Published: September 22, 2011 r2011 American Chemical Society 11491 dx.doi.org/10.1021/jf202596q | J. Agric. Food Chem. 2011, 59, 11491–11500 ARTICLE pubs.acs.org/JAFC Wastes Generated during the Storage of Extra Virgin Olive Oil as a Natural Source of Phenolic Compounds Jesus Lozano-S  anchez, †,§ Elisa Giambanelli, # Rosa Quirantes-Pin e, †,§ Lorenzo Cerretani,* ,# Alessandra Bendini, # Antonio Segura-Carretero, †,§ and Alberto Fern  andez-Guti  errez †,§ † Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Fuentenueva s/n, E-18071 Granada, Spain § Functional Food Research and Development Center, Health Science Technological Park, Avenida del Conocimiento s/n, E-18100 Granada, Spain # Departament of Food Science, Alma Mater Studiorum, University of Bologna, Piazza Goidanich 60, I-47521 Cesena (FC), Italy b S Supporting Information ABSTRACT: Phenolic compounds in extra virgin olive oil (EVOO) have been associated with beneficial effects for health. Indeed, these compounds exert strong antiproliferative effects on many pathological processes, which has stimulated chemical characteriza- tion of the large quantities of wastes generated during olive oil production. In this investigation, the potential of byproducts generated during storage of EVOO as a natural source of antioxidant compounds has been evaluated using solidliquid and liquidliquid extraction processes followed by rapid resolution liquid chromatography (RRLC) coupled to electrospray time-of- flight and ion trap mass spectrometry (TOF/IT-MS). These wastes contain polyphenols belonging to different classes such as phenolic acids and alcohols, secoiridoids, lignans, and flavones. The relationship between phenolic and derived compounds has been tentatively established on the basis of proposed degradation pathways. Finally, qualitative and quantitative characterizations of solid and aqueous wastes suggest that these byproducts can be considered an important natural source of phenolic compounds, mainly hydroxytyrosol, tyrosol, decarboxymethyl oleuropein aglycone, and luteolin, which, after suitable purification, could be used as food antioxidants or as ingredients in nutraceutical products due to their interesting technological and pharmaceutical properties. KEYWORDS: olive oil, storage, byproduct, polyphenols, degradation pathways, antioxidant natural source, RRLC, MS/MS fragmentation ’ INTRODUCTION Phenolic compounds are an important class of natural anti- oxidants. The term “phenolic compound” includes a large number of secondary plant metabolites that differ in chemical structure and reactivity, ranging from simple compounds to highly polymerized molecules. When present in small amounts in food, phenolic compounds are capable of preventing or retarding the oxidation of oils and fats. The presence of these compounds in fats, oils, and lipid- based foods is very important to reduce oxidative reactions that can lead to a decrease in both the nutritional value and sensory quality. 1 Polyphenols belonging to many chemical classes have been described in extra virgin olive oil (EVOO), and in particular phenolic acids and alcohols, including p-coumaric acid, ferulic acid, vanillic acid, vanillin, 3,4-(dihydroxyphenyl)ethanol (hydroxytyrosol), and p-hydro- xyphenylethanol (tyrosol), have been described. However, the main phenolic compounds are secoiridoid derivatives of oleuropein and ligstroside, such as the decarboxymethylated form of elenolic acid linked to either hydroxytyrosol or tyrosol (oleocanthal). 24 The phenolic profile has been used to evaluate the quality of EVOO as well as the presence of these compounds as they differentiate olive oil from other edible vegetable oils as the most hydrophilic phenols in EVOO are not common to other oils or fats. 5 There are many technological factors that can influence the content of phenolic compounds, and in this regard the effects of the extraction process as well as the changes in minor compounds of EVOO during storage have been evaluated. 69 Furthermore, olive oil production, an agroindustrial activity of vital economic signi ficance for many Mediterranean countries, is asso- ciated with the generation of large quantities of wastes. Polyphenolic content has been assessed in these byproducts due to the biological and pharmaceutical interest in olive oil phenolic compounds. 10,11 Indeed, the polyphenolic activity on di fferent cancer cells is also well- known 4,12 and has stimulated research on the profile of phenolic compounds in the di fferent parts of the olive tree, and new methods have been developed to extract these compounds. 13 Wastewater from olive oil mill wastes is characterized by a high content of phenolic alcohols (mainly hydroxytyrosol and tyrosol). 14 Oleuropein has been described as a major compound in olive leaves and branches, which also contain high concen- trations of glucosylated flavones such as luteolin-7-glucoside and apigenin-7-glucoside. 15 Additionally, branches are char- acterized with an elevated amount of verbascoside, a precursor of hydroxytyrosol. 16,17 The byproducts generated during the filtration process of EVOO have been also evaluated, and different classes of hydrophilic phenolic compounds are retained in filter aids, including phenolic acids, phenolic alcohols, secoiridoids, lignans, and flavones. 18 Received: June 29, 2011 Revised: September 21, 2011 Accepted: September 22, 2011