Quantification of 19-nortestosterone sulphate and boldenone sulphate in urine from male horses using liquid chromatography/tandem mass spectrometry.
Abstract: Following administration of the anabolic steroid 19-nortestosterone or its esters to the horse, a major urinary metabolite is 19-nortestosterone-17beta-sulphate. The detection of 19-nortestosterone in urine from untreated animals has led to it being considered a naturally occurring steroid in the male horse. Recently, we have demonstrated that the majority of the 19-nortestosterone found in extracts of 'normal' urine from male horses arises as an artefact through decarboxylation of the 19-carboxylic acid of testosterone. The aim of this investigation was to establish if direct analysis of 19-nortestosterone-17beta-sulphate by liquid chromatography/tandem mass spectrometry (LC/MS/MS) had potential for the detection of 19-nortestosterone misuse in the male horse. The high concentrations of sulphate conjugates of the female sex hormones naturally present in male equine urine were overcome by selective hydrolysis of the aryl sulphates using glucuronidase from Helix pomatia; this was shown to have little or no activity for alkyl sulphates such as 19-nortestosterone-17beta-sulphate. The 'free' phenolic steroids were removed by solid-phase extraction (SPE) prior to LC/MS/MS analysis. The method also allowed for the quantification of the sulphate conjugate of boldenone, a further anabolic steroid endogenous in the male equine with potential for abuse in sports. The method was applied to the quantification of these analytes in a population of samples. This paper reports the results of that study along with the development and validation of the LC/MS/MS method. The results indicate that while 19-nortestosterone-17beta-sulphate is present at low levels as an endogenous substance in urine from 'normal' male horses, its use as an effective threshold substance may be viable.
Publication Date: 2008-09-09 PubMed ID: 18777513DOI: 10.1002/rcm.3698Google Scholar: Lookup
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Summary
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The research article details an investigation into the misuse of the anabolic steroid 19-nortestosterone in male horses. The researchers created and tested a method for detecting 19-nortestosterone in horse urine, by analysing a substance created when the steroid is metabolized, and assessing the possibility of using this as a threshold substance to detect steroid misuse.
Research Goals
- The primary objective of this study was to develop and test a method of using liquid chromatography/tandem mass spectrometry (LC/MS/MS) to directly analyse 19-nortestosterone-17beta-sulphate, a metabolite of the anabolic steroid 19-nortestosterone.
- The researchers also aimed to investigate whether this method could be useful in detecting the misuse of 19-nortestosterone in male horses.
Methods and Approaches
- The researchers addressed the high concentration of the sulphate conjugates of female sex hormones in male equine urine, which could interfere with the analysis, by using a selective hydrolysis technique. They used glucuronidase extracted from Helix pomatia, a species of snail, which showed little to no activity for alkyl sulphates, including 19-nortestosterone-17beta-sulphate.
- The phenolic steroids were then removed, and the remaining substances were analysed using the LC/MS/MS method.
- This method also allowed for the quantification of the sulphate conjugate of boldenone, another anabolic steroid found naturally in male horses, which also has potential for abuse in sports.
Results
- The study found that 19-nortestosterone-17beta-sulphate is present in male horse urine as an endogenous substance – one that originates from within the organism.
- However, the researchers found that the levels of this substance in the urine of “normal” (i.e., non-steroid-using) horses were low.
- This indicates that, despite this substance’s presence as a naturally occurring compound, it might be possible to use its concentration levels as a threshold substance for detecting steroid misuse in male horses.
- The paper presents the results of this study, along with a detailed account of the development and validation of the LC/MS/MS method for detecting 19-nortestosterone-17beta-sulphate and boldenone sulphate in horse urine.
Cite This Article
APA
Grace PB, Drake EC, Teale P, Houghton E.
(2008).
Quantification of 19-nortestosterone sulphate and boldenone sulphate in urine from male horses using liquid chromatography/tandem mass spectrometry.
Rapid Commun Mass Spectrom, 22(19), 2999-3007.
https://doi.org/10.1002/rcm.3698 Publication
Researcher Affiliations
- HFL Sport Science, Quotient Bioresearch Ltd., Fordham CB7 5WW, UK. pgrace@hfl.co.uk
MeSH Terms
- Animals
- Chromatography, High Pressure Liquid / methods
- Doping in Sports / prevention & control
- Horses / urine
- Male
- Nandrolone / urine
- Reproducibility of Results
- Sensitivity and Specificity
- Spectrometry, Mass, Electrospray Ionization / methods
- Substance Abuse Detection / methods
- Sulfates / urine
- Testosterone / analogs & derivatives
- Testosterone / urine
- Urinalysis / methods
Citations
This article has been cited 2 times.- Iwona MŻ, Barbara W, Alicja K, Sebastian W, Katarzyna S, Andrzej P. Control of Anabolic Hormone Residues in Tissues of Slaughter Animals in Poland During the Period of 2011-2015.. J Vet Res 2017 Mar;61(1):69-79.
- Yuan M, Breitkopf SB, Asara JM. Serial-omics characterization of equine urine.. PLoS One 2017;12(10):e0186258.
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