The objective of the research work, presented in this Ph.D. thesis, was the development of fast and “green” analytical methods using advanced chromatographic techniques and innovative sample preparation protocols for the study of food, environmental and biological samples. These techniques have been used to deepen the knowledge regarding the composition of some foods and to investigate the possible presence of contaminants (residues of plant protection products) present in food and environmental samples. Plant protection products are used to destroy or control any harmful organism and preventing the damage that these organisms can cause in the production. The persistence of plant protection products in foods after they have been placed on the market can be harmful to the consumer, and an excessive use of these products can lead to pollution of the ecosystem and groundwater. For this reason, the use of plant protection products is strictly regulated at various levels by various national and European directives which indicate and limit their uses and place residual limits on the products placed on the market. Different techniques have been studied and employed, such as: conventional and fast gas chromatography coupled to mass spectrometry (GC-MS); two-dimensional comprehensive gas chromatography coupled to triple quadrupole mass spectrometry (FM GC×GC-QqQMS) and flame ionization detector (FID); high performance liquid chromatography coupled to diode array spectrophotometric detector (HPLC-DAD). Particular attention was paid to the development of extraction and separation methods following the guidelines of Green Analytical Chemistry which has various objectives, including elimination or reduction of the use of organic solvents (harmful to both humans and the environment), reduction of analysis times, miniaturization of extraction processes also reducing the amount of sample used, robotization of sample preparation. Conventional techniques such as liquid-liquid or liquid-solid extraction often make use of large quantities of sample and reagents causing both economic expenditure and laboratory waste pollution. In this regard, during the PhD course, various protocols were developed for the extraction of contaminants in environmental and food matrices using solid phase microextraction techniques (SPME) and miniaturized and scaled techniques.6 The PhD has foreseen a six-monthly collaboration with the corporate partner of the PON project, Shimadzu Italia S.r.l. (Milan). The period in the company took place under the supervision of the company tutor Mattia Casanova, during which analyzes were carried out through the application of head space SPME protocols and separation using FM GC×GC-MS, for the elucidation of the volatile fraction of tomato sauce, basil pesto and honey samples. Furthermore, these analyzes had the purpose of monitoring the possible presence of volatile compounds prohibited by current legislation through a simple and solvent-free but at the same time robust preparation technique. It is interesting to report how from the analyzes conducted on the honey samples the presence of benzaldehyde was found in some of these. Benzaldehyde is a compound with a strong smell of bitter almonds and is used in beekeeping to keep the bees away from the comb. The European community has repeatedly advised against the use of benzaldehyde in the production of honey. Furthermore, I spent a semester at the “Vienna University of Technology” (Vienna), European partner of the PON project, under the supervision of Prof. Erwin Rosenberg. During the semester, studies were conducted on pollutants in environmental waters and fruit juices, focusing mainly on the optimization of extraction protocols through solid phase microextraction techniques. Other studies involved the use of the SPME Arrow coupled to a GC×GC equipped with a cryogenic modulator and single quadrupole mass spectrometer for the exploration of the volatile fraction of alcoholic and non-alcoholic beverages. More in detail, the applications regarding the sphere of pollutants are reported. A reduced scale QuEChERS protocol was developed for monitoring 88 plant protection product residues in cucumbers, tomatoes, iceberg lettuce and red peppers. The chromatographic separations were carried out by the use of a FM GC×GCQqQMS. The aim of the research was to demonstrate the possibility of using flow modulators for the analysis of trace compounds [5]. The capsule phase microextraction (CPME) method was applied for the extraction and preconcentration of organophosphorus pesticides (OPPs) from environmental water samples using monolithic sol-gel octadecyl siloxane (sol-gel C18) absorbent encapsulated within porous polypropylene capsules. Identification was7 conducted through the use of a GC-MS system. The method has been optimized to ensure high sensitivity [6]. Two extraction protocols of benzoylurea-based plant protection products were optimized and validated in environmental waters (river, lake, pond, tap) and apple juices. Given the polar nature of the compounds examined, the determination was carried out through the use of an HPLC-DAD system. The extractions were conducted through two different techniques, magnet-integrated fabric phase sorptive extraction (MI-FPSE) for water samples, while the apple juices were subjected to extraction by CPME. Both protocols allowed to obtain high preconcentration factors of the analytes from the starting sample [7,8]. With regard to untargeted analyses, the extraction of volatile organic compounds of various whiskies was obtained through the use of the SPME Arrow fiber. The analytes separation and determination were performed by the use of twodimensional comprehensive gas chromatography equipped with a cryogenic modulator (CM GC×GC) coupled to mass spectrometry. After optimization, the method was used for the analysis of Irish whisky, single malt Scotch whiskey and blended Scotch whisky. The use of SPME Arrow fiber combined with GC×GC-MS has proven to be a powerful analytical tool for the exploration of the volatile profile of complex samples, allowing to identify differences in important flavor compounds for the three different whiskeys studied [9]. Additionally, I was involved in the optimization of an automated sample preparation and fast gas chromatography-mass spectrometry (GC-MS) method for the analysis of fatty acid methyl esters (FAMEs) in human blood and dietary supplement samples. FAME separation was achieved in approximately 11 minutes using a 15 m × 0.10 mm ID × 0.10 µm df microbore column with a polyethylene glycol stationary phase. The novelty of the research consists in reducing the analysis times by using the fast GC-MS method with greater reliability and identification sensitivity. The accuracy of the method was evaluated by analyzing certified reference materials.
L’obiettivo del mio lavoro di ricerca, presentato in questo lavoro di tesi di dottorato, è stato quello di sviluppare e validare metodiche analitiche rapide e green attraverso l’uso di tecniche cromatografiche avanzate e protocolli innovativi di preparazione del campione per lo studio di campioni alimentari, ambientali e biologici. Tali tecniche sono state usate per approfondire le conoscenze in merito alla composizione di alcuni alimenti di particolare interesse commerciale e per indagare l’eventuale presenza di contaminanti (residui di prodotti fitosanitari) presenti in campioni alimentari e ambientali. I prodotti fitosanitari vengono usati per distruggere o tenere sotto controllo qualsiasi organismo nocivo (tra cui microrganismi e piante infestanti), impedendo o prevenendo i danni che tali organismi possono causare nelle fasi di produzione, raccolta e commercializzazione dei prodotti alimentari. La persistenza dei prodotti fitosanitari negli alimenti dopo l’immissione in commercio può essere dannosa per il consumatore, inoltre un uso spropositato di tali prodotti può portare ad inquinamento dell’ecosistema e delle falde acquifere. Per tale motivo l’uso dei prodotti fitosanitari è strettamente regolamentato a diversi livelli da diverse direttive nazionali ed europee che ne indicano e limitano gli usi e ne pongono i limiti residuali sui prodotti posti in commercio. Inoltre, è stata posta attenzione anche allo studio di integratori alimentari e campioni biologici (plasma e sangue) per lo studio della composizione in acidi grassi di queste matrici attraverso l’uso di tecniche automatizzate di preparazione del campione e di separazioni cromatografiche veloci. Sono state studiate e impiegate tecniche quali: gascromatografia convenzionale e veloce accoppiata a spettrometria di massa (GC-MS); gascromatografia bidimensionale comprehensive con modulatore a flusso accoppiata a spettrometria di massa a triplo quadrupolo (FM GC×GC-QqQMS) e rivelatore a ionizzazione di fiamma (FID); cromatografia liquida ad alte prestazioni