Mycotoxins are significant contaminants in food and feeds. These molecules have demonstrated serious effects in both human and animal health. Different studies showed the presence of a multitude of fungal metabolites that contaminate food and feed. Recently, the international health authorities have expressed concerns about the presence of "emerging" mycotoxins in foodstuffs. The term “emerging mycotoxins” is commonly referred to those compounds that are currently under the spotlight of the scientific community and the policy-makers, due to their toxicological profile. Among the fungal species responsible for the metabolite production, Alternaria species have the ability to produce more than 70 toxins, such as Alternariol (AOH), Alternariol monomethyl ether (AME), Tenuazonic Acid (TeA) e Tentoxin (TEN), found in cereals, tomato products, olive oil, fresh fruits and vegetables. These mycotoxins have been recently covered by scientific opinions from the European Food Safety Authority (EFSA), who has expressed concern about the low availability of incidence data in food for human and animal consumption. There is, indeed, a high degree of uncertainty related to the representativeness of the food currently tested because it contains too many inaccuracies. Furthermore, the lack of information on the analytical method used further contributes to the uncertainty of the reported Alternaria toxin levels. More comprehensive studies are required to update the risk evaluation by the EFSA and, therefore, the specific aim of this project was focussed on the development of accurate LC-MS/MS and LC/HRMS methods for the analysis of emerging Alternaria toxins. The protocol was employed in collaboration with the Istituto Zooprofilattico Sperimentale della Sicilia. LC-MS/MS method was used for the target analysis of emerging mycotoxins in food matrices. In particular, the following activities were carried out: • optimisation of the method parameters for simultaneous screening of Alternariol (AOH), Alternariol monomethyl ether (AME), Tenuazonic Acid (TeA), Ochratoxins and Zearalenone (ZEA), Fumonisin B1, B2, B3, T-2 toxin, HT-2 toxin and deoxynivalenol (DON) (vomitoxin); • development of a sample preparation protocol, apply to cereals and dry fruits; the sample preparation protocol was based on a double extraction (Liquid extraction) and purification through solid-phase extraction (SPE). • validation of the method according to EU guidelines (Commission Decision 2002/657/EC); the method was performed by evaluating the following parameters: specificity, precision, instrumental linearity, recovery, decision limit (CCα), detection capability (CCβ), matrix effect and ruggedness. The ultrahigh-performance chromatography coupled to quadrupole-orbitrap high-resolution mass spectrometry (UHPLC-Q-HRMS) was applied to the sensitive and specific determination of the emerging Alternaria toxins. Performances were compared to those obtained by high-performance liquid chromatography detection. All two methods showed good linearity and repeatability. The Q-Exactive mass spectrometer is said to be better suitable for trace detection than state-of-the-art MS/MS methods based on the triple quadrupole, because, despite having performance comparable, it has better selectivity. The research project should have provided a broad investigation of the emerging mycotoxin content in various food products, contributing to an estimate of mycotoxin risk exposure. The difficulties linked to the pandemic in progress made difficult research, initially planned within this project. Despite everything, the purpose of this work was to fill the data gap and provide relevant information to improve the safety of local products.

Le micotossine sono contaminanti significativi negli alimenti e nei mangimi e sono causa di gravi effetti tossici sulla salute umana e animale. Diversi studi hanno dimostrato l’esistenza di una moltitudine di metaboliti fungini che contaminano alimenti e mangimi. Recentemente, le autorità sanitarie internazionali hanno espresso preoccupazione per la presenza di micotossine "emergenti" negli alimenti. Tra le specie fungine responsabili della produzione di tali metaboliti, la specie Alternaria ha la capacità di produrre più di 70 tossine, come Alternariol (AOH), Alternariol monomethyl ether (AME), Tenuazonic Acid (TeA) e Tentoxin (TEN), presenti nei cereali, in prodotti a base di pomodoro, nell’olio d'oliva, nella frutta e nella verdura fresca. Queste micotossine sono state recentemente oggetto di pareri scientifici dell'Autorità Europea per la Sicurezza Alimentare (EFSA), che ha espresso preoccupazione per la scarsa disponibilità di dati sull'incidenza negli alimenti destinati al consumo umano e animale. Esiste, infatti, un alto grado di incertezza legato alla rappresentatività degli alimenti attualmente testati. Inoltre, la mancanza di informazioni sul metodo analitico utilizzato contribuisce ulteriormente a creare incertezza sui livelli di tossine da Alternaria. Sono necessari ulteriori studi per aggiornare la valutazione del rischio da parte dell'EFSA e, pertanto, l'obiettivo specifico di questo progetto era incentrato sullo sviluppo di metodi LC-MS/MS e LC/HRMS accurati per l'analisi delle tossine da Alternaria. Il progetto è stato sviluppato in collaborazione con l'Istituto Zooprofilattico Sperimentale della Sicilia. Il metodo LC-MS/MS è stato utilizzato per l'analisi target delle micotossine emergenti nelle matrici alimentari. In particolare sono state svolte le seguenti attività: • ottimizzazione dei parametri del metodo per lo screening simultaneo di Alternariol (AOH), Alternariol monometiletere (AME), Acido tenuazonico (TeA), Ocratossine e Zearalenone (ZEA), Fumonisina B1, B2, B3, tossina T-2, HT-2 tossina e deossinivalenolo (DON) (vomitossina); • sviluppo di un protocollo di preparazione del campione, da applicare a cereali e frutta secca; il protocollo di preparazione del campione si è basato su una doppia estrazione (estrazione liquida) e purificazione mediante estrazione in fase solida (SPE). • validazione del metodo secondo le linee guida comunitarie (Decisione della Commissione 2002/657/CE); il metodo è stato ottimizzatoo valutando i seguenti parametri: specificità, precisione, linearità strumentale, recupero, limite di decisione (CCα), capacità di rilevamento (CCβ), effetto matrice e robustezza. La cromatografia accoppiata alla spettrometria di massa ad alta risoluzione (UHPLC-Q-HRMS) è stata utilizzata per la determinazione, sensibile e specifica, delle tossine da Alternaria. Le prestazioni ottenute sono state confrontate con quelle ottenute mediante cromatografia liquida ad alte prestazioni. Tutti e due i metodi hanno mostrato una buona linearità e ripetibilità. Si dice che lo spettrometro di massa Q-Exactive sia più adatto per il rilevamento in tracce rispetto ai metodi MS/MS basati sul triplo quadrupolo, perché, nonostante abbia prestazioni comparabili, ha una migliore selettività. Il progetto di ricerca avrebbe dovuto fornire un'ampia indagine sul contenuto emergente di micotossine in vari prodotti alimentari, contribuendo a una stima dell'esposizione al rischio di micotossine. Le difficoltà legate alla pandemia in corso hanno reso difficile la ricerca, inizialmente prevista in questo progetto. Nonostante tutto, lo scopo di questo lavoro è stato colmare il vuoto di dati e fornire informazioni rilevanti per migliorare la sicurezza dei prodotti locali.

Identificazione e quantificazione delle Micotossine prodotte da Alternaria in alimenti e mangimi, mediante LC-MS/MS e LC/HRMS / Vita Giaccone , 2022 Apr 22. 34. ciclo, Anno Accademico 2020/2021.

Identificazione e quantificazione delle Micotossine prodotte da Alternaria in alimenti e mangimi, mediante LC-MS/MS e LC/HRMS.

GIACCONE, VITA
2022

Abstract

Mycotoxins are significant contaminants in food and feeds. These molecules have demonstrated serious effects in both human and animal health. Different studies showed the presence of a multitude of fungal metabolites that contaminate food and feed. Recently, the international health authorities have expressed concerns about the presence of "emerging" mycotoxins in foodstuffs. The term “emerging mycotoxins” is commonly referred to those compounds that are currently under the spotlight of the scientific community and the policy-makers, due to their toxicological profile. Among the fungal species responsible for the metabolite production, Alternaria species have the ability to produce more than 70 toxins, such as Alternariol (AOH), Alternariol monomethyl ether (AME), Tenuazonic Acid (TeA) e Tentoxin (TEN), found in cereals, tomato products, olive oil, fresh fruits and vegetables. These mycotoxins have been recently covered by scientific opinions from the European Food Safety Authority (EFSA), who has expressed concern about the low availability of incidence data in food for human and animal consumption. There is, indeed, a high degree of uncertainty related to the representativeness of the food currently tested because it contains too many inaccuracies. Furthermore, the lack of information on the analytical method used further contributes to the uncertainty of the reported Alternaria toxin levels. More comprehensive studies are required to update the risk evaluation by the EFSA and, therefore, the specific aim of this project was focussed on the development of accurate LC-MS/MS and LC/HRMS methods for the analysis of emerging Alternaria toxins. The protocol was employed in collaboration with the Istituto Zooprofilattico Sperimentale della Sicilia. LC-MS/MS method was used for the target analysis of emerging mycotoxins in food matrices. In particular, the following activities were carried out: • optimisation of the method parameters for simultaneous screening of Alternariol (AOH), Alternariol monomethyl ether (AME), Tenuazonic Acid (TeA), Ochratoxins and Zearalenone (ZEA), Fumonisin B1, B2, B3, T-2 toxin, HT-2 toxin and deoxynivalenol (DON) (vomitoxin); • development of a sample preparation protocol, apply to cereals and dry fruits; the sample preparation protocol was based on a double extraction (Liquid extraction) and purification through solid-phase extraction (SPE). • validation of the method according to EU guidelines (Commission Decision 2002/657/EC); the method was performed by evaluating the following parameters: specificity, precision, instrumental linearity, recovery, decision limit (CCα), detection capability (CCβ), matrix effect and ruggedness. The ultrahigh-performance chromatography coupled to quadrupole-orbitrap high-resolution mass spectrometry (UHPLC-Q-HRMS) was applied to the sensitive and specific determination of the emerging Alternaria toxins. Performances were compared to those obtained by high-performance liquid chromatography detection. All two methods showed good linearity and repeatability. The Q-Exactive mass spectrometer is said to be better suitable for trace detection than state-of-the-art MS/MS methods based on the triple quadrupole, because, despite having performance comparable, it has better selectivity. The research project should have provided a broad investigation of the emerging mycotoxin content in various food products, contributing to an estimate of mycotoxin risk exposure. The difficulties linked to the pandemic in progress made difficult research, initially planned within this project. Despite everything, the purpose of this work was to fill the data gap and provide relevant information to improve the safety of local products.
Identification and quantification of Mycotoxins produced by Alternaria in food and feed by LC-MS/MS and LC/HRMS.
22-apr-2022
PULVIRENTI, Andrea
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