title: Employment of High-Performance Thin-Layer Chromatography for the Quantification of Oleuropein in Olive Leaves and the Selection of a Suitable Solvent System for Its Isolation with Centrifugal Partition Chromatography.
abstract: A high-performance thin-layer chromatographic methodology was developed and validated for the isolation and quantitative determination of oleuropein in two extracts of Olea europaea leaves. OLE_A was a crude acetone extract, while OLE_AA was its defatted residue. Initially, high-performance thin-layer chromatography was employed for the purification process of oleuropein with fast centrifugal partition chromatography, replacing high-performance liquid-chromatography, in the stage of the determination of the distribution coefficient and the retention volume. A densitometric method was developed for the determination of the distribution coefficients, KC = CS/CM. The total concentrations of the target compound in the stationary phase (CS) and in the mobile phase (CM) were calculated by the area measured in the high-performance thin-layer chromatogram. The estimated Kc was also used for the calculation of the retention volume, VR, with a chromatographic retention equation. The obtained data were successfully applied for the purification of oleuropein and the experimental results confirmed the theoretical predictions, indicating that high-performance thin-layer chromatography could be an important counterpart in the phytochemical study of natural products. The isolated oleuropein (purity > 95%) was subsequently used for the estimation of its content in each extract with a simple, sensitive and accurate high-performance thin-layer chromatography method. The best fit calibration curve from 1.0 µg/track to 6.0 µg/track of oleuropein was polynomial and the quantification was achieved by UV detection at λ 240 nm. The method was validated giving rise to an efficient and high-throughput procedure, with the relative standard deviation % of repeatability and intermediate precision not exceeding 4.9% and accuracy between 92% and 98% (recovery rates). Moreover, the method was validated for robustness, limit of quantitation, and limit of detection. The amount of oleuropein for OLE_A, OLE_AA, and an aqueous extract of olive leaves was estimated to be 35.5% ± 2.7, 51.5% ± 1.4, and 12.5% ± 0.12, respectively. Statistical analysis proved that the method is repeatable and selective, and can be effectively applied for the estimation of oleuropein in olive leaves' extracts, and could potentially replace high-performance liquid chromatography methodologies developed so far. Thus, the phytochemical investigation of oleuropein could be based on high-performance thin-layer chromatography coupled with separation processes, such as fast centrifugal partition chromatography, showing efficacy and credibility.
authors.affiliation: Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens, Greece.
authors.forename: Vasiliki-Ioanna
authors.initials: VI
authors.lastname: Boka
authors.affiliation: Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens, Greece.
authors.forename: Aikaterini
authors.initials: A
authors.lastname: Argyropoulou
authors.affiliation: Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens, Greece.
authors.forename: Evangelos
authors.initials: E
authors.lastname: Gikas
authors.affiliation: Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens, Greece.
authors.forename: Apostolis
authors.initials: A
authors.lastname: Angelis
authors.affiliation: Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens, Greece.
authors.forename: Nektarios
authors.initials: N
authors.lastname: Aligiannis
authors.affiliation: Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens, Greece.
authors.forename: Alexios-Leandros
authors.initials: AL
authors.lastname: Skaltsounis
mesh_terms: D002498:Centrifugation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002855:Chromatography, Thin Layer; D057888:Iridoid Glucosides; D039823:Iridoids / Q000302:isolation & purification*; D031658:Olea / Q000737:chemistry*; D018515:Plant Leaves / Q000737:chemistry*; D012997:Solvents
chemical_list: D057888:Iridoid Glucosides; D039823:Iridoids; D012997:Solvents; C002769:oleuropein