HPLC-DAD Analysis of Phytochemicals

The concentrations of active substances were measured by using the HPLC-DAD (Dionex Thermoline Fisher Scientific, Waltham, MA, USA) with Chromeleon software version 7.0. Separations were performed on a LiChrospher RP-18 column, 5 μm particle size, 250 mm × 4 mm (Merck, Darmstadt, Germany). The detection was done with a diode array detector at a wavelength maxima (λ_max) of 270 and 360 nm, depending on active compounds. The mobile phase was composed of formic acid 0.1% in water (A) and acetonitrile (B) with a gradient elution: 0–35 min, 2–20% B; 35–55 min, 20–70% B; 55 min, 2% B; 55–60 min, 2% B, with mobile phase flow set at 1.0 mL/min. The column temperature was kept at 30 °C.
The presence of 11 active compounds (phenolic acids: caffeic acid, ellagic acid, gallic acid, syringic acid; flavonols: quercetin, kaempferol, and their glycosides: rutin, isoquercetin, hyperoside; flavon-3-ols: catechin, epicatechin) in the extracts was confirmed by comparison of retention time and UV spectra of particular substances; whereas, the quantitative assessment of the content included 7 actives (caffeic acid, ellagic acid, quercetin, kaempferol, rutin, hyperoside, epicatechin).
In terms of selectivity, linearity, intra- and inter-day accuracy, limits of detection (LOD), and quantitation, the HPLC-DAD method was validated according to the International Conference on Harmonization Guideline Q2 (LOQ) [15 ].