accoppiata a rivelatore spettrofotometrico a serie di diodi (HPLC-DAD). Durante il corso del triennio particolare attenzione è stata posta allo sviluppo di metodi estrattivi e separativi in linea con le linee guida della Green Analytical Chemistry che si pone diversi obiettivi, tra i quali l’eliminazione o la riduzione dell’uso di solventi organici nocivi sia per l’uomo che per l’ambiente, la riduzione dei tempi di analisi, la miniaturizzazione dei processi estrattivi riducendo anche la quantità di campione usata, la robotizzazione della preparazione dei campioni. Tecniche convenzionali come l’estrazione liquido-liquido o liquido-solido fanno spesso uso di un elevata quantità di campione e reattivi causando sia un dispendio economico sia un inquinamento da scarti di laboratorio. A tal proposito durante il triennio di dottorato sono state messe a punto diversi protocolli per l’estrazione di contaminanti in matrici ambientali e alimentari facendo ricorso a tecniche di microestrazione in fase solida (SPME) e tecniche miniaturizzate ed a scala ridotta. Il dottorato ha previsto una collaborazione semestrale con il partner aziendale del progetto PON, Shimadzu Italia S.r.l. (Milano). Il periodo in azienda si è svolto sotto la supervisione del tutor aziendale Mattia Casanova, durante il quale sono state effettuate analisi attraverso l’applicazione di protocolli SPME nello spazio di testa (HS) e separazione tramite FM GC×GC-MS, per l’elucidazione della frazione volatile di campioni di salsa di pomodoro, pesto di basilico e miele. Inoltre, tali analisi avevano lo scopo di monitorare l’eventuale presenza di composti volatili vietati dalla normativa vigente attraverso una tecnica preparativa semplice e solvent-free ma al tempo stesso robusta. È interessante riportare come dalle analisi condotte sui campioni di miele è stata rilevata presenza di benzaldeide in alcuni di questi. La benzaldeide è un composto con forte odore di mandorle amare e viene usata in apicoltura per allontanare le api dal favo. La comunità europea ha più volte sconsigliato l’uso di benzaldeide nella produzione di miele. Inoltre, ho trascorso un semestre presso la “Vienna University of Technology” (Vienna), partner europeo del progetto PON, sotto la supervisione del Prof. Erwin Rosenberg. Durante il semestre sono stati condotti studi su inquinanti in acque ambientali e succhi di frutta soffermandomi principalmente sull’ottimizzazione dei protocolli di estrazione attraverso tecniche di microestrazione in fase solida. Altri studi hanno riguardato l’uso della SPME Arrow accoppiata a GC×GC con modulatore criogenico e spettrometro di massa a singolo quadrupolo per l’esplorazione della frazione volatile di bevande alcoliche e non alcoliche. Più nel dettaglio, vengono riportate le applicazioni per quanto riguarda la sfera degli inquinanti. È stato sviluppato un protocollo QuEChERS a scala ridotta per il monitoraggio di 88 residui di prodotti fitosanitari in cetrioli, pomodori, lattuga iceberg e peperoni rossi. Le separazioni cromatografiche sono state condotte tramite FM GC×GC-QqQMS. Lo scopo della ricerca è stato quello di dimostrare la possibilità di utilizzo dei modulatori a flusso per analisi di composti in tracce. È stato applicato il metodo capsule phase microextraction (CPME) per l'estrazione e la preconcentrazione di pesticidi organofosforici (OPP) da campioni di acqua ambientale usando l'assorbente monolitico sol-gel ottadecil silossano (sol-gel C18) incapsulato all'interno di capsule porose di polipropilene. L’identificazione è stata condotta attraverso l’uso di un GC-MS. Il metodo è stato ottimizzato per garantire un'elevata sensibilità ed è stato completamente validato. Sono stati ottimizzati e validati due protocolli di estrazione di prodotti fitosanitari a base di benzoilurea, in acque ambientali (fiume, lago, stagno, rubinetto) e succhi di mela. Vista la natura polare dei composti presi in esame la determinazione è stata effettuata attraverso l’uso di un HPLC-DAD. Le estrazioni sono state condotte attraverso due diverse tecniche, magnet-integrated fabric phase sorptive extraction (MI-FPSE) per i campioni di acqua, mentre i succhi di mela sono stati sottoposti ad estrazione tramite CPME. Entrambi i protocolli hanno consentito di ottenere elevati fattori di preconcentramento degli analiti dal campione di partenza. permettendo di ottenere buoni risultati in termini di limiti di rilevabilità. In merito ad analisi di tipo