The objective of this thesis is to develop a qualitative analytical method for detecting tropane alkaloids (TAs) and their precursors in plant-based foods, using Direct Analysis in Real Time combined with High-Resolution Mass Spectrometry (DART-HRMS) as the analytical technique. The method was applied to various species of the Solanaceae family, including Datura stramonium, Datura metel, and Mandragora autumnalis, which are notably associated with food contamination by TAs. The invasive nature of these species, combined with their botanical and morphological similarities, facilitates cross-contamination in plant-based foods, significantly impacting consumer health. The European Food Safety Authority (EFSA) has set an acute reference dose (ARfD) of 0.016 μg/kg, above which the consumption of TAs can cause serious toxic effects. EU regulations set limits for TAs concentrations in many foods. However, leafy vegetables are currently excluded from this legislation. Therefore, the implementation of rapid methods represents an opportunity for detecting TA even in foods not considered at risk under the current legislation. This allows for the timely identification of the cause of a clinical food poisoning event and ensures a quick and prompt response. The use of DART-HRMS offers significant advantages over conventional techniques. These advantages lie in the rapidity of use and the minimal sample preparation; however, the technique is affected by matrix effects, which reduce repeatability and selectivity. The results obtained from the study of TAs demonstrate that the proposed method is capable of qualitatively detecting tropine, atropine, scopolamine, and other secondary metabolites in foods such as spinach, chard, and chicory when contaminated. The findings indicates that DART-HRMS is a promising tool for the rapid screening of multiple samples due to its speed, ease of use, and lack of sample preparation, making it a viable alternative to conventional techniques. However, its effectiveness varies depending on the plant matrix. Calibration curves for tropine, a precursor of many TAs, were also developed to assess the analytical technique's capability in quantitative analysis. In conclusion, DART-HRMS has proven to be a user-friendly and fast analytical tool that require minimal sample preparation, making it a promising solution for detecting TAs in the ready-to-eat food industry. However, challenges related to environmental conditions and matrix effects must be addressed to improve the accuracy of the results. Therefore, further experimental studies are required to confirm the method's capability to identify trace levels of TAs with high sensitivity. Although the results confirm the potential of DART-HRMS for identifying and quantifying these target metabolites, additional optimizations are necessary to improve its sensitivity and reproducibility.
L'obiettivo di questa tesi è lo sviluppo di un metodo analitico qualitativo per rilevare alcaloidi tropanici e i loro precursori in alimenti vegetali, utilizzando l’analisi diretta in tempo reale abbinata alla spettrometria di massa ad alta risoluzione (DART-HRMS) come tecnica analitica. Il metodo è stato applicato a diverse specie della famiglia delle Solanacee, tra cui Datura stramonium, Datura metel e Mandragora autumnalis, notoriamente associate alla contaminazione alimentare da alcaloidi tropanici (AT). La natura invasiva di queste specie, unita alla loro somiglianza botanica e morfologica, facilita la cross-contaminazione in alimenti di origine vegetale con un impatto significativo sulla salute dei consumatori. L’ Autorità europea per la sicurezza alimentare (EFSA) ha fissato un valore di dose acuta di riferimento (ARfD) di 0.016 μg/kg oltre il quale il consumo di AT può causare gravi effetti tossici. I regolamenti UE stabiliscono dei limiti per la concentrazione di AT in molti alimenti; tuttavia, i prodotti vegetali a foglia sono attualmente esclusi dalla normativa. Pertanto, l’implementazione di metodi rapidi rappresenta un’opportunità per il rilevamento di AT anche in alimenti non considerati a rischio dalla normativa vigente, consentendo di individuare tempestivamente la causa di un evento clinico di intossicazione alimentare, garantendo un intervento rapido e tempestivo. L’utilizzo del DART-HRMS offre vantaggi significativi rispetto alle tecniche analitiche convenzionalmente usate. Questi vantaggi risiedono nella rapidità d'uso e nella minima preparazione del campione, tuttavia risente degli effetti della matrice, che riducono la ripetibilità e la selettività. I risultati ottenuti dallo studio