Micro-method to analyze polar phenolic substances in extra-virgin olive oil by HPLC-DAD-MS

Introduction: Polar phenolic substances play an important role in the definition of the quality of extra virgin olive oil, as supported also by the health claim allowed by EFSA[1]. In this study, a method to extract and analyze olive oil polar phenolic substances has been micro-scaled in order to reduce solvent volumes needed, and thus environmental impact, and time requested for sample preparation. Method: The extraction procedure previously developed[2] has been ten times downscaled and thus applies to 0.5 g of oil dissolved in 0.5 ml of hexane, added with internal standard and extracted 4 times each with 0.5 ml of MeOH/H2O 6/4. The final extract is washed with hexane, concentrated to 0.25 ml and analyzed by HPLC-DAD-MS. Recovery, sensitivity, linearity, precision of the method are evaluated. Results: The method allows the quantification of tyrosol, hydroxytyrosol, vanillic, ferulic, p-coumaric acid, the main secoiridoid derivatives, pinoresinol, acetoxypinoresinol, luteolin and apigenin with high sensitivity, since they are found always in amounts above the limit of quantifications (0.008-0.350 mg kg-1). Recoveries were in the range 64-100% and interday and intraday repeatability, were in the range 0.7-4.6% in terms of relative standard deviation %. In oils from the large scale retail distribution, the total content of phenolic substances was in the range 150-450 mg kg-1. In 4 out of 10 oils from the large scale retail distribution, the selected phenolic substances allowing to acknowledge the health claim, were found in concentrations above the minimum requested (250 mg kg-1) to apply the health claim. The percentage was much higher in oils purchased directly from small producers. Conclusions: The ten times down-scaled extraction method allowed to process a high number of samples in a much shorter time reducing solvents required and environmental impact. The validation of the method and the application to real samples demonstrated its efficiency and applicability to assess if an olive oil can be acknowledged with the health claim on olive oil polyphenols. References 1. Commission Regulation (EU) 432/2012, Off. J. Eur. Union, 136, 1–40 (2012). 2. Ricciutelli M., Marconi S., Boarelli M. C., Caprioli G., Sagratini G., Ballini R., Fiorini D., J. Chromatogr. A, 1481, 53–63 (2017).