Alma Mater Studiorum – Università di Bologna DOTTORATO DI RICERCA IN SCIENZE VETERINARIE Ciclo XXX Settore Concorsuale: 07/H2 Settore Scientifico Disciplinare: VET/04 MYCOTOXIN DETERMINATION IN NON CONVENTIONAL MATRICES: DEVELOPMENT OF MASS SPECTROMETRY BASED ANALYTICAL METHODS Presentata da: Dott.ssa Adele Repossi Coordinatore Dottorato Supervisore Chiar.mo Prof. Arcangelo Gentile Chiar.ma Prof.ssa Teresa Gazzotti Esame finale anno 2018 ABSTRACT Mycotoxins are low-molecular-weight natural products produced, as secondary metabolites, by filamentous fungi. These molecules represent a wide chemical group with different toxicity effects in human and other animals. These toxins can accidentally occur in food and feed, due to a direct or indirect contamination. The aim of this work was to develop a method for the first time to quantitatively determine zearalenone and its metabolites (α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol, zearalanone) in bovine and human hair using LC-MS/MS. Once the method was set-up for bovine hair, it was successfully validated according to Decision 657/2002/CE on three analytes, with satisfying performances. Moreover the applicability of the method was tested on human hair in a one-day validation with reasonable performances. This method could be a useful tool to evaluate natural feed contamination or detect illegal use of α- zearalanol in bovines and to perform a first inventory of the occurrence of these molecules in bovine and human hair, as biomarkers for zearalenone exposure in future studies. Another purpose of this work was to make a preliminary screening on mycotoxins contamination (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, deoxynivalenol, zearalenone, fumonisin B1, fumonisin B2, ochratoxin, T-2 toxin and HT-2 toxin) in different pet food types for cats. This research showed that mycotoxin occurrence in pet food for cats can represent an issue to put under control. Since pets are fed with the same type of pet food for long periods of their life, the necessity of evaluating mycotoxin presence in feed is evident. Summary 1. GENERAL INTRODUCTION ............................................................................ 1 1.1 MYCOTOXIN PRODUCTION .................................................................................... 3 1.2 CLASSIFICATION ........................................................................................................ 4 1.2.1 Aflatoxins ..................................................................................................................... 5 1.2.2 Trichothecenes ............................................................................................................ 7 1.2.3 Fumonisins .................................................................................................................. 8 1.2.4 Zearalenone ................................................................................................................ 10 1.2.5 Ochratoxins ................................................................................................................ 10 1.3 EXPOSURE AND HEALTH EFFECTS .................................................................... 12 1.4 PREVENTION AND DECONTAMINATION STRATEGIES ............................... 14 1.5 LEGISLATION ............................................................................................................ 17 1.6 METHODS OF ANALYSIS ........................................................................................ 23 1.6.1 Sampling .................................................................................................................... 23 1.6.2 Analytical Techniques ............................................................................................... 25 2. AIM OF THE THESIS .......................................................................................... 28 3. ZEARALENONE AND METABOLITES ...................................................... 29 3.1 PROJECT INTRODUCTION .................................................................................... 29 3.1.1 Hair composition and development .......................................................................... 29 3.1.2 Mechanisms of xenobiotics incorporation into hair .................................................. 31 3.1.3 Hair preparation and decontamination .................................................................... 33 3.1.4 Hair biomonitoring ................................................................................................... 35 3.1.5 Bovine hair analysis ................................................................................................... 35 3.1.6 Zearalenone biotransformation and exposure .......................................................... 37 3.1.7 Analytical challenges and current state-of-the-art literature ..................................... 41 3.2 MATERIALS AND METHODS ................................................................................ 44 3.2.1 List of abbreviations .................................................................................................. 44 3.2.2 Sampling .................................................................................................................... 45 3.2.3 Glassware and consumables ..................................................................................... 46 3.2.4 Lab Equipment ......................................................................................................... 46 3.2.5 Solvents and reagents ................................................................................................ 47 3.2.6 Sample preparation and sample series...................................................................... 50 3.2.7 Analytical conditions ................................................................................................. 53 3.2.8 Method validation in bovine hair .............................................................................. 56 3.2.9 Method development in bovine hair ......................................................................... 67 I 3.2.9.1 Extraction/Clean-up set-up ................................................................................... 67 3.2.9.2 LC conditions set-up .............................................................................................. 85 3.2.10 Applicability of the method on human hair ............................................................ 88 3.2.11 Method validation on human hair ........................................................................... 97 3.3 RESULTS AND DISCUSSION .................................................................................. 99 3.3.1 Set-up and validation method in bovine hair ............................................................ 99 3.3.2 Method development in bovine hair ........................................................................ 108 3.3.3 Set-up and one day validation on human hair ......................................................... 117 3.3.4 Applicability of the method for the analysis of human hair samples ...................... 121 4. MYCOTOXINS IN PET FOOD FOR CATS ................................................ 122 4.1 PROJECT INTRODUCTION ................................................................................... 122 4.1.1 Pet food for cats and mycotoxin risk ........................................................................ 122 4.1.2 Legislation ................................................................................................................ 124 4.2 MATERIALS AND METHODS ............................................................................... 126 4.2.1 List of abbreviations ................................................................................................. 127 4.2.2 Sampling ................................................................................................................... 127 4.2.3 Glassware and consumables .................................................................................... 128 4.2.4 Lab equipment ......................................................................................................... 128 4.2.5 Solvents and reagents ............................................................................................... 128 4.2.6 Sample preparation .................................................................................................. 130 4.2.7 Analytical conditions ................................................................................................ 132 4.2.8 Statistical analysis .................................................................................................... 136 4.3 RESULTS AND DISCUSSION ................................................................................. 137 4.3.1 Measurement range .................................................................................................. 137 4.3.2 Concentration of mycotoxins in commercial pet food for cats ............................... 138 5. CONCLUSIONS ..................................................................................................... 149 6. BIBLIOGRAPHY ................................................................................................... 151 II 1. GENERAL INTRODUCTION Mycotoxins are low-molecular-weight natural products (250 to 720 Da) produced as secondary metabolites by filamentous fungi belonging to the phylum Ascomicota and mainly