Application of different extraction techniques and HPLC-PDA-ESI/MS methods to the analysis of phenolic compounds in food samples

Phenolic compounds encompass a major group of secondary plant metabolites that display a wealth of structural variety and a large diversity of significant biological activities. Over the last 15, years researchers and food manufactures have become increasingly interested in phenolic compounds. The chief reason for this interest is the recognition of their positive effects on human health, like antioxidant activity and their probable role in the prevention of various diseases associated with oxidative stress, such as cancer and cardiovascular and neurodegenerative diseases, and their great abundance in our diet. The present thesis study was based in the development and validation of quali-quantitative analysis methods of phenolic bioactive compounds by high performance liquid chromatography (HPLC) coupled to UV-visible detector (PDA) and mass spectrometry (MS). Two food samples were investigated: Goji berry (Lycium barbarum L.) and Hazelnut kernels (Coryllus avellana L.). Goji berry fruits (Lycium barbarum L.) have generated particular interest in recent times for their potential beneficial effects on human health, such as antioxidant, anti-inflammatory and antitumor activities. It is believed that phenolic compounds are responsible for these effects. The study on Goji berry fruits was divided in two sections. A first study was based on the investigation of the effects of solvent type on the extraction of phenolic compounds through a solid-liquid method. Methanol, methanol 3% formic acid, ethyl acetate, ethyl acetate/methanol 3% formic acid (50:50 v/v) and water/methanol (90:10 v/v) were tested, in order to study their effect on the content of total phenolic compounds and on antioxidant activity, by Trolox antioxidant capacity (TEAC) assay, in Goji berry extracts. HPLC-PDA-ESI-QMS was employed for separation, identification and quantification of phenolic compounds in Goji berry samples. The analytical method was fully validated in terms of retention time and peak area precision, expressed as %RSD, limit of detection (LOD) and limit of quantitation (LOQ) and linearity range employing nine phenolic standard compounds belonging to phenolic acid and flavonoid classes as representative of each class of phenolics. The validated method was then applied in Goji berry extracts obtained by using different extraction solvents. Seven phenolic compounds, belonging to phenolic acid and flavonoid classes, were detected and tentatively identified in Goji berry extracts. Methanol 3% formic acid was the solvent presenting the highest amount of the seven phenolic compounds detected (18,32 µg/100 g of dried weight, DW) and the highest TEAC value (56,71 µmol TE/g DW), while ethyl acetate was the lowest one with an amount of phenolic compounds of 1,13 µg/100 g DW and a TEAC value of 0,56 µmol TE/g DW. A second study was based on the study of pressurized-liquid extraction (PLE) method by a design of experiments (DOE) based on response surface methodology (RSM) for the extraction of phenolic compounds from Goji fruits. The global yield (% w/dw, weight/dry-weight), total phenolic content (TPC), total flavonoid (TF) and antioxidant activity (determined via ABTS assay, expressed as TEAC value) were used as response variables to study the effects of temperature (50–180°C) and green solvent composition (mixtures of ethanol/water). The analysis of phenolic compounds were performed by HPLC-PDA-MS/MS. PLE optimum conditions (180°C and 86% ethanol in water) were obtained using the commercial sample as representative matrix. Once the experimental design was validated for commercial fruit samples, the optimum conditions were applied to three different varieties of fruit samples (Selvatico mongolo, Bigol and Polonia). Nine phenolic compounds were tentatively identified in these extracts, including phenolic acids and their derivatives, and flavonols. The optimized PLE conditions were compared to a conventional solid-liquid extraction (SLE), demonstrating that PLE is a useful alternative to extract phenolic compounds from Goji berry. Hazelnut (Corylus avellana L.) is one of the nuts most consumed in many countries including Turkey, Italy, Spain and United States. It is a rich source of dietary fibers and beneficial nutrients such as lipids, proteins, but also significant micronutrients like essential minerals, vitamin E, B complex vitamins and phenolic compounds, which contribute to its organoleptic properties such as astringent and bitter taste. The content of phenolic compounds may be a significant parameter in the assessment of hazelnuts quality. In fact, it depends on several factors like cultivar, geographical origin and processing condition such as roasting. 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. The individual phenolic compounds in hazelnut kernels of different cultivars were analyzed by HPLC-PDA-MS/MS. The best extraction conditions in terms of the highest value of total phenolic compounds extracted together to other parameters like simplicity and cost were selected for method validation and individual phenolic compounds analysis. 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. The analytical method was developed and then validated using a mixture of six different phenolic acids and flavonoids standards. RSD % for intra-day e inter-day 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.