Phd in PHARMACEUTICAL SCIENCES

Thesis title: Emerging strategies in antidoping analysis: new procedures, new biomarkers, new matrices

The primary purpose of anti-doping authorities is to protect the integrity of sports, ensuring that athletes can participate in clean competitions while safeguarding their health. The doping control system is tasked with developing and validating analytical procedures to detect substances and methods of the World Anti-Doping Agency (WADA) Prohibited List. This thesis aims to investigate and solve some of the analytical challenges faced by anti-doping laboratories. In detail, the first part of the thesis describes the development of two multi-analyte methods for the detection in the urine of substances belonging to two different classes of emerging drugs: selective androgen receptor modulators (SARMs) and hypoxia-inducible factor activating agents (HIFs) listed in section S1 as "other anabolic agents" and section S2 as "peptide hormones, growth factors, related substances, and mimetics," respectively. For this purpose, each substance's chromatographic behavior and characteristic fragmentation profile were studied. In addition, in vitro studies using human liver microsomes (HLM) were conducted to select the optimal intake markers. As a result, the optimal markers for SARMs and HIF activating agents were established, and the analytical procedures developed and optimized were validated according to ISO 17025 and WADA requirements for the accredited laboratories. The second part focused on the reduced availability of blood samples for drug testing. According to WADA's 2020 report, less than 10% of the samples collected for doping control tests are venous blood samples. For this reason, WADA is encouraging the development of analytical methods that detect prohibited substances and methods in new blood matrices characterized by less invasiveness, such as capillary blood or dried blood spots (DBS). More specifically, an analytical method was developed and successfully validated for detecting a substance minimally excreted in the urine, IGF-1. IGF-1 is an endogenously produced substance, an in vivo effector of the action of GH, but is also taken exogenously given its anabolic properties for sports performance enhancement. Unfortunately, it is impossible to detect IGF-1 doping, as we cannot discriminate between the endogenous and exogenous origin of IGF-1 as they are chemically identical. Therefore, the method was applied to evaluate the circadian and longitudinal fluctuations of IGF-1 in male and female volunteers. Part of this investigation was conducted in collaboration with the UniL Centre de recherche et d’expertise des sciences anti-dopage (REDs). In detail, the aim was to plan a longitudinal study to determine individual profiles of IGF-1 of elite athletes and non-athletes to evaluate the implementation of the athlete biological passport (ABP) with its monitoring in the Endocrinological Module. Indeed, abnormal trends in basal IGF-1 levels could be an indicator of exogenous IGF-1 intake or related growth factors. The use of alternative matrices, such as capillary blood, whether whole or spotted/adsorbed on filter media, can be beneficial not only in the case of detection of substances detectable only and exclusively in blood but also as a complementary analysis in cases of controversial positive results: an analytical method for the detection of small molecules in Dried Blood Spot/ Dried Plasma Spot by UHPLC-HRMS, comparing different support was developed and validated.