A simple HPLC-fluorescence screening method for rapid determination of oleuropein in olive leaves; cross validation with an UPLC-MS-UV method and analysis of leaves of French olive varieties

ABSTRACT Olive leaves are an abundant but under-exploited by-product of the olive oil industry, comprising 10 % of the total mass processed for olive oil. They are a rich source of oleuropein, the most abundant polyphenol in olive leaves, whose bioactive properties are widely documented. If olive leaves are to be seriously considered as a readily-available source of oleuropein, simple analytical methods are needed to identify varieties and growing conditions conducive to its formation. HPLC-fluorescence detection and UPLC-UV-MS methods on different instruments were developed and cross-validated using blank (oleuropein-free) leaves produced in-house to establish matrix standards in the concentration range 0.4 to 4.8 mg/g. For the HPLC-FLD method, mean repeatability and intermediate precision were respectively 3.64% and 4.76% relative standard deviation; the coefficient of determination was generally >0.9999 and linearity was demonstrated by the Fischer test. There was a close linear relationship (R2 > 0.999) between back-calculated concentrations of both spiked placebos and authentic standards. Analysis of the same spiked placebos by the three chromatographic methods showed no significant differences in the validation results as confirmed by ANOVA. Cross-validation of the three methods indicate that selective quantification can equally be carried out on standard HPLC equipment available to most laboratories or by more sophisticated UPLC-MS techniques in larger structures. The determination of oleuropein in “Olivière” olive variety is reported here for the first time, and leaves collected in 2022 contained about 40 mg/g oleuropein, almost twice the amount found in Picholine and three times more than in other varieties.