Abstract
Abstract : Introduction Olive leaves, abundant by‐products of the olive oil industry, are a rich source of oleuropein, an important polyphenol in olive leaves. So far, no published methods have been validated using matrix standards for oleuropein quantification in olive leaves. Objectives : The study aimed to develop an HPLC method for oleuropein determination in olive leaves using spiked matrix standards prepared from a blank olive leaf matrix, to validate the method with respect to aqueous standards, and cross‐validate the HPLC method with UPLC‐MS and UPLC‐UV techniques. Methodology : Oleuropein was extracted into methanol and analysed by HPLC with fluorescence detection (FLD; excitation and emission wavelengths 281 and 316 nm, respectively) and by UPLC‐MS‐UV. For validation, calibration curves of spiked matrix standards (0.4 to 4.8 mg/g) were analysed by the three methods over several days. Oleuropein was then analysed in French olive varieties. Results : For the HPLC‐FLD method, repeatability and intermediate precision were less than 5% RSD and linearity was demonstrated by the Fischer test. Differences in results of the spiked placebos by the three methods were non‐significant, as confirmed by ANOVA. Extraction recovery was >90%, and there was a strong linear relationship between authentic and spiked matrix standards. The determination of oleuropein in French olive varieties is reported, including analysis in “Olivière” cultivar for the first time, leaves of which contained twice the amount of oleuropein compared with “Picholine”. Conclusion : Accurate quantification of oleuropein is possible using aqueous standards. Cross‐validation indicates that selective analysis can equally be carried out by HPLC or by UPLC‐MS techniques.