untargeted, attraverso l'utilizzo della fibra SPME Arrow è stata ottenuta l’estrazione dei composti volatili, importanti per le caratteristiche organolettiche di diversi whisky, la cui determinazione è stata eseguita mediante gascromatografia bidimensionale comprehensive con modulatore criogenico (CM-GC×GC) accoppiata alla spettrometria di massa. Dopo l'ottimizzazione, il metodo è stato usato per l'analisi di whisky irlandese, whisky scozzese single malt e whisky scozzese blended. L’uso della fibra SPME Arrow combinata con GC×GC-MS si è dimostrato un potente strumento analitico per l'esplorazione del profilo volatile di campioni complessi, consentendo di identificare differenze in importanti composti aromatici per i tre diversi tipi di whisky studiati. Inoltre, mi sono dedicato all'ottimizzazione di una preparazione automatizzata del campione e di un metodo rapido di gascromatografia accoppiato a spettrometria di massa (GC-MS) per l'analisi degli esteri metilici degli acidi grassi (FAME) in campioni di sangue e integratori alimentari, con l'obiettivo primario di riduzione del tempo di analisi. La separazione dei FAME è stata ottenuta in circa 11 minuti utilizzando una colonna a microbore di dimensioni 15 m × 0,10 mm ID × 0,10 μm df con una fase stazionaria di polietilenglicole. La novità della ricerca consiste nel ridurre i tempi di analisi utilizzando il metodo GC-MS veloce con maggiore affidabilità e sensibilità di identificazione. L'accuratezza del metodo è stata valutata analizzando materiali di riferimento certificati.
Sviluppo di metodi analitici “green” e innovativi per la caratterizzazione generale dei composti volatili e per la determinazione di residui fitosanitari in campioni alimentari e ambientali Development of “green” and innovative analytical methods for the general profiling of volatile compounds and the determination of phytosanitary residues in food and environmental samples
FERRACANE, ANTONIO
2022-12-14
Abstract
The objective of the research work, presented in this Ph.D. thesis, was the development of fast and “green” analytical methods using advanced chromatographic techniques and innovative sample preparation protocols for the study of food, environmental and biological samples. These techniques have been used to deepen the knowledge regarding the composition of some foods and to investigate the possible presence of contaminants (residues of plant protection products) present in food and environmental samples. Plant protection products are used to destroy or control any harmful organism and preventing the damage that these organisms can cause in the production. The persistence of plant protection products in foods after they have been placed on the market can be harmful to the consumer, and an excessive use of these products can lead to pollution of the ecosystem and groundwater. For this reason, the use of plant protection products is strictly regulated at various levels by various national and European directives which indicate and limit their uses and place residual limits on the products placed on the market. Different techniques have been studied and employed, such as: conventional and fast gas chromatography coupled to mass spectrometry (GC-MS); two-dimensional comprehensive gas chromatography coupled to triple quadrupole mass spectrometry (FM GC×GC-QqQMS) and flame ionization detector (FID); high performance liquid chromatography coupled to diode array spectrophotometric detector (HPLC-DAD). Particular attention was paid to the development of extraction and separation methods following the guidelines of Green Analytical Chemistry which has various objectives, including elimination or reduction of the use of organic solvents (harmful to both humans and the environment), reduction of analysis times, miniaturization of extraction processes also reducing the amount of sample used, robotization of sample preparation. Conventional techniques such as liquid-liquid or liquid-solid extraction often make use of large quantities of sample and reagents causing both economic expenditure and laboratory waste pollution. In this regard, during the PhD course, various protocols were developed for the extraction of contaminants in environmental and food matrices using solid phase microextraction techniques (SPME) and miniaturized and scaled techniques.6 The PhD has foreseen a six-monthly collaboration with the corporate partner of the PON project, Shimadzu Italia