June 2012

nabolic androgenic steroids (AAS) and other anabolic agents are widely abused as performance-enhancing drugs by athletes of all abilities—from elite competitors to participants in amateur sports to high-school students. AAS and other anabolic compounds are banned by most amateur sports anti-doping programs, the World Anti-Doping Agency (WADA), and many professional sports organizations (1). In 2010, 60% of all adverse analytical and atypical findings reported by WADA were for AAS or related substances (2). Although anecdotal evidence suggested AAS use was prevalent among athletes in the early 1970s, inadequate testing methods limited efforts to detect their use. A breakthrough came in the mid-1970s with the introduction of AAS testing using radioimmunoassay (RIA) (3,4). Not long thereafter, AAS testing by gas chromatography-mass spectrometry (GC-MS) began (5). By 1975, the International Olympic Committee (IOC) banned the use of AAS. The first official AAS testing occurred during the 1976 Olympic Games in Montreal, where RIA was used to screen urine specimens and GC-MS was used for confirmation (6). GC-MS quickly became the gold standard for steroid testing and has been used for both screening and confirmatory analyses. Recently, however, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been used increasingly for the detection of analytically challenging AAS. In modern anti-doping laboratories both GC-MS(/MS) and LC-MS/MS are used to detect and confirm AAS use. Currently, anti-doping scientists are faced with two major challenges regarding the detection of AAS use. The first challenge is to differentiate administration of endogenous steroids from those naturally produced; the second is to detect the use of synthetic “designer steroids.” Unequivocal proof of synthetic AAS administration can be demonstrated by using MS coupled with separation by GC or LC to identify the steroid, its metabolites, or both in urine. However, none of these techniques can readily distinguish exogenous use of natural steroids, such as testosterone and its precursors, from endogenous steroids. Similarly, the use of synthetic designer compounds cannot be determined easily using routine MS screening and confirmation methods unless the compound has been previously identified and the method has been programmed to detect it. The doping agents and techniques used by athletes are constantly evolving, requiring creative approaches for detection and presenting ongoing challenges for anti-doping scientists.