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Applications  |  Authentication: Endogenous versus Exogenous Drugs


Tracing the presence of exogenous (epi)testosterone in urine of sportsmen.

Introduction

Administration of exogenous testosterone (T; Figure 1) is prohibited in professional sports. This also applies for the naturally occurring epitestosterone (E), also called cis-testosterone. Endogenous concentrations of both substances in human urine are usually quite similar, with a T/E ratio of 1:1 or 2:1. An increased T/E ratio above 4 is considered as an indication of exogenous testosterone administration. A method to mask illegal testosterone application in the T/E ratio test is to self-administer epitestosterone to rebalance this ratio (Dehennin, 1994). Although this practice will of course result in increased absolute concentrations in urine of both testosterone and epitestosterone, the wide concentration range of these steroids in urine often prevents unequivocal conclusions whether exogenous testosterone and epitestosterone have been administered.


Figure 1. Testosterone.

Stable Isotope Solution: Gas Chromotography - Combustion - Isotope Ratio Mass Spectrometry (GC-C-IRMS)

Endogenous testosterone in human beings originates from cholesterol present in vegetables and meat. Plant species often have different 13C levels, with so-called C3 plants (e.g. wheat, soy) having lower levels than C4 plants (e.g. maize, sugar cane). The 13C level of endogenous testosterone therefore reflects the 13C level in the diet. Exogenous testosterone or epitestosterone usually have lower 13C levels than their endogenous counterparts, because they usually originate from soy. Administration of exogenous testosterone or epitestosterone therefore significantly lowers the 13C-values of these steroids in blood and urine samples. GC-C-IRMS is the technique to determine the 13C levels of purified steroids from urine samples and has been accepted as a reliable method to determine testosterone abuse (Aguilera et al, 2002).

Eye-catching Result

On July 27, 2006, The Times Online reported that Floyd Landis (Figure 2), winner of the Tour de France 2006, had tested positive for unusual levels of testosterone in his urine after his remarkable comeback in stage 17 of the Tour. The test showed that the testosterone to epitestosterone ratio (T/E ratio) in Landis' urine was between 4.4:1 (1st test) to 11.8:1 (2nd test), well above the World Anti-Doping Agency's allowable limit of 4:1. Since such values are considered as suspicious, a GC-C-IRMS test (the so-called Carbon Isotope Ratio test, CIR) was performed to determine the 13C-level in the steroid (in fact, the 13C/12C ratio expressed as a d13C‰-value was determined). The test showed a large difference in Landis' testosterone compared with the reference. Unless serious errors have been made during urine sample processing, as was extensively argued by Landis' defenders, the difference leads to a statistically significant conclusion that exogenous testosterone was present in his urine (P<0.001). On September 20, 2007, finally Floyd Landis was officially found guilty of doping by a U.S. arbitration panel. Floyd Landis found out by early 2008 that his final attempt to regain his 2006 Tour de France title was not successful at the Court of Arbitration for Sport (CAS)— the top court in sports. On May 19, 2010, The Wall Street Journal published emails in which Landis confessed the use of performance-enhancing drugs like epo, testosterone, human growth hormone, blood transfusions, and female hormones, thus confirming the reliability of the GC-C-IRMS analyses.

Figure 2.

References

Aguilera R, CK Hatton, DH Catlin. 2002.
Detection of epitestosterone doping by Isotope Ratio Mass Spectrometry.
Clinical Chemistry 48: 629–636.

Becchi M, R Aguilera, Y Farizon, M-M Flament, H Casabianca, P James. 1994.
Gas chromatography/combustion/isotope-ratio mass spectrometry analysis of urinary steroids to detect misuse of testosterone in sport.
Rapid Communications in Mass Spectrometry 8: 304-308.

Dehennin L. 1994.
Detection of simultaneous self-administration of testosterone and epitestosterone in healthy men.
Clinical Chemistry 40: 106 –109.

 

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