Development, optimization and validation of conventional and non-conventional analytical methods for phenolic compounds in foods and food by-products

Phenolic compounds represent a group of highly heterogeneous secondary metabolites which in the last 15 years have been arousing strong interest especially for their beneficial effects for humans. Traditionally, these molecules are extracted by using techniques such as solid-liquid extraction, liquid-liquid extraction, solid-phase extraction, using organic solvents, such as methanol, ethanol, methanol and water mixtures as extractive solvents. Phenolic compounds extracts are typically analyzed by high performance liquid chromatography coupled with UV-Vis spectrophotometry and mass spectrometry (HPLC/PDA-ESI/MS). With the advent of green chemistry, more and more attention has been paid to the search for sustainable extractive solvents, among these deep eutectic solvents (DESs) are having great success. The present thesis study was based in the development, optimization and validation of conventional and non-conventional, extraction with deep eutectic solvent, analytical methods for the analysis of phenolic compounds in foods and food wastes using HPLC/PDA-ESI/MS system. Specifically, two food matrices were considered being extra virgin olive oil (EVOO) and hazelnuts and two waste products from the food supply chain being spent coffee ground and hazelnut skin. EVOO and hazelnuts are famous for their high content in phenolic compounds. A very large quantity of spent coffee ground and hazelnut skin are produced annually following the production of the coffee beverage and the consumption of hazelnuts respectively. These by-products are characterized by a high content of phenolic compounds, the same present in the food matrices from which they derive. In the first project phenolic compounds in EVOO were analyzed using an HPLC/PDAESI/MS method. Thirty samples from four different Italian regions were analyzed. 15 phenolic compounds belonging to the following classes have been identified: secoiridoids, phenolic alcohols, flavonoids, and phenolic acid classes. Quantitative analysis was performed by UV detection considering eight standard phenolic compounds. In all samples secoiridoids were the main compounds ranging from 85 to more than 99% (w/w) of the total concentration of detected phenolic compounds while phenolic acids accounted for the lowest percentage (0.1-0.6%, w/w). Intra-day and inter-day retention time precision, limit of detection, limit of quantification, and linearity were considered for the validation of the optimized analytical method. Finally, total concentration of phenolic compounds and antioxidant activity were determined with different chemical assays. A good correlation was found between antioxidant activity and total phenolic content. It was not possible to cluster the samples according to the regions but statistically significant differences were found between the Puglia and Sicilia samples. The second project involved the optimization and validation of an HPLC/DAD-ESI/MS method for the analysis of the most representative phenolic compounds in EVOO samples but using a green extraction approach based on deep eutectic solvents (DESs) at room temperature. Ten DESs based on choline chloride and betaine in combination with different hydrogen bond donors comprising six alcohols, two organic acids, and one urea were considered for the study. To evaluate the extraction capacity of each DES five phenolic compounds, secoridoids and phenolic alcohols were considered. The optimization of the extraction method involved the study of the quantity of water to be added to the DES and evaluation of the sample-to-solvent ratio optimal condition. Betaine-based DES with glycerol (molar ratio 1:2), added of thirty percent of water considering for the extraction a sample to solvent ratio 1:1 (g/mL) are resulted the best conditions for the extraction. The chromatographic method was validated by studying LOD, LOQ, intraday and interday retention time precision, and linearity range using two standard molecules. Good results about recovery (5 and 100 μg/g concentration) were obtained being in the range of 75.2% and 98.7%. Other two work involved the development, optimization and validation of a conventional HPLC-DAD/ESI-MS method, studying best conditions of the extraction, for the analysis of phenolic compounds in hazelnuts, in the first case and in the second case the development of a green approach for the extraction of phenolic compounds in hazelnut skin, using DESs. For the first one, phenolic compounds were extracted from hazelnut kernels employing two extraction techniques: ultrasound-assisted solid-liquid extraction (UA-SLE) and solid-phase extraction (SPE). Different extraction solvents were tested evaluating total phenolic content, total flavonoids and antioxidant capacity. Different protocols were performed using commercial hazelnut kernels. The UA-SLE protocol performed using 0.1 g of defatted sample and 15 mL of extraction solvent (methanol/water/methanol 0.1 % HCOOH/acetonitrile (1:1:8:5, v/v/v/v)) was selected as best extraction conditions. Six standard molecules were selected for the method validation and RSD % for intra-day e interday of retention time, LOD and LOQ were evaluated. The accuracy of the extraction was also assessed. Calibration curves were constructed with a good linearity and satisfactory determination coefficients R2 for quantitative analysis. Finally, the method was applied to the analysis of phenolic compounds in three different hazelnut kernel varieties. The second project involved the study of fifteen DESs as extractive solvents for phenolic compounds in hazelnut skin. Eighteen phenolic compounds were identified in the extracts, using HPLC/DAD-ESI/MS optimized method. Total phenolic content in the extracts obtained using tested DESs was evaluated through FOLIN ASSAY with the aim to understand which DES guaranteed the best extraction. DES ChCl/lactic acid (1:2) resulted better than the other DESs tested and also compared to an organic solvent. Extraction technique was then optimized considering the quantity of water to add to the DES to reduce its viscosity, time of extraction and sample-to-solvent ratio. The optimal conditions for obtain the best extraction of phenolic compounds from hazelnut skin are resulted: use of DES ChCl/lactic acid (1:2) as extraction solvent, without addition of water, extraction time of 30 min and quantity of matrix/ volume of solvent ratio of 0.2 g matrix/5mL of solvent. Application of DESs was also tested for the extraction of chlorogenic acids, phenolic compounds, from another food by-product, spent coffee ground (SCG). HPLC/PDA-ESI/MS method has been developed for the analysis of the most representative CGAs in SCG samples. Fifteen DESs based on choline chloride and betaine as hydrogen bond acceptor in combination with different hydrogen bond donors were considered to evaluate their extraction efficiency. Fifteen CGAs were detected, identified and quantified into the different extracts obtained using tested DESs with the aim to evaluate which of these was the best. A betaine-based DES with triethylene glycol (molar ratio 1:2) extracted the higher content in phenolic compounds also compared to the conventional extraction using organic solvents. Thirty percent of water added to DES and sample to solvent ratio 1:15 (g/mL) were selected as the best extraction conditions. The chromatographic method was validated studying LOD, LOQ, retention time and peak area precision, and linearity range. Good results of repeatability intra-day and inter-day were determined obtaining RSDs values below 0.63 % for retention time and 8.51 % for and peak area and below 0.58 % for retention time and 8.80 % for peak area, respectively.