Diode array detector

The HPLC analysis was performed using an HP1100 HPLC equipped with a diode-array detector (Hewlett-Packard, Palo Alto, CA, USA) and a reversed phase column, LiChrospher RP-18. The compounds were eluted with a gradient system of 0.4% formic acid (solvent A): methanol (solvent B) at a flow rate of 1.0 mL/min. The column temperature was set at 25 °C. The UV detection was carried out at 270 nm. The injection volume was 10 µL. The gradient system started from 0 min (100%A) to 2 min (95%A), 5 min (70%A), 8 min (66%A), 11 min (45%A), 14 min (45%A), 17 min (100%A) and maintained at this ratio until 20 min.

In order to identify and quantify the main components of the extracts, a series of HPLC-DAD analyses were performed. The HPLC system consisted of a quaternary pump (HP 1100 gradient pump), a degasser (HP 1100), an autosampler (Agilent Infinity 1260), and a Diode Array Detector (Hewlett Packard, Waldbronn, Germany). A ZORBAX Eclipse XDB-C18 column (5 μm, 250 × 4.6 mm, Agilent, Santa Clara, CA, USA) was used at room temperature, while the samples were injected after filtration (0.45 μm, PVDF syringe filters, Teknokroma, Barcelona, Spain). The gradient method, including three solvents (water, methanol, acetonitrile, acidified with TFA 0.2% v/v), has been extensively described in previous papers [81 (link),84 (link),85 (link)]. The detection was performed at 230, 280, and 360 nm, and the elaboration of chromatographic data was performed on a ChemStation for LC 3D software version B.04.06 (Agilent Technologies, Santa Clara, CA, USA). Hydroxytyrosol (Extrasynthese, France) and luteolin (Extrasynthese, France) standards were used for the development of the respective calibration curves for the quantification of the compounds in extracts.