pubs.acs.org/JAFC Published on Web 07/26/2010 © 2010 American Chemical Society J. Agric. Food Chem. 2010, 58, 9129–9136 9129 DOI:10.1021/jf101847e Separation and Identification of Phenolic Compounds of Extra Virgin Olive Oil from Olea europaea L. by HPLC-DAD-SPE-NMR/MS. Identification of a New Diastereoisomer of the Aldehydic Form of Oleuropein Aglycone MI ´ RIAM PE ´ REZ-TRUJILLO,* ,† ANA MARI ´ A GO ´ MEZ-CARAVACA, ‡ ANTONIO SEGURA-CARRETERO, ‡ ALBERTO FERNA ´ NDEZ-GUTIE ´ RREZ, ‡ AND TEODOR PARELLA † † Servei de Resson ancia Magn etica Nuclear (SeRMN), Universitat Aut onoma de Barcelona (UAB), 08193 Bellaterra, Spain, and ‡ Departamento de Quı´mica Analı´tica, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain The phenolic fraction of a monovarietal extra virgin olive oil (EVOO) from Olea europaea L. var. Cornezuelo was studied by the hyphenated HPLC-DAD-SPE-NMR/MS techniques. This survey led to the identification of 25 main compounds. One was identified as a new diastereoisomer of the aldehydic form of oleuropein aglycone (AOA) and characterized by 1D and 2D NMR techniques. The relative configuration of this new AOA was determined as 5R*,8S*,9S* on the basis of the results obtained from the combination of NOE experiments and Monte Carlo conformational search calculations. Assuming, as for the described diastereoisomers, that the new AOA comes from the natural oleuropein aglycone (OA), the absolute configuration was proposed as 5S,8R,9R. KEYWORDS: Olea europaea L.; olive oil; phenolic compounds; aldehydic form of oleuropein aglycone; HPLC-DAD-SPE-NMR/MS INTRODUCTION Olive oil represents the typical lipidic source of the Mediterranean diet, and its consumption is related to lower death rates, higher life expectancy, and a low incidence of several pathologies, inclu- ding cardiovascular diseases and neurological disorders ( 1 -3 ). In recent years its consumption has also spread remarkably outside the Mediterranean basin. The growing interest in olive oil may be partly related to its unique taste; however, particular interest is due to its nutritional properties. Phenolic compounds from olive oil are widely known due to their fundamental importance for its nutritional properties, sensory characteristics, and shelf life ( 4 ); because they have high antioxidant properties, they can confer a marked bitter taste or a sweet taste typical of some virgin olive oils ( 5 ). However, the biological properties of virgin olive oil’s constituents have only been investigated in the past decade. Olive oil phenolics have beneficial biological activities such as altered lipid composition, platelet and cellular function, and microbio- logical activity, as well as a reduction in oxidative damage and inflammation in both human and animal (in vivo and in vitro) studies ( 6 ). Besides, some effects against cancer have been seen; that is, treatment of human colon adenocarcinoma cells with olive oil phenolics inhibits initiation, promotion, and metastasis of the colon carcinogenesis process ( 7 ). Also, some studies have been carried out in breast cancer, revealing antiproliferative effects by the action of crude extra virgin olive oil (EVOO) phenolic extracts and suggesting that the stereochemistry of EVOO-derived lignans and secoiridoids might provide an excellent and safe platform for the design of new anti-breast cancer drugs ( 8 ). With regard to seco- iridoids and specifically oleuropein aglycones, Paiva-Martins et al. ( 9) have demonstrated recently the noteworthy protective role against oxidative injury in human cells of an oleuropein aglycone, specifi- cally the 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde. Considering all this, detailed knowledge of the phenolic com- pounds of olive oil becomes of high interest. Over the years the determination of the phenolic fraction of EVOO has been widely studied, and the development of methodo- logies for its determination has been discussed extensively in the literature. Because of the need to carry out an individual identifi- cation of each phenolic compound present in the extract, spectro- photometric methods were replaced with separative techniques: GC ( 10 ), capillary electrophoresis (CE) ( 11 , 12 ), and, in particular, HPLC ( 13 ) coupled to different detectors such as ultraviolet (UV-vis) ( 14 ) electrochemical ( 15 ), fluorescence ( 16 ), or MS detection ( 17 , 13 ). In some cases, MS and MS/MS data cannot give detailed and conclusive information. Specifically, MS detec- tion can hardly ever differentiate between isomers ( 18 ). However, NMR spectroscopy is a powerful technique that provides valuable structural information of molecules, leads to the complete char- acterization of compounds, and more importantly can distinguish between structural isomers and between diastereoisomers. The hyphenation of HPLC and NMR spectroscopy allows the complete assignment and structure determination of analytes in complex mixtures, becoming an important technique in a lot of *Corresponding author (phone 00-34-935813785; fax 00-34-935813784; e-mail miriam.perez@uab.cat).