Validation of Simple UPLC-MS-UV and HPLC-Fluorescence Methods for the Determination of Oleacein in Olive Mill Wastewater. Application in the Analysis of Oleacein in French Cultivars

Olive oil production results in a highly polluting aqueous byproduct, known as olive mill wastewater (OMW), which occurs in volumes up to 1200 L per ton of crushed olives. OMW contains 98% of olive phenolic compounds, so there is rapidly growing interest in the bioactive properties of secoiridoids such as oleacein in this waste product. The aim of the study presented here was first to validate rapid UPLC-MS, UPLC-UV, and HPLC-fluorescence methods for the analysis of oleuropein in OMW and secondly to apply these methods to the determination of factors influencing the concentration of oleacein in five French olive varieties. Spiked matrix calibration standards containing 10.5 to 90 mg/g oleacein were used for validation and were prepared in-house by adding known amounts of oleacein to the OMW of a variety with a low amount of oleacein. The spiked matrix standards were extracted twice into 2 × 2 mL of dichloromethane by rotary mixing for 15 min. Following centrifugation, the combined supernatants were evaporated under a stream of nitrogen at 30 °C. The evaporated residues were reconstituted in methanol and diluted 8-fold for analysis by UPLC-MS-UV and by HPLC-fluorescence detection on a different instrument. The three techniques were both individually validated and cross-validated by repeat analysis and comparison of aqueous and extracted matrix standards. Repeatability and intermediate precision of spiked standards, expressed as the mean (n = 5) coefficient of variation, were 2.89 and 3.79%, respectively, for UPLC-MS and 2.14 and 2.88%, respectively, for UPLC-UV, with corresponding values of 3.80 and 4.78% for HPLC-fluorescence. The coefficients of correlation (R²) were generally greater than 0.999, and for each of the three methods, the “lack of fit” test showed that deviation from linearity was nonsignificant. The methods were applied to investigate how variety, harvest year, and ripening stage influence the amount of oleacein in five French olive cultivars including Picholine and Olivière whose concentrations of this compound were not previously reported. Picholine contained up to five times more oleacein than the other cultivars, and while the ripening stage combined with the harvest year affected overall concentration, it was not significantly impacted by the ripening stage or harvest year individually.