2001;25(8):685-690.
    doi: 10.1093/jat/25.8.685google scholar: lookup
  40. Dehennin L, Bonnaire Y, Plou P. Detection of nandrolone administration to the entire male horse by a provisional concentration threshold for urinary oestranediol determined by gas chromatography-mass spectrometry. Equine Vet J 2007;39(2):186-188.
    doi: 10.2746/042516407x170418google scholar: lookup

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