Performance characteristics of a carbon isotope ratio method for detecting doping with testosterone based on urine diols: controls and athletes with elevated testosterone/epitestosterone ratios.
BACKGROUND
Carbon isotope ratio methods are used in doping control to determine whether urinary steroids are endogenous or pharmaceutical.


METHODS
Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was used to determine the delta(13)C values for 5 beta-androstane-3 alpha,17 beta-diyl diacetate (5 beta A), 5 alpha-androstane-3 alpha,17 beta-diyl diacetate (5 alpha A), and 5 beta-pregnane-3 alpha,20 alpha-diyl diacetate (5 beta P) in a control group of 73 healthy males and 6 athletes with testosterone/epitestosterone ratios (T/E) >6.


RESULTS
The within-assay precision SDs for 5 beta A, 5 alpha A, and 5 beta P were +/- 0.27 per thousand, +/- 0.38 per thousand, and +/- 0.28 per thousand, respectively. The between-assay precision SDs ranged from +/- 0.40 per thousand to +/- 0.52 per thousand. The system suitability and batch acceptance scheme is based on SDs. For the control group, the mean delta(13)C (SD) values were -25.69 per thousand (+/- 0.92 per thousand), -26.35 per thousand (+/- 0.68 per thousand), and -24.26 per thousand (+/- 0.70 per thousand), for 5 beta A, 5 alpha A, and 5 beta P, respectively. 5 beta P was greater than 5 beta A and 5 alpha A (P <0.01), and 5 beta A was greater than 5 alpha A (P <0.01). The means - 3 SD were -28.46 per thousand, -28.39 per thousand, and -26.37 per thousand for 5 beta A, 5 alpha A, and 5 beta P, respectively. The maximum difference between 5 beta P and 5 beta A was 3.2 per thousand, and the maximum 5 beta A/5 beta P was 1.13. Three athletes with chronically elevated T/Es had delta(13)C values consistent with testosterone administration and three did not.


CONCLUSIONS
This GC-C-IRMS assay of urine diols has low within- and between-assay SDs; therefore, analysis of one urine sample suffices for doping control. The means, SDs, +/-3 SDs, and ranges of delta(13)C values in a control group are established. In comparison, testosterone users have low 5 beta A and 5 alpha A, large differences between 5 beta A or 5 alpha A and 5 beta P, and high 5 beta A/5 beta P and 5 alpha A/5 beta P ratios.
Aguilera
R
R
UCLA Olympic Analytical Laboratory, Department of Molecular and Medical Pharmacology, University of California at Los Angeles, Los Angeles, CA 90025, USA. rodrigoa@ucla.edu
Chapman
T E
TE
Starcevic
B
B
Hatton
C K
CK
Catlin
D H
DH
D015113:Androstane-3,17-diol / Q000652:urine*; D002247:Carbon Isotopes; D004300:Doping in Sports; D004845:Epitestosterone / Q000652:urine*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008297:Male; D011276:Pregnanediol / Q000652:urine*; D015813:Substance Abuse Detection / Q000379:methods*; D013739:Testosterone / Q000652:urine*
D002247:Carbon Isotopes; D015113:Androstane-3,17-diol; D013739:Testosterone; C016836:allopregnane-3,20-diol; D004845:Epitestosterone; D011276:Pregnanediol