Development and validation of a simple reversed-phase HPLC-UV method for determination of oleuropein in olive leaves

A simple, precise, accurate, and selective method is developed and validated for the determination of oleuropein, which is the main phenolic compound in olive leaves. Separation was achieved on a reversed-phase C₁₈ column (5 μm, 150 × 4.6 mm inner diameter) using a mobile phase consisting of acetonitrile/phosphate buffer pH 3.0 (20:80, v/v), at a flow rate of 1.0 mL/minute and UV detection at 280 nm. This method is validated according to the requirements for new methods, which include accuracy, precision, selectivity, robustness, limit of detection, limit of quantitation, linearity, and range. The current method demonstrates good linearity over the range of 3–1000 ppm of oleuropein, with r² > 0.999. The recovery of oleuropein in olive leaves ranges from 97.7% to 101.1%. The method is selective, in that oleuropein is well separated from other compounds of olive leaves with good resolution. The method is also precise—the relative standard deviation of the peak areas of replicate injections of oleuropein standard solution is <1%. The degree of reproducibility of the results obtained as a result of small deliberate variations in the method parameters and by changing analytical operators has proven that the method is robust and rugged. The low limit of detection and limit of quantitation of oleuropein when using this method enable the detection and quantitation of oleuropein at low concentrations.