DEVELOPMENT AND VALIDATION OF METHODS FOR THE DETECTION OF RESIDUES IN UNCONVENTIONAL AND INNOVATIVE MATRICES THROUGH LC-MS/MS ANALYSES FOR SAFETY OF FOOD OF ANIMAL ORIGIN.

Successful animal growth depends on a combination of many factors related to health, management and nutrition. The use of veterinary drugs in food-producing animals for therapeutic purposes is regulated (corticosteroids, antibiotics) or banned (anabolic steroids) in the European Union; however, their use as growth promoters cannot be excluded. Moreover, the eventual presence of residues in food constitutes a fraud and a health issue for the consumers. For these reasons the need to find new accumulation matrices and new sensitive, specific and robust methods that are able to reveal the presence of drug residues is essential, based on the fact that there is a low percentage of non-conformity in the final reports of the National Residues Plan in recent years, although the threat of a disproportionate use of these substances is increasingly on the rise. In the light of these facts, there is the need to implement the framework of controls aimed to food safety, due to the inefficiency of tools for the study of these substances. Often, the use of conventional matrices, such as urine, liver or muscle, recommended for the official controls of illegal treatment are not completely satisfactory due to the fast elimination rate of the compounds or to the difficulties arising from the compounds characterised also by a pseudoendogenous nature. The debate about the presence of β-boldenone II phase metabolites and prednisolone in urine samples, owing to endogenous or illicit treatment, is currently ongoing within the European Union. These compounds have been appropriately defined “grey-zone substances”, for their double origin. The simple detection of some steroids in urine is currently considered to provide insufficient evidence of illicit treatment. Parameters such as cut-off levels, the presence of metabolites, or both, must be accounted for. As regards antibiotics, the overuse, over the last decades, as growth promoters in food producing animal have caused favorable condition about the threat of bacterial resistance. The antibiotics can directly affect the consumer in the form of residues from the food chain, or by accumulation in the environment via the application of manure to land as organic fertiliser, via sludge storage or by direct contamination of illicitly additivated water and feed. The main challenge is to monitor contemporally different antibiotic classes, in different steps of the food chain, trying to control this phenomenon. On the other hand, food contamination by new environmental contaminants should not be neglected. In particular, perfluoroalkyl substances (PFASs) have recently aroused great scientific interest and concern for public health, due to the fact they have been found in appreciable concentrations in human serum. On the basis of EFSA requestes and of analytical problems associated with their determination many studies are recommended to monitor their presence, building a database on PFASs in food, evaluate the contamination levels of the individual compound and finally draw up a reliable risk assesstment of European population. This work was born with the aim to detect residues of the most commonly used drugs in broad sense, and then extended over time, also following requests from public and private entities, based on realistic situations of risk. Therefore, based on the mentionated issues, the development, optimisation and validation of multiresidual methods and the direct application on real unconventional matrices allowed us to have a greater amount of information in terms of number, frequency, and concentration of different classes of veterinary drugs than in conventional matrices. We confirmed the presence of pseudoendogenous compounds and their precursors in the unconventional matrix bile, for example. The study of the unconventional matrices, e.g. bovine teeth, has also allowed us to detect esterified forms of some drugs, discriminating them from the active free forms that could have a double, exogenous and endogenous, origin. Finally, this work demonstrates the utility of an eventual introduction, through the food of animal origin chain, of several monitoring points of different types of residues, consisting of non-edible matrix analyses that are not destructive of the product intended for the consumer. On the other hand, the sensitivity and good performance of the developed LC-HRMS methods for the emerging PFASs, could help further studies and also EFSA to increase the number of quantifiable data useful to extend a risk assessment in its final reports.