Investigation on the origin of prednisolone in urine and adrenal glands of cows.

Prednisolone is a steroid belonging to the corticosteroid group. The results obtained in the application of the 2008 and 2009 Italian Residue Control Plans show the frequent detection of prednisolone traces in cow's urine. Since most of the positive samples were detected at the slaughterhouse, the researchers hypothesised that, together with an increase of cortisol concentration, traces of prednisolone could be produced endogenously during stressful situations due to transport and handling before slaughter. In the present trial, 52 lactating cows housed in seven different farms in Lombardy, Italy, were studied. Urine samples were collected at the farm (after urethral catheterisation) and immediately after slaughter (from urinary bladder) together with 40 adrenal gland samples belonging to the same animals. All the samples were analysed for the determination of prednisolone and cortisol by LC/MS(n). The results demonstrated that prednisolone can be endogenously produced in dairy cows and, furthermore, its endogenous presence in bovine urine seems to be strongly related to a state of stress in the animals (at the farm and at the slaughterhouse). The data from adrenal glands do not, however, clarify if the endogenous production occurs, partially or totally, in this organ.

Analytical investigations about the presence of prednisolone in cow urine.

Since 2008, the analyses carried out in the Lombardia region as part of National Residue Control Plans have evidenced unexpected frequent detection of the corticosteroid prednisolone (PRED) in cow urine samples taken to the slaughterhouse. Considering the scarce plausibility of these high frequent findings, analytical investigations were started to ascertain the real presence of this corticosteroid. The applied confirmatory method involved liquid-chromatography low-resolution tandem mass spectrometry (triple quadrupole) as instrumental technique, and it was validated in compliance with the requirements of the Commission Decision 2002/657/EC. However, recently some criticism regarding Commission Decision 2002/657/EC identification criteria has been pointed out, experimentally demonstrating false positive results (wrong identification) although these criteria have been strictly observed. Therefore, considering the serious implications (i.e. the possibility that PRED could be considered endogenous in particular animal conditions), studies were carried out to investigate the reliability of PRED identification through the change of the chromatographic conditions (mobile phases, gradient and analytical column) of the confirmatory procedure routinely applied. Further confirmation came from the application of high-resolution mass spectrometry technique (MS(2) and MS(3) experiments) to analyze incurred cow urines samples. All the obtained results confirmed definitively the real presence of this corticosteroid excluding false-positive findings in routine analysis. In addition, other experiments demonstrated that high-resolution mass spectrometers (Time of Flight and Orbitrap technologies) could be successfully applied to routine determination of steroid residues in biological fluids at very low concentrations (< 1 µg L(-1)).