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

Challenges for Today & the Near Future: From GC/MS to LC/MS

In the search for the ultimate invisible substances, cheaters finally developed an apparently winning strategy: to design, synthesize – and use – previously unknown molecules. Designer steroids like norbolethone, tetrahydrogestrinone and desoxymethyl T (Madol) were designed and synthesized, out of the channels of the official pharmaceutical industry, with the specific goal of obtaining an anabolic effect without the risk of being caught at the test. The reply from the testers came, once again, from MS, this time used in combination with liquid chromatography: LC/MS- and LC/MS-MS-based techniques, previously used mainly as advanced confirmation techniques, entered from the main door in the anti-doping laboratories to identify the presence in the urine also of designer steroids.[10–12] Norbolethone, tetrahydrogestrinone and Madol were followed by other designer steroids, but the period of invisibility of these newly developed doping agents became shorter and shorter. The labs were alerted: the monitoring activity of suspicious signals by the accredited laboratories further reduced the risk of abuse of unknown steroids. LC/MS-MS systems, also equipped with the new generation of mass spectrometers, can be the ultimate tool for the anti-doping analyst and for the detection of previously noncharacterized target compounds [13–15]; in particular, LC/MS systems equipped with Q-TOF-based detectors also allow the storage of full-scan MS data for each relevant analytical signal, making possible the a posteriori evalutation of data: in other words, these techniques allow the storage of data today to identify an unknown molecule whose identity will be revealed later on, without the need of re-analyzing the sample.[16]

At present, the recourse to LC/MS-MS is progressively increasing among the WADA-accredited laboratories for a number of reasons, and not only to face the new challenges of doping analysis. LC/MS allows the drastic simplification of the overall analytical procedures in the case of low-molecular weight xenobiotics, also reducing the complexity of the pre-treatment steps. In this sense, the WADA-accredited laboratories are extending the range of applications of LC/MS-based methods in their daily routine work.

But perhaps the most striking feature of LC/MS and LC/MS-MS is that of allowing the analysis of peptides and proteins with a higher selectivity with respect to the traditional immunological techniques. Indeed, the 1990s had also opened a new frontier in doping and anti-doping: in that period the abuse of peptide hormones and, more generally, of biologically active macromolecules, started to accompany the traditional low-molecular-weight doping substances, due to the increased availability of the synthetic macromolecules, produced mainly by recombinant DNA technologies (reviewed in [17]). human chorionic gonadotropin, erythropoietin and human growth hormones were reportedly illicitly used by the athletes respectively to alter the urinary steroid profile (making more problematic the detection of the intake of T and/or its precursors), to improve the oxygen carrying capacity of the blood, and to achieve a generic anabolic effect without the risk of being caught at the test. Other macromolecules, like modified polysaccharides (e.g., dextran and hydroxyethylstarch) used as plasma volume expanders, could also be abused in combination with the administration of erythropoietin and with other blood doping methods, including hemoglobin-based oxygen carriers.

Although advanced immunological techniques have been for a long time the analytical point of reference in the detection of polypeptides and proteins, the contribution of MS-based detection methods is progressively increasing also in these areas. Techniques based on LC/MS-MS are currently used by WADA-accredited laboratories for the confirmation analysis of plasma volume expander and hemoglobin-based oxygen carriers (reviewed in [18]). In addition to this, a considerable number of studies on the suitability of LC/MS-MS for the detection of previously undetectable peptide hormones (e.g., insulins, synachten and other synthetic peptide hormones) have also been published (reviewed in [19]).


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