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New test makes the drug cheats easier to detect

By Dr Matt Wilkinson , 27-Jun-2008

US researchers have developed a more sensitive method of detecting whether athletes have taken drugs to artificially boost testosterone which increases physical performance.

The research, published in a special issue of the Journal of Mass Spectrometry concentrating on the detection of performance enhancing drugs in sports, details the development of a faster and more sensitive method of detecting abnormally high levels of testosterone. One of the major biological roles of the male hormone testosterone is to increase muscle size and strength, and therefore taking testosterone, or a chemical that can create extra testosterone is banned by the World Anti-Doping Agency (WADA). Following a positive drug test for unusually high levels of testosterone, 2006 Tour de France winner Floyd Landis was stripped of his title. In protesting his innocence, Landis attacked the procedures used by the laboratory to determine his T/E ratio which is one of 34 anti-doping laboratories in WADA's international network. Exact levels of testosterone vary considerably between different people, so simply measuring the total testosterone in an athlete's urine does not show whether he or she has deliberately taken extra. However, a second chemical in the body called epitestosterone is normally present in approximately equal proportions to testosterone. Comparing the ratio of testosterone to epitestosterone (T/E) can indicate whether testosterone or a precursor has been taken. However measuring the concentrations of these two substances is not easy as they are only present at very low concentrations in urine. WADA currently allows a maximum T/E ratio of 4:1, but according to Landis' personal physician, his levels were as high as 11:1. The new test uses liquid chromatography tandem mass spectrometry (LC/MS/MS) to determine the relative concentrations of testosterone and epitestosterone more accurately than ever before. Typically, quantitative analysis is conducted using gas chromatography / mass spectrometry (GC/MS) techniques. However, sensitivity issues make unambiguous identification difficult. This has led to the use of Isotope Ratio Mass Spectrometric (IRMS) analysis to confirm the use drugs to boost testosterone levels. According to Dr Jonathan Danaceau of the University of Utah and lead author of the report, the new method "means that we can determine the T/E ratio in a sample with greater confidence, and therefore be in a better position to spot doping violations without falsely accusing innocent athletes." The researchers used an Agilent 1100 series HPLC (high performance liquid chromatography) instrument connected to an Applied Biosystems QStar-XL tandem MS equipped with a heated electrospray ion source. The technique enables Girard's Reagent P (1-(Hydrazinocarbonylmethyl)pyridinium Chloride) derivatised testosterone and epitestosterone to be quantitatively analysed at concentrations as low as 1 ng/ml. Giraud's Reagent P enables a dramatic increase in the ability to analyse poorly ionising compounds such as testosterone and other steroids. "Not only is the test more sensitive, it is also faster to perform," said co-author Scott Morrison. Indeed the researchers needed less than 16 minutes to run a single analysis using the new method, compared to 25 minutes using standard GC/MS techniques. "Having this sort of test available makes cheating harder and lets us take one more step towards enabling free and fair competition," said Laboratory Director Dr Matthew Slawson.

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