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.

中文翻译：

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验证简单的 UPLC-MS-UV 和 HPLC-荧光方法测定橄榄厂废水中的油酸素。在法国品种中油酸苷分析中的应用

橄榄油生产会产生一种高度污染的水性副产品，称为橄榄厂废水 (OMW)，每吨压碎的橄榄产生的废水量高达 1200 升。OMW 含有 98% 的橄榄酚类化合物，因此人们对这种废品中的环烯醚萜类化合物（例如油酸素）的生物活性特性的兴趣迅速增长。本研究的目的首先是验证 OMW 中橄榄苦苷分析的快速 UPLC-MS、UPLC-UV 和 HPLC-荧光方法，其次是将这些方法应用于测定影响五种物质中橄榄苦苷浓度的因素。法国橄榄品种。含有 10.5 至 90 mg/g 油酸素的加标基质校准标准品用于验证，并通过将已知量的油酸素添加到具有少量油酸素的品种的 OMW 中来内部制备。通过旋转混合 15 分钟，将加标基质标准品两次萃取到 2 × 2 mL 二氯甲烷中。离心后，将合并的上清液在30°C的氮气流下蒸发。将蒸发的残留物在甲醇中重新溶解并稀释8倍，用于在不同仪器上通过UPLC-MS-UV和HPLC-荧光检测进行分析。通过重复分析和比较水性和提取的基质标准品，这三种技术均经过单独验证和交叉验证。加标标准品的重复性和中间精密度（以平均 ( n = 5) 变异系数表示）对于 UPLC-MS 分别为 2.89 和 3.79%，对于 UPLC-UV 分别为 2.14 和 2.88%，相应值为HPLC-荧光为 3.80 和 4.78%。相关系数 ( R ² ) 一般大于 0.999，对于三种方法中的每一种，“失拟”检验表明线性偏差不显着。该方法用于研究品种、收获年份和成熟阶段如何影响五个法国橄榄品种（包括 Picholine 和 Olivière）中的油酸苷含量，这些品种的这种化合物的浓度以前未曾报道过。甲基胆碱的油精素含量比其他品种高出五倍，虽然成熟阶段和收获年份会影响整体浓度，但单独成熟阶段或收获年份对其的影响并不显着。