Method M1
Determination of total hydroxytyrosol and tyrosol content in olive oil fit for the purpose of the health claim by UHPLC
The M1 method was developed within the frame of the project task “Increasing consumer’s confidence on EVOOs health properties: development of methods to determine phenolic compounds” is UHPLC determination of total hydroxytyrosol (Htyr) and tyrosol (Tyr) content in olive oil fit for the purpose of the health claim introduced by the EC regulation 432/2012 for “olive oil polyphenols”. It aims at quantifying the total hydroxytyrosol (Htyr) and tyrosol (Tyr) content in olive oil polar fraction before and after acidic hydrolysis of their bound forms. This content can be accurately quantified using commercially available standards and is expressed as total Htyr and Tyr (mg/20 g of oil) after correction for molecular weight differences between free and bound forms. UHPLC has been used to expedite the analysis (Tsimidou et al., 2019, Molecules, 24(6), 1044; https://doi.org/10.3390/molecules24061044).
The M1 method which is graphically depicted in Fig. 1 has been in-house validated in terms of specificity, linearity, precision, recovery and robustness, including LOD and LOQ determination of the target analytes. The extraction procedure was not validated as it was the one proposed by IOC (COI/T.20/Doc. No. 29, 2009) without the addition of the internal standard.
Fig. 1 In house validated UHPLC protocol fit for the purpose of the health claim in olive oil
The results of the M1 method have been then compared to those using the COI method (Method COI/T.20/DOC. 29/Rev. 1 – 2017, Determination of biophenols in olive oils by HPLC) as shown in a recent publication (Tsimidou et al., 2019, Molecules, 24(13), 2429; https://doi.org/10.3390/molecules24132429).
According to OLEUM project procedures, the M1 method was selected for full validation that is carried out in two circles. In the pre-trial study thirteen laboratories from the EU, China, the US and Australia participated between September and December 2019. The participants received two different extra virgin olive oil samples and were asked to analyse them following a standard operation procedure (SOP) prepared by the method developers. Participants also had the opportunity to attend a knowledge transfer workshop in October 2019 in Seville, in the course of which they asked questions and provided feedback about the application of the analytical protocol and the SOP information in particular.
Meta data analysis of the chromatograms and the respective quantitative results identified the hydrolysis step as a critical one if the exact conditions described in the SOP are not followed. The weak points raised in the pre-trial study were addressed in the revised version of the SOP sent to the participants of the trial-proper. Moreover, an audio-video of the M1 method including explanations on calculations by examples that was also produced by the developers is uploaded in YouTube (https://www.youtube.com/watch?v=-nr45PnLnF4) through OLEUM to facilitate the training of the interested stakeholders. Trial proper is now going on.
For the Aristotle University M1 developing team
Maria Z Tsimidou (Professor) and Nikolaos Nenadis (Assistant Professor)