Toward a Harmonized and Standardized Protocol for the Determination of Total Hydroxytyrosol and Tyrosol Content in Virgin Olive Oil (VOO). Extraction Solvent

The determination of the total hydroxytyrosol (Htyr) and tyrosol (Tyr) content of virgin olive oil is of utmost interest for the International Olive Council (IOC), food authorities, producers, and distributors after the issuing of a health claim that “olive oil polyphenols contribute to the protection of blood lipids from oxidative stress.” To address the need of a harmonized and standardized protocol the present study focuses on the extraction solvent of the polar fraction. Aqueous mixtures of methanol and acetonitrile of equal polarity are prepared and comparatively examined. Different analytical approaches (Folin‐Ciocalteu assay, reversed phase chromatography‐diode array‐fluorescence detection, LC‐Orbitrap, LC‐TOF, LC‐TQd, ¹H‐NMR) are applied to highlight the extracting efficiency of the tested mixtures regarding the phenolic content and composition. The use of acetonitrile does not have a clear positive effect that could compensate its higher cost, commercial availability, and toxicity. The findings justify further why methanol:water, 80:20 v/v should be retained in a future IOC protocol for the accurate and repeatable determination of total Htyr and Tyr content, which is necessary to support the health claim for “olive oil polyphenols.” Practical Applications: The development of a harmonized and standardized methodology for the determination of total Htyr and Tyr content in an explicit manner is a request of the olive oil sector. A tool that can be introduced easily in the olive industry and official laboratories for the control of the label that bears a health claim for “olive oil polyphenols,” is also requested by the consumers and the IOC. In this view, the present study contributes to the cornerstone that is the standardization of the extraction solvent. Methanol:water 80:20 v/v, should be retained in an International Olive Council protocol for the accurate and repeatable determination of total hydroxytyrosol and tyrosol content necessary to support the health claim for “olive oil polyphenols”. If the total hydroxytyrosol and tyrosol content is determined after hydrolysis of the polar fraction no effect of artifact formation in the quantitation should be expected.