Green analytical strategies in food analysis: deep eutectic solvents applications in the extraction of bioactive molecules and contaminants from food and food waste matrices

In the following thesis, some aspects and critical issues related to using deep-eutectic solvents and the miniaturization of some extraction techniques in food analysis to recover bioactive molecules and detect contaminants, have been explored. The manuscript is divided into a first introductory section consisting of two chapters (Chapter 1-2) describing the state of the art of the Green Analytical Chemistry and the Deep Eutectic Solvents, followed by an experimental section in which firstly a brief overview of contaminants and bioactive molecules in food is provided (Chapter 4 and 6) followed by the experimental results of the developed techniques (Chapters 5 and 7). The first chapter deals with the state of the art regarding Green Analytical Chemistry and Green Sample Preparation to give an overview of what are the requirements taken into account during the development of extraction methods and some of the metrics used to quantify the greenness of the methods developed. In Chapter 2 deep eutectic solvents are presented and described as an innovative class of green solvents that find various applications in the field of analytical chemistry. The main physical- chemical properties and the main limitations they present in analytical applications are also described. Before the experimental section, the third chapter gives an overview of the aim of the thesis. The experimental section starts with Chapter 4 in which the use of pesticides in food and the health risks associated with the consumption of contaminated food are briefly described. Furthermore, in the same chapter, the principal innovative sample preparation methods that utilize deep eutectic solvents and the instrumental techniques most often used for both qualitative and quantitative analysis of pesticides are presented. Chapter 5, deals with the first two experimental works, presenting the results obtained in the application of deep eutectic solvent to the MSPD sample preparation technique for the determination of pesticide residues from tomato samples. Matrix solid-phase dispersion is an alternative technique to simultaneously extract and clean up pesticides from food matrices. Specifically, in paragraph 5.2 the application of choline chloride-ethylene glycol in a molar ratio of 1:2 DES as an extraction solvent in the MSPD procedure for the determination of triazole fungicides from tomato samples is presented. One of the drawbacks of deep eutectic solvents is the impracticality of their evaporation, leading to higher limits of quantification. To overcome this drawback, a deep eutectic solvent-based extraction followed by liquid-liquid back-extraction was carried out using alumina as dispersant sorbent in a 1-to-5 w/w sample-to-dispersant ratio. Volumes of 4.5 ml of deep eutectic solvents and 2 mL ethyl acetate were the best choices for back-extraction. The method was fully validated. The limit of quantification was in the range of 5–11 ng/g. Intra-day and inter-day precision ranged between 3 % and 14 %, respectively. Extraction recoveries varied from 61 % to 116 %. The matrix effect was also evaluated. In paragraph 5.3, an environmental-friendly method, based on a miniaturized matrix solid-phase dispersion followed by high-performance liquid chromatography-tandem mass spectrometry, for the analysis of fourteen pesticides in tomatoes, was described. For the recovery of pesticides from tomato samples, a low transition temperature mixture (LTTM), composed of choline chloride and sesamol 1:3 molar ratio, was employed. Extraction parameters like sample-to- dispersant ratio, extraction solvent volume and LTTM volume were optimized through a Box- Behnken design. A value of 1:4 sample-to-dispersant ratio, 900 μL of ethanol as extraction solvent and 50 μL of LTTM guaranteed the best result in terms of the mean of extracted pesticides' peak areas. The optimized analytical method was validated obtaining the following results: linearity range was between LOQ and 5 mg kg-1 with a minimum R2 of 0.9944 for tebufenozide. Maximum values of 0.20 and 0.06 mg kg-1 were obtained for LOQ and LOD respectively, corresponding to difenoconazol while peak areas intra-day and inter-day repeatability were maximum of 10.19 and 9.15 %, respectively. The optimized and validated method was then applied to real samples considering a whole tomato pool, a tomato pulp pool, and a tomato peel pool. Chapter 6 describes the applications of deep eutectic solvents for the recovery of bioactive molecules from food waste. Also included is a description of the main classes of phenolic compounds and their antioxidant properties that are beneficial for human health. In conclusion, some of the innovative methods of recovery of phenolic compounds from food matrices based on the use of deep eutectic solvent and spectrophotometric and chromatographic analyses commonly used for their quantification and identification are described. Following is the presentation of the