THE DOUBLE BOND IN Δ1-2 POSITION OF STEROIDS: A MATTER OF CONTROVERSY IN THE CONTROL OF ILLICIT TREATMENTS OF FARM ANIMALS

Two tight correlated topics were dealt in this thesis: - Bovine urine can result positive for boldenone, an androgenic steroid with the structure of testosterone with a double bond in Δ1-2 position of ring A, and androstadienedione, its 17-oxidized form, in consequence of faecal contamination, mainly through a high neo production in faeces-contaminated urine. A simple in vitro method was developed – incubation of faecal matter suspended in 0.9% saline – to induce steroid transformations in faeces, and analyzed the products by liquid chromatography/tandem mass spectrometry, without the need for prior extraction. Androstenedione, androstadienedione, alfa and beta boldenone, testosterone and epitestosterone transformations were investigated. Mutual interconversions of steroids were observed, although 17 beta hydroxy steroids had lower stability if compared with 17 alfa hydroxy and 17-keto steroids. This simple in vitro system proved to be an effective way of studying hormone transformations in faeces and in faeces-contaminated urine. An analogous method, i.e. the direct injection, for LC/MS-MS analysis, of diluted equine urine, without prior extraction, was carried out to evaluate the stability of phase II metabolites of boldenone, testosterone and epitestosterone (glucuronides and sulphates) in intentionally poorly stored urine. Both 17 beta and 17 alfa-glucuronide steroids hydrolyzed to their correspondent hydroxysteroids, but only the 17 beta epimers oxidized to the keto forms. The sulphate compounds showed a complete stability. The transformations were temperature dependent: faster at room temperature than at 4°C, while did not occur in frozen urines. This indicated that proper storage conditions of biological samples are fundamental to avoid microbiological contamination that can cause chemical modifications of androgen steroids. - After a two-year period of the frequent detection of prednisolone-positive bovine urine samples in the Italian region of Lombardy, studies were initiated to investigate the source. The problem of modifications in biological fluids was therefore evaluated also regarding a possible transformation of cortisol and cortisone into prednisolone and prednisone in cattle faeces. The transformation of the II phase metabolite cortisol glucuronide was studied, too. The method consisted once more of incubation of bovine faecal matter suspended in 0.9% saline spiked with cortisol, cortisone and cortisol glucuronide (400 ng/ml). The deconjugation of the conjugated form the and the dehydrogenation in ring A occurred. It was so concluded that also urine contaminated with faeces may be positive for prednisone and prednisolone in the same way as they are positive for boldenone, i.e. as a result of microbiological dehydrogenase activity on cortisol and cortisone. The successive step was to assess the transformation of cortisol and cortisone in urine collected from the bladder at the slaughterhouse (i.e. without faecal contamination) and incubated at 37°C for 24 hours. The influence of deconjugation with beta-glucuronidase from Helix pomatia was assessed, too. Only urine where prednisolone and prednisone were absent and cortisol was higher than 80 ng/mL were used. In this study a liquid-liquid extraction with a mixture of tert-Buthylmethylether:Ethylacetate (80:20) was performed, to concentrate the sample, so raising the sensitivity of the method. The analyses were carried out by LC MS3. It was demonstrated that a relatively high temperature (e.g. due to inappropriate storage conditions or to enzymatic deconjugation), could give results that do not represent the effective presence and concentration of the abovementioned corticosteroids at the moment of the sample collection. However, because of the stressful conditions of the slaughterhouse, the by-production of prednisolone was supposed, together with the increase of cortisol and cortisone. The urines of three dairy cows were therefore collected and analyzed before and after a pharmacological induced stress (intra muscular administration of tetracosactide hexaacetate, an adrenocorticotropic hormone analogue), and before and after slaughter. Prednisolone was occasionally detected in the non-stressful state, but was consistently found in the urine of stressed cows (from 1.01 to 4.08 ng/mL), together with the raise in concentration of urinary cortisol and cortisone, so indicating a possible endogenous production of prednisolone in stressful situations.