sugli AT dimostrano che il metodo proposto è in grado di rilevare qualitativamente tropina, atropina, scopolamina e altri metaboliti secondari in alimenti come spinaci, bieta e cicoria, quando contaminati. I risultati indicano che il DART-HRMS è uno strumento promettente per lo screening rapido di più campioni, grazie alla sua velocità, facilità d'uso e all'assenza di preparazione del campione, rendendolo una valida alternativa alle tecniche convenzionali. Tuttavia, la sua efficacia varia a seconda della matrice vegetale. Sono state inoltre sviluppate curve di calibrazione per la tropina, un precursore di molti AT, per valutare la capacità della tecnica analitica nell'analisi quantitativa. In conclusione, il DART-HRMS si è dimostrato uno strumento analitico rapido e di facile utilizzo, che richiede una preparazione minima del campione, rendendolo una soluzione promettente per il rilevamento degli AT nell'industria degli alimenti pronti al consumo. Tuttavia, è necessario affrontare le problematiche legate alle condizioni ambientali e agli effetti della matrice per migliorare la precisione dei risultati. Pertanto, sono necessari ulteriori studi sperimentali per confermare la capacità del metodo di identificare tracce di AT a concentrazioni molto basse. Sebbene i risultati confermino il potenziale del DART-HRMS per l'identificazione e la quantificazione dei metaboliti target, sono necessarie ulteriori ottimizzazioni per migliorarne la sensibilità e la riproducibilità.
Analysis of metabolic fingerprinting of toxic plants (Datura stramonium, Datura metel, Mandragora autumnalis) in plant-based foods via DART-HRMS
FABRIS, SAMUELE
2023/2024
Abstract
The objective of this thesis is to develop a qualitative analytical method for detecting tropane alkaloids (TAs) and their precursors in plant-based foods, using Direct Analysis in Real Time combined with High-Resolution Mass Spectrometry (DART-HRMS) as the analytical technique. The method was applied to various species of the Solanaceae family, including Datura stramonium, Datura metel, and Mandragora autumnalis, which are notably associated with food contamination by TAs. The invasive nature of these species, combined with their botanical and morphological similarities, facilitates cross-contamination in plant-based foods, significantly impacting consumer health. The European Food Safety Authority (EFSA) has set an acute reference dose (ARfD) of 0.016 μg/kg, above which the consumption of TAs can cause serious toxic effects. EU regulations set limits for TAs concentrations in many foods. However, leafy vegetables are currently excluded from this legislation. Therefore, the implementation of rapid methods represents an opportunity for detecting TA even in foods not considered at risk under the current legislation. This allows for the timely identification of the cause of a clinical food poisoning event and ensures a quick and prompt response. The use of DART-HRMS offers significant advantages over conventional techniques. These advantages lie in the rapidity of use and the minimal sample preparation; however, the technique is affected by matrix effects, which reduce repeatability and selectivity. The results obtained from the study of TAs demonstrate that the proposed method is capable of qualitatively detecting tropine, atropine, scopolamine, and other secondary metabolites in foods such as spinach, chard, and chicory when contaminated. The findings indicates that DART-HRMS is a promising tool for the rapid screening of multiple samples due to its speed, ease of use, and lack of sample preparation, making it a viable alternative to conventional techniques. However, its effectiveness varies depending on the plant matrix. Calibration curves for tropine, a precursor of many TAs, were also developed to assess the analytical technique's capability in quantitative analysis. In conclusion, DART-HRMS has proven to be a user-friendly and fast analytical tool that require minimal sample preparation, making it a promising solution for detecting TAs in the ready-to-eat food industry. However, challenges related to environmental conditions and matrix effects must be addressed to improve the accuracy of the results. Therefore, further experimental studies are required to confirm the method's capability to identify trace levels of TAs with high sensitivity. Although the results confirm the potential of DART-HRMS for identifying and quantifying these target metabolites, additional optimizations are necessary to improve its sensitivity and reproducibility.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/80390