results of 4 works which concerned the development of extractions for the recovery of phenolic compounds from food waste in Chapter 7. The last chapter of the experimental section (Chapter 7) presents four different applications of deep eutectic solvents as extraction media in UA-SLE procedures for the recovery of phenolic compounds from food waste matrices. UA-SLE has been selected as it is considered green and easily scalable for possible industrial applications. In the first work (paragraph 7.2) the use of the DES composed of choline chloride and lactic acid in a molar ratio of 1:2 for extracting phenolic compounds from Hazelnut Skin is presented. Hazelnut (Corylus avellana L.) is one of the most cultivated tree nuts worldwide. The skin represents one of the major byproducts and it is a rich source of phenolic compounds like flavan-3-ols, flavonols, dihydrochalcones, and phenolic acids. Fifteen DESs based on choline chloride and betaine, with different compositions, were studied to test their phenolic compounds extraction efficiency through the determination of their total concentration via the Folin–Ciocalteu assay. A qualitative analysis of extracted phenolic compounds was assessed by HPLC with UV and MS detection. Using the DES with the best extraction efficiency, a new ultrasound-assisted solid-liquid extraction (UA-SLE) method was optimized through the response surface methodology. Efficient recovery of extracted phenolic compounds was achieved using a 35 % water solution of choline chloride and lactic acid (molar ratio 1:2) as an extraction solvent, working at 80 ◦C and with a solid-to-solvent ratio of 1:25 g mL−1. The optimized conditions made it possible to recover 39 % more phenolic compounds compared to a classic organic solvent. In the second work (paragraph 7.3) deep eutectic solvents are applied for the recovery of bioactive molecules from the waste of malt production in the brewery industrial processes (barley malt rootlets). Several natural deep eutectic solvents were tested as extractive medium and, among them, the best results in terms of total phenolic content were obtained using a choline chloride-malic acid (1:2 molar ratio) based mixture. The optimization through a Box- Behnken experimental design guaranteed the extraction of 9.51 ± 0.83 gallic acid equivalent/g of barley malt rootlets, under the following optimal extraction conditions: 1:21 solid-to-liquid ratio, 80 °C as extraction temperature, 43 min as the time of extraction, and 29 % as a percentage of added water in the natural deep eutectic solvents. Phenolic acids and flavonoids were detected in the barley malt rootlets extract through HPLC-PDA/MS analysis. In paragraph 7.4 two systems of deep eutectic solvent, an acid and an alcoholic have been studied as possible extraction solvents applied to the recovery of phenolic compounds from artichoke outer bracts. The value of artichoke waste for extracting bioactive molecules is of great interest, due to the high percentage of vegetable biomass discarded post-harvest (85 %) and the presence of phenolic acids and flavonoids. Through a D-Optimal design, some of the main parameters that affect the extraction were studied being the nature of the solvent, the time of extraction, the amount of solvent and the amount of water to be added in the deep eutectic solvent. Moreover, a PCA analysis revealed differences in the responsiveness of the two different deep eutectic solvents studied under the different extraction conditions tested. Under the optimal extraction condition, 5 mL of ChCl-LA DES added with 60 % of water were used as solvent in a 60 min extraction. 3.28 ± 0.2 g GAE/100 g of DM, 35.44 ± 1.7 mg TroloxE/g DM, 51664 ± 5233 μg/g DM and 11940 ± 583 μg/g DM were obtained as values of TPC, DPPH, phenolic acids and flavonoids amounts, respectively. Through high-performance liquid chromatography coupled with photodiode array and mass spectrometry detection, some flavonoids and phenolic acids were identified in the artichokes' outer bracts extracts. Finally, paragraph 7.5 describes the results obtained from the extraction and analysis of phenolic compounds from avocado peel. Avocado (Persea americana Mill.) is a tropical fruit grown in different areas of the world. Avocado pulp is of interest to food companies while seed and peel are wasted. Twelve deep eutectic solvents were tested as extraction solvents considering the provided total phenolic content. Among these, ChCl-lactic acid-based DES has been selected considering the extraction results and its low toxicity. Then, to obtain the highest amount of phenolic compounds, the most relevant factors affecting solid-liquid extraction were evaluated. The matrix-to-solvent ratio of 1:30 (w/v), 25 °C and 15 min as temperature and time of extraction, respectively, were found as optimal conditions to guarantee an extracted amount of phenolic compounds of 8.29 ± 0.07 g GAE/100g of dry avocado peel. Through high- performance liquid chromatography coupled with photodiode array and mass spectrometry detection, some flavonoids and phenolic acids were identified in the avocado peel extract.