Phenolic Compounds in Virgin Olive Oil. 2. Reappraisal of the Extraction, HPLC Separation, and Quantification Procedures

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Abstract
The extraction procedures (solid/liquid SPE and liquid/liquid LLE) and HPLC separation and quantification methods of polyphenolic compounds have been checked in virgin olive oils in order to explain the differences in content reported in the literature. The work has been carried out on oils prepared from one cultivar and produced under the same protocol. The extraction methods are practically equivalent, but the SPE technique is more favorable because it is faster and simpler. It has been proved that the chromatographic features and the method of chemical expression of the concentrations may greatly affect the final values. Thus, under the same analytical method, the total concentration values of polyphenols of the same oil show variations from 18% to 80%, according to the formality of expression as gallic acid, caffeic acid, or tyrosol equivalents. The role of the nature and spectrophotometric features of the phenols and of the internal standard is also discussed, and it was found to be an important source of reported variation. A gradient separation with an eluent mixture acetonitrile−sulfuric acid (0.1 mol/L), detection at 225 nm, and quantitative calculation of polyphenolic compounds in oils (expressed as tyrosol equivalents, THYeq) is proposed. Keywords: Olive oil; polyphenols; HPLC; quantification