Analytical and Bioanalytical Chemistry

, Volume 407, Issue 15, pp 4385–4396 | Cite as

A validated UHPLC-MS/MS method to quantify low levels of anabolic-androgenic steroids naturally present in urine of untreated horses

  • Anneleen Decloedt
  • Ludovic Bailly-Chouriberry
  • Julie Vanden Bussche
  • Patrice Garcia
  • Marie-Agnes Popot
  • Yves Bonnaire
  • Lynn VanhaeckeEmail author
Research Paper
Part of the following topical collections:
  1. Hormone and Veterinary Drug Residue Analysis


Doping control is a main priority for regulatory bodies of both the horse racing industry and the equestrian sports. Urine and blood samples are screened for the presence of hundreds of forbidden substances including anabolic-androgenic steroids (AASs). Based on the suspected endogenous origin of some AASs, with β-boldenone as the most illicit candidate, this study aimed to improve the knowledge of the naturally present AAS in horse urine. To this extent, a novel ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated according to the Association of Official Racing Chemists (AORC) and European Commission (EC) guidelines, proving the power of this new method. Low limits of detection (0.2 ng/mL), good reproducibility (percentage of standard deviation (%RSD) < 10 %), high recovery (94.6 to 117.1 %), selectivity and specificity, and a linear response (confirmed with R 2 > 0.99 and lack-of-fit analysis) were obtained for all included AASs. With this method, urine samples of 105 guaranteed untreated horses (47 geldings, 53 mares, and 5 stallions serving as a control) were screened for β-boldenone and five related natural steroids: androstadienedione (ADD), androstenedione (AED), alpha-testosterone (αT), beta-testosterone (βT), and progesterone (P). Progesterone, β-testosterone, and α-testosterone were detected in more than half of the horses at low concentrations (<2 ng/mL). Occasionally, not only testosterone and progesterone but also low concentrations of AED, ADD, and boldenone (Bol) were found (0.5–5 ng/mL).

Graphical Abstract

A sensitive, new and fully validated UHPLC-MS/MS method has been developed that is able to quantify low levels of anabolic-androgenic steroids naturally present in urine of untreated horses (mares and geldings)


AAS Drug monitoring/screening Urine Equine UHPLC-MS/MS 



This work was funded by the Fédération Nationale des Courses Françaises (FNCF) and executed at the Laboratory of Chemical Analysis (Department of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University). Special thanks to student Tijs Van Hevel and colleagues Lieselot Hemeryck and Julie Kiebooms for their practical support.

Conflict of interest

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript, apart from those disclosed. No writing assistance was utilized in the production of this manuscript.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Anneleen Decloedt
    • 1
  • Ludovic Bailly-Chouriberry
    • 2
  • Julie Vanden Bussche
    • 1
  • Patrice Garcia
    • 2
  • Marie-Agnes Popot
    • 2
  • Yves Bonnaire
    • 2
  • Lynn Vanhaecke
    • 1
    Email author
  1. 1.Faculty of Veterinary Medicine, Department of Veterinary Public Health and Food Safety, Laboratory of Chemical AnalysisGhent UniversityMerelbekeBelgium
  2. 2.L.C.H., Laboratoire des Courses HippiquesVerrières-le-BuissonFrance

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