INNOVATIVE ANALYTICAL METHODS FOR THE EVALUATION OF ILLEGAL ADMINISTRATION OF “GROWTH PROMOTING” SUBSTANCES IN SPORTS AND LIVESTOCK

Development and optimisation of novel analytical methods (capillary electrophoresis and high performance liquid chromatography coupled with UV/VIS and Mass Spectrometry) for the determination of anabolic androgenic steroids (AASs) and peptide hormones in biological fluids. In particular:  investigation of natural and synthetic AASs and their main metabolites in urine by HPLC-MS/MS developing easy, quick and highly specific multiresidue analyses; allowing to determine excretion profiles and the ratios between conjugated and free fractions of the target compounds.  development of direct methods for the determination of exogenous human Growth Hormone (hGH) administration in human biological fluids, using CE- UV/Vis for screening purposes and Mass Spectrometry (with Q/ToF system) for confirmatory purposes. The CE-UV/Vis method could be based on the determination of concentration ratio between the major pituitary isoforms (22 kDa and 20 kDa) of the hormone and on the changes induced by an injection of recombinant hGH, which is composed only by the 22 kDa isoform. The research will be subdivided in three fractions: I. The development and validation of a Multiresidue High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) method for the determination and quantification of natural hormones and their Phase I and II metabolites (5 endogenous steroids and their respective sulphate and glucuronide forms) in human and bovine urine, in order to obtain relevant data to describe natural physiological excretion patterns on the basis of species, age and sex. II. The development and validation of a Multiresidue High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) method for the determination and quantification 17 β-boldenone, a synthetic androgen steroid hormone with anabolic effects widely used for fraudulent purposes, its Phase I metabolites (5 β-androst-1- en-17β-ol-3-one and 17α-boldenone) and Phase II (the same hormones already cited as sulphates and glucuronides) in urine. The analytical technique used, alternative to the routinely used technique in official laboratories (GC-MS with hydrolysis of the sample prior to determination of the total fraction of the excreted hormone), is the HPLC coupled to Tandem Mass Spectrometry (MS/MS). This technique is extremely selective in Selected Reaction Monitoring (SRM) mode and allows certain identification of the investigated molecules. The developed methods will allow to simultaneously analyze the Phase I (free fraction) and Phase II (conjugated fraction) metabolites of these hormones in a single chromatographic run without manipulation of the sample (no hydrolysis and derivatisation) apart from an early stage of clean up. This translates into significant cost and time savings. III. The study of the electrophoretic behaviour of the human Growth Hormone (hGH) and optimization of the analytical conditions, using Capillary Zone Electrophoresis (CZE) coupled with spectrophotometric UV-Vis detector. This research will be followed by the evaluation of some validation parameters such as linearity, the limit of detection (LOD) and the limit of quantification (LOQ), the repeatability (run to run and day to day). Studies on the qualitative determination of the exact masses of the major isoforms of human GH will be carried out by hybrid Quadrupole/Time of Flight (Q/TOF) mass spectrometric measurements on standards of hGH (extracted from pituitary gland) and on pharmaceutical preparations of hGH obtained by recombinant DNA technology.