New analytical methodologies for doping control – detection of anabolic androgenic steroids in human urine

Dissertation submitted to Faculdade de Ciências e Tecnologia - Universidade Nova de Lisboa in fulfilment of the requirements for the degree of Doctor of Philosophy (Biochemistry - Biotechnology)The use of anabolic androgenic steroids (AAS) and other banned substances to enhance athletic performance has important health and social implications. The AAS are a major group included in the prohibited list of the world anti-doping agency (WADA) as well as of major sports authorities. This class of drugs, along with other anabolic agents, represent 64,9 % of all adverse analytical findings reported by WADA accredited laboratories, as stated in the WADA statistic report for 2009. The AAS are a class of hormones that include the natural male sex hormone, testosterone, and its many synthetic derivatives. They exert multiple actions affecting both the physiology of the human body and the individual behaviour. Under intensive training, the AAS induce the synthesis of proteins in muscle and bone causing an accelerated growth of these organs. Furthermore, during acute endurance workout, as well as during competition, androgen’s action seems to be critical to enhance the performance capacity, since they affect the production of red blood cells and increase neural conduction. In addition, after intense exercises, androgens are thought to prevent muscle catabolism and exhaustion and to speed up the recovery process. In general, the normal proceeding for AAS determination includes chromatographic separation coupled to mass spectrometry detectors. The use of GC-MS methodologies is the most employed strategy for AAS control. However, over the last years, with the development of suitable LC-MS and LC-MS/MS systems, some AAS presenting poor chromatographic properties for GC-MS analysis, even after derivatisation, are being analysed by LC-MS(/MS) procedures. The aim of the research programme presented in this thesis was, primarily, the development of a new screening method based on mass spectrometry (MS) using the soft ionisation technique matrix-assisted laser desorption/ionisation (MALDI). The major goals to be achieved were the development of an accurate, sensitive and robust methodology able to improve the screening of AAS for doping control in both analysis time and sample throughput. Additionally, the developed method should be capable to overcome the GC-MS limitation related to thermo-labile and polar AAS, so that the initial screening method could be extended to all AAS included in the prohibited list. In parallel with the development of a screening procedure based on MALDI-MS(/MS) techniques, and applying the deep expertise of the research group on reaction enhancement by delivery energy based techniques, the improvement of the global sample preparation for the analysis of AAS by anti-doping control laboratories was also included in the research programme.Fundação para a Ciência e a Tecnologia”, for financial support through the grant SFRH/BD/31652/200