Unusual observations during steroid analysis.
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.
Kwok
Wai Him
WH
Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China. wh.kwok@hkjc.org.hk
Leung
David K K
DK
Leung
Gary N W
GN
Tang
Francis P W
FP
Wan
Terence S M
TS
Wong
Colton H F
CH
Wong
Jenny K Y
JK
D045930:Anabolic Agents / Q000652:urine*; D000735:Androstenedione / Q000652:urine*; D000738:Androsterone / Q000652:urine*; D000818:Animals; D016477:Artifacts; D004300:Doping in Sports; D053593:Forensic Toxicology; D008401:Gas Chromatography-Mass Spectrometry; D006736:Horses; D066298:In Vitro Techniques; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D015203:Reproducibility of Results; D015813:Substance Abuse Detection / Q000379:methods*; D016482:Urinalysis
D045930:Anabolic Agents; D000735:Androstenedione; D000738:Androsterone