Title: (empty line)
Author: Herr Dr. Amoneit
Last saved by: Saritas, Sevim
Company: Universidad de Castilla-La Mancha

Searching a Suitable Method to Determine the Phenolic Content of Olive Oils in order to Bear the Health Claim Introduced by Commission Regulation 432/2012
P. Reboredo-Rodríguez^a, E. Valli^b, A. Bendini^b,c, T. Gallina Toschi^b,c, J. Simal-Gándara^a
^a Analytical and Food Chemistry Department, Nutrition and Bromatology Group, University of Vigo, Ourense, Spain
^b Interdepartmental Centre for Agri-Food Industrial Research, Alma Mater Studiorum- University of Bologna, Cesena (FC), Italy
^c Department of Agricultural and Food Sciences, Alma Mater Studiorum- University of Bologna, Cesena (FC), Italy

In European Union, the Commission Regulation 432/2012 introduced the possibility to report the health claim “Olive oil polyphenols contribute to the protection of blood lipids from oxidative stress” if the olive oil contains at least 5 mg of hydroxytyrosol (HTyr) and its derivatives (e.g. oleuropein complex and tyrosol (Tyr) per 20 g of product). 
The purpose of this work was to find a simple and suitable method −among the most widely employed− to guarantee the proper determination and quantification of the olive oil phenolic content. Several protocols were employed: total phenolics by Folin-Ciocalteu (FC) and o-diphenolics by the colorimetric molibdate method (MbM) versus phenols by high-performance liquid chromatography-diode array detection (HPLC-DAD), being the HPLC-DAD the most sensitive and specific method for evaluating the phenolic content. All protocols were performed before and after an acid hydrolysis; acid hydrolysis involves a break of the bound forms of HTyr and Tyr and quantification of their total free forms. Different phenolics were used in the calibration curves of the quantification process: oleuropein, gallic acid and/or caffeic acid only for spectrophotometric determinations, while HTyr and Tyr for spectrophotometric and HPLC determinations.
The concentrations obtained for 10 oil samples with very different phenolic profiles were statistically compared by means of two-tailed paired t-tests. In this way, results obtained by FC assay (quantified by gallic acid) before and/or after acid hydrolysis were statistically comparable with results obtained by acid hydrolysis-HPLC-DAD and quantified by external calibration curves of HTyr and Tyr.