Mass Spectrometry and Illicit Drug Testing: Analytical Challenges of the Anti-doping Laboratories

Francesco Botrè


Expert Rev Proteomics. 2008;5(4):535-539. 

In This Article

The "Middle Age": Detection of Testosterone & Its Precursors

In the "cops and robbers" interplay between testers and cheaters, the next challenge was represented by the detection of the abuse of testosterone (T) itself and, later on, of some among its metabolic precursors. The direct detection of T and its endogenous analogs was initially considered almost impossible at that time, since the molecular structure of the synthetic steroids is identical to that of the endogenous hormone. Furthermore, the urinary concentration of endogenous hormones is not constant, even within the same subject, so that it would have been unreasonable to develop an approach based on a "threshold" value. The detection strategy was consequently based on the measurement of indirect or relative parameters, mainly the concentration ratio between T and its naturally present epimer, epitestosterone (E). The T/E concentration ratio was measured to be around 1 in a reference Caucasian population,[7] so that preliminary evidence of a possible intake of T could be based on the measurement of the T/E ratio. A T/E ratio threshold value was originally fixed at 6, to be subsequently raised to 10 and then lowered again to 6, where it stayed until 2004, when it was finally lowered to 4.

The first pilot tests to detect the abuse of synthetic T were carried out at the beginning of 1980, and became part of the normal laboratory practice in the late 1980s and 1990s. The consensus procedure that was developed in the mid 1990s was that of considering a T/E ratio greater than 6 simply as a possible index of T abuse: since cases of physiologically elevated T/E ratios are statistically possible, a T/E value greater than 6 (nowadays 4) was (is) not itself a proof of doping, but simply preliminary evidence which should promote further investigation, including repeated, no-advance-notice retesting of the athlete.

This approach, although the only one to be reasonably followed at that time, had two main limitations:

  • The number of false-negative cases could be significant: the T/E ratio values depends on many parameters, and also on ethnicity: an individual with baseline values of the T/E ratio of 0.1 or lower could intake relatively high amounts of synthetic T and still remain with a T/E value smaller than the threshold;

  • Other substances capable of lowering the T/E ratio could be used as "masking agents": these included E, but also peptide hormones like the gonadotropins, and primarily among them human chorionic gonadotropin.

While the second problem was quickly dealt with by adding the potential masking agents or T/E value modulators to the list of prohibited substances, the first limitation still stood. The problem was apparently without solution because of the apparent lack of easily detectable differences between the molecular structure of synthetic hormones with respect to that of the endogenously produced hormones. The solution came in the late 1990s and it was, once again, based on a MS technique, again combined with GC: at the turn of the millennium the final instrumental evidence of a doping offense with synthetic T was based on GC coupled to combustion (C) isotopic ratio MS (IRMS; GC-C-IRMS). IRMS is indeed a technique that measures the relative abundance of stable isotopes in a sample. In the case of carbon-IRMS, the isotopes are the two stable isotopes of carbon (12C and 13C). If coupled to GC, IRMS can be used to measure the relative abundance of 12C and 13C in organic compounds present in complex matrices, including biological fluids. In the anti-doping laboratories, GC coupled to IRMS could therefore be used to discriminate between the endogenous and synthetic origin of naturally produced steroids, mainly T and/or its precursors and metabolites, since the synthetic compounds have less 13C than their endogenous homologues (reviewed in[8–9]). The limitation of this technique is that it is not sensitive enough to detect all cases of synthetic T administration: the most promising strategy, presently in the final stages of its development, is based on the combination of IRMS with longitudinal testing, aimed to build up reference values for each athlete, therefore putting the focus on the development of individual, rather than population, threshold values of reference parameters (including, but not limited to, the value of the T/E ratio).


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