Forensic application of isotope ratio mass spectrometry: development and validation of GC-C-IRMS based methods in doping analysis

The isotope ratio mass spectrometry coupled to gas chromatography (GC-C-IRMS) is a mandatory procedure in anti-doping analyses performed on urinary samples in which the concentration and/or the ratio concentrations of specific steroid profile markers overcome the limits defined by the World Anti-Doping Agency (WADA) or deviate from the population and individual reference range. It is a confirmatory investigation to establish whether these alterations, if not caused by physiological reasons, can be attributed to a synthetic steroids’ intake. Indeed, the determination of their 13C composition, which is typically more depleted for pharmaceutical preparations compared to their physiologically produced counterparts, allows disclosing the exogenous or endogenous origin of the steroid compounds. In this Thesis, novel approaches have been implemented to simplify, standardize the current operating protocols, and reduce the risk of misinterpreting the analytical findings. The benefits of performing a large volume injection by a programmed temperature vaporizer inlet have been proved in analyzing steroids showing low urinary levels and new methods for detecting the prednisolone/prednisone, and 19-norsteroids abuse have been developed and fully validated. In order to provide a detailed overview of the IRMS technique, some criticalities have been also emphasized, proposing, whenever possible, suitable troubleshooting