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Unusual observations during steroid analysis.
Abstract: In September 2005, our laboratory detected the presence of 4-androstene-3,17-dione and androsterone in a standard steroid screen of a post-race gelding urine sample received from an overseas authority. All other urine samples from the same batch tested negative. Subsequent gas chromatography/mass spectrometry (GC/MS) confirmatory analyses, however, repeatedly failed to detect any amount of 4-androstene-3,17-dione and androsterone in the suspicious sample. On the other hand, identical results were obtained when the initial GC/MS screening method was repeated on the suspicious sample as well as on the other samples of the same batch, showing the presence of 4-androstene-3,17-dione and androsterone only in the suspicious sample. These unusual and contradictory findings between the screening and confirmatory procedures were investigated, leading to the unequivocal conclusion that the 4-androstene-3,17-dione and androsterone observed during screening were artefacts from the internal standards, [16,16,17-d3]-testosterone and [16,16,17-d3]-5alpha-androstane-3alpha,17beta-diol. The two deuterated internal standards were thought to have undergone first an enzymatic oxidation of the 17beta-hydroxyl group to a 17-keto function by the enzyme 17beta-hydroxysteroid dehydrogenase; complete deuterium-hydrogen exchange at C16 during the methanolysis deconjugation step would then produce the two artefacts. The findings from this study highlight the potential problem of using internal standards in qualitative confirmatory analyses, which may lead to undesirable false positive results.
Publication Date: 2008-02-06 PubMed ID: 18247405DOI: 10.1002/rcm.3420Google 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
Summary
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The research article explores an anomaly observed in the steroid analysis of a urine sample from a racehorse, where certain steroid substances were detected initially but couldn’t be confirmed in further analyses, leading to an examination of potential complications arising from the use of internal standards in such tests.
Initial Investigation and Finding
- The research began with a detection of 4-androstene-3,17-dione and androsterone in a horse’s urine sample in a standard steroid screening test.
- The sample came from a gelding after a race and was provided by an overseas authority.
- Interestingly, the same substances were not found in any of the other samples from the same batch as the suspicious one.
- Subsequent confirmatory analyses using gas chromatography/mass spectrometry (GC/MS) were conducted, but neither of the steroids were found in the suspicious sample during these tests.
- When the initial GC/MS screening method was repeated, the results mirrored the original findings, indicating the presence of the two steroids only in the suspicious sample.
Investigating The Discrepancy
- Such conflicting results between the screening and confirmatory tests spurred a deeper investigation into the matter.
- The conclusion found was that the 4-androstene-3,17-dione and androsterone observed during the initial screening were actually artifacts from the internal standards used in the process.
- These internal standards, [16,16,17-d3]-testosterone and [16,16,17-d3]-5alpha-androstane-3alpha,17beta-diol, appear to have undergone enzymatic oxidation and complete deuterium-hydrogen exchange at the C16 position during the methanolysis deconjugation phase, thus producing the two artifacts.
Conclusions and Implications
- The findings of this study emphasize the potential problems with using internal standards in confirmatory analyses for steroid tests.
- The process can potentially lead to false positive results, which might have significant implications and consequences, especially in fields like sports where drug tests form a critical part of the competition integrity management.
Cite This Article
APA
Kwok WH, Leung DK, Leung GN, Tang FP, Wan TS, Wong CH, Wong JK.
(2008).
Unusual observations during steroid analysis.
Rapid Commun Mass Spectrom, 22(5), 682-686.
https://doi.org/10.1002/rcm.3420 Publication
Researcher Affiliations
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China. wh.kwok@hkjc.org.hk
MeSH Terms
- Anabolic Agents / urine
- Androstenedione / urine
- Androsterone / urine
- Animals
- Artifacts
- Doping in Sports
- Forensic Toxicology
- Gas Chromatography-Mass Spectrometry
- Horses
- In Vitro Techniques
- Male
- Microsomes, Liver / metabolism
- Reproducibility of Results
- Substance Abuse Detection / methods
- Urinalysis
Citations
This article has been cited 2 times.- Jalabert C, Shock MA, Ma C, Bootsma TJ, Liu MQ, Soma KK. Ultrasensitive Quantification of Multiple Estrogens in Songbird Blood and Microdissected Brain by LC-MS/MS. eNeuro 2022 Jul-Aug;9(4).
- de Jong FA, Beecher C. Addressing the current bottlenecks of metabolomics: Isotopic Ratio Outlier Analysis™, an isotopic-labeling technique for accurate biochemical profiling. Bioanalysis 2012 Sep;4(18):2303-14.
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