Development and validation of a liquid chromatography method for anthocyanins in strawberry (Fragaria spp.) and...

A RPLC-DAD method for the analysis of eight anthocyanins was developed, validated and applied to strawberry extracts. The chromatographic method was conducted under gradient elution in acidulated water-methanol