Evaluation of Advanced Analytical Methodologies for the Control of Pesticides and Natural Toxins in Waters, Food and Nutraceuticals

Abstract

In this Thesis, different analytical strategies using different techniques have been proposed for the determination of residues of fipronil insecticide and its metabolites and cyanotoxins in food products and environmental waters. Fipronil is an insecticide derived from the phenylpirazole family which is well-known for disturbing the γ-aminobutyric acid (GABA)-gated chloride channels from the cell membranes in the central nervous system. Disruption of the GABA receptors by fipronil prevents the uptake of chloride ions resulting in excess neuronal stimulation and death of the insect. Even though it is known that this insecticide has higher binding affinity for insect receptor complexes, sometimes its effects are negative on other organisms such as vertebrates and humans, thus, it is authorized in the European Union (EU) as a antiparasitic drug for pet animals, but not for food producing animals. In addition, fipronil-sulfone, the primary biological metabolite of fipronil, is reported to be twenty times more active at mammalian chloride channels than at insect ones. In the case of foodstuff, current legislation establishes a maximum residues limit (MRL), which must not be surpassed to guarantee the quality of the product and to permit its distribution and consumption. The approval of fipronil as an agricultural insecticide expired in 2017 and thus, the Commission Regulation (EU) 2019/1792 modified its MRL (as sum of fipronil and fipronil-sulfone) established for foodstuffs, set in the analytical determination limit of 0.005 mg/kg. However bad practices or handling errors sometimes cause that fipronil or its metabolite residues can be found in foodstuffs at levels above this value. If this happens, alerts about their presence in products of animal origin destined to human consumption are notified by the Rapid System of Food and Feed RASFF) portal, which provides EU authorities a tool for the exchange of information on the measures taken when a risk is detected in feed or food. This happened with the health alert incident occurred in 2017 in Europe as a result of fipronil contamination of chicken eggs from poultry farms in Netherland and Belgium intended for human consumption. On the other hand, the presence of not only pesticides, but emerging natural toxins is a matter of interest and awareness. Cyanotoxins are toxic secondary metabolites produced by some genera of cyanobacteria, also known as blue-green algae. Cyanobacteria is a group of oxygenic prokaryotes which habits in a wide variety of freshwater and marine ecosystems and, under certain environmental conditions, such as high levels of nutrients, they reproduce exponentially forming blooms. As a result, cyanotoxins present in water can negatively affect different species and accumulate in animals such as fish and shellfish causing shellfish poisoning through the food chain. In fact, cyanotoxins can be present in different food products such as dietary supplements based on cyanobacteria. Cyanotoxins include potent neurotoxins, hepatotoxins, cytotoxins, compounds with a wide variety of structures and physicochemical properties. However, cyanotoxicity has not been closely studied and therefore the World Health Organization (WHO) has established a drinking water guideline of 1 μg/L only for the most explored cyanotoxin, microcystin-LR. Considering these concerns, it is obvious that sensitive, selective and effective analytical methods are required for the determination of these compounds in the fields of food and environmental safety to: a) Monitor the concentration of pesticide residues, particularly the insecticide fipronil and its metabolite fipronil-sulfone, to ensure the quality and safety of food products and to fulfill the current European legislation. b) Study and evaluate the presence and potential health risks associated with less studied or emerging natural toxic compounds, such as cyanotoxins. In general, to face with the analytical challenges abovementioned several techniques have been taken into consideration. The proposed methods are based on miniaturized separation techniques, such as capillary electrophoresis (CE) and capillary liquid chromatography (CLC) as they involve low sample volume, low solvent consumption and low waste generation, being recommended as green analytical techniques. Furthermore, the use of high efficiency techniques such as ultra-high performance liquid chromatography (UHPLC) has also been investigated as it offers a lower solvent consumption than in conventional LC methods, higher efficiency and shorter analysis time. Besides UV-Vis detection, mass spectrometry (MS) has been considered in this Thesis due to its advantages such as higher sensitivity and selectivity and its capability to identify unequivocally a large number of compounds. Finally, the coupling of ion mobility spectrometry (IMS) to LC-High Resolution Mass Spectrometry (LC-HRMS) workflow has been assessed as it provides complementary information by means of the collision cross section (CCS) parameter, which can be derived from such a technique. At the same time, suitable sample treatment methods have been investigated to achieve the extraction and preconcentration of the analytes. It is worthy to mention that this Thesis has sought to obtain novel contributions by exploring alternative methodologies to conventional ones to address these problems. Therefore, it has been developed the first application of CE method for the determination of fipronil and its metabolites in egg samples. Furthermore, it is addressed for the first time the joint determination of cyanotoxins from different families, and therefore, with different characteristics, and the characterization in terms of CCS of a high number of cyanotoxins employing travelling wave ion mobility (TWIMS) coupled to quadrupole time-of-flight high resolution mass spectrometry (QTOF-HRMS). The present Thesis has been divided into two different parts. Part I presents an introduction including relevant aspects related with the insecticides under study and the experimental work developed. The following chapters are included in this part: • Chapter 1 describes a method based on CE in its micellar electrokinetic chromatography mode (MEKC) with ultraviolet detection (UV) as a novel approach for the determination of fipronil and its main metabolite, fipronilsulfone in chicken egg samples. With this purpose, a sample treatment based on salting out liquid-liquid extraction (SALLE) was developed to efficiently extract the analytes. • Chapter 2 involves the employ of CLC coupled to UV as a miniaturized technique for the separation and determination of fipronil and two of its main metabolites, fipronil-sulfone and fipronil-sulfide in chicken egg samples. SALLE was the extraction technique employed to extract the target analytes from the matrix of interest. On the other hand, in Part II an introduction with valuable information about cyanobacteria, cyanotoxins and analytical methods is provided as well as the methods developed in this Thesis involving those analytes. The following chapters can be found in this part: • Chapter 3 deals with a comprehensive study of the determination of cyanotoxins from different families in reservoir water samples using hydrophilic interaction liquid chromatography (HILIC) coupled to tandem MS detection (MS/MS). To achieve the simultaneous extraction of the analytes a novel sample treatment method based on tandem solid phase extraction (SPE) was developed. • Chapter 4 consists of the development of a method based on HILIC-MS/MS to determine cyanotoxins from different families in cyanobacteria based dietary supplements. A sample treatment based on solid-liquid extraction (SLE) followed by a tandem-SPE was employed. The method was applied to different dietary supplements samples to study the occurrence of cyanotoxins and evaluate the potential health risk. • Chapter 5 presents the research carried out with the supervision of the Professors Dr. Chiara Dall’Asta and Dr. Laura Righetti during the predoctoral research stay at the University of Parma (Parma, Italy). The work consisted of the development of a CCS database as a complementary parameter to characterize 20 cyanotoxins and one marine biotoxin employing travelling wave ion mobility coupled to quadrupole time-of-flight (TWIMS- QTOF) method. The obtained results were discussed, and then, a screening of cyanotoxins was carried out in several dietary supplement samples to evaluate their occurrence.