Oleuropein Quantification in Olive Leaf Extracts

Olive leaf extracts were analyzed to determine the content of oleuropein by HPLC-UV analysis using a previous method with slight modifications [52 (link)]. The extracts were reconstituted in MeOH, and HPLC-UV analyses were performed on an UltiMate 3000 instrument equipped with an RS-3000 Diode Array Detector operating at 280 nm. Xcalibur software was used to acquire and process the data. A Kinetex Polar C18 column (150 × 2.1 mm, 2.6 µm, 100 Å; Phenomenex, Bologna, Italy) with a SecurityGuard C18 guard column (2 × 2.1 mm), thermostated at 35 ± 1 °C, was used for the chromatographic separation. The solvents used for the elution gradient were A (H₂O containing 0.1% formic acid) and B (acetonitrile containing 0.1% formic acid); the linear gradient was eluted from 5% to 50% of B (0–10 min), and from 50% to 95% of B (10–15 min). The injection volume was 3 μL, and the flow rate of 0.4 mL/min. The quantitative determination of oleuropein was carried out via external calibration. The calibration curve was acquired by injecting standard solutions in the linearity range of concentrations, 5–100 mg/L, obtaining the equation y = 1930.8x, R² = 0.9998. The LOD and LOQ were also determined as 1.00 and 3.00 mg/L, respectively. All samples were extracted and analyzed in triplicate (n = 9), and the results are expressed in milligrams per gram as mean concentration ± standard deviation.