S.r.l. (Milan). The period in the company took place under the supervision of the company tutor Mattia Casanova, during which analyzes were carried out through the application of head space SPME protocols and separation using FM GC×GC-MS, for the elucidation of the volatile fraction of tomato sauce, basil pesto and honey samples. Furthermore, these analyzes had the purpose of monitoring the possible presence of volatile compounds prohibited by current legislation through a simple and solvent-free but at the same time robust preparation technique. It is interesting to report how from the analyzes conducted on the honey samples the presence of benzaldehyde was found in some of these. Benzaldehyde is a compound with a strong smell of bitter almonds and is used in beekeeping to keep the bees away from the comb. The European community has repeatedly advised against the use of benzaldehyde in the production of honey. Furthermore, I spent a semester at the “Vienna University of Technology” (Vienna), European partner of the PON project, under the supervision of Prof. Erwin Rosenberg. During the semester, studies were conducted on pollutants in environmental waters and fruit juices, focusing mainly on the optimization of extraction protocols through solid phase microextraction techniques. Other studies involved the use of the SPME Arrow coupled to a GC×GC equipped with a cryogenic modulator and single quadrupole mass spectrometer for the exploration of the volatile fraction of alcoholic and non-alcoholic beverages. More in detail, the applications regarding the sphere of pollutants are reported. A reduced scale QuEChERS protocol was developed for monitoring 88 plant protection product residues in cucumbers, tomatoes, iceberg lettuce and red peppers. The chromatographic separations were carried out by the use of a FM GC×GCQqQMS. The aim of the research was to demonstrate the possibility of using flow modulators for the analysis of trace compounds [5]. The capsule phase microextraction (CPME) method was applied for the extraction and preconcentration of organophosphorus pesticides (OPPs) from environmental water samples using monolithic sol-gel octadecyl siloxane (sol-gel C18) absorbent encapsulated within porous polypropylene capsules. Identification was7 conducted through the use of a GC-MS system. The method has been optimized to ensure high sensitivity [6]. Two extraction protocols of benzoylurea-based plant protection products were optimized and validated in environmental waters (river, lake, pond, tap) and apple juices. Given the polar nature of the compounds examined, the determination was carried out through the use of an HPLC-DAD system. The extractions were conducted through two different techniques, magnet-integrated fabric phase sorptive extraction (MI-FPSE) for water samples, while the apple juices were subjected to extraction by CPME. Both protocols allowed to obtain high preconcentration factors of the analytes from the starting sample [7,8]. With regard to untargeted analyses, the extraction of volatile organic compounds of various whiskies was obtained through the use of the SPME Arrow fiber. The analytes separation and determination were performed by the use of twodimensional comprehensive gas chromatography equipped with a cryogenic modulator (CM GC×GC) coupled to mass spectrometry. After optimization, the method was used for the analysis of Irish whisky, single malt Scotch whiskey and blended Scotch whisky. The use of SPME Arrow fiber combined with GC×GC-MS has proven to be a powerful analytical tool for the exploration of the volatile profile of complex samples, allowing to identify differences in important flavor compounds for the three different whiskeys studied [9]. Additionally, I was involved in the optimization of an automated sample preparation and fast gas chromatography-mass spectrometry (GC-MS) method for the analysis of fatty acid methyl esters (FAMEs) in human blood and dietary supplement samples. FAME separation was achieved in approximately 11 minutes using a 15 m × 0.10 mm ID × 0.10 µm df microbore column with a polyethylene glycol stationary phase. The novelty of the research consists in reducing the analysis times by using the fast GC-MS method with greater reliability and identification sensitivity. The accuracy of the method was evaluated by analyzing certified reference materials.File | Dimensione | Formato | |
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