3.4.2. Analysis and Quantification of Flavonoids by HPLC–DAD

Flavonoids were quantified directly from the ethanolic extracts. A Jasco-LC-Net II ADC liquid chromatograph system equipped with a DAD was used. Separation was carried out in a Mediterranea Sea C18 reverse-phase analytical column (25 cm length × 4.6 mm i.d., 5 µm particle size; Teknokroma, Barcelona, Spain). A gradient of solvent A (water with 1% formic acid) and solvent B (acetonitrile with 1% formic acid) was used: the proportion of solvent B was increased from 0% to 20% in 20 min, then to 21% over the next 8 min, maintained at 21% for 2 min, then to 30% over the next 10 min, and to 100% over the next 5 min, maintained at 100% B for 5 min and finally returned to the initial conditions over the next 5 min. The flow rate was 1 mL/min and the column temperature was 30 °C. Spectra from all peaks were recorded in the 200–600 nm range and the chromatograms were acquired at 360 nm for flavonoid glycosides and 370 nm for their aglycones.

The quantification of flavonoids was carried out as described by Fuentes-Alventosa et al. [42]. The identification of individual flavonoid glycosides was based on their retention times (tR) and spectroscopic data. The quantification of individual flavonoid monoglycosides and flavonoid diglycosides was directly performed by HPLC-DAD using an eight-point regression curve in the range of 0–250 μg on the basis of standards. Results were calculated from the mean of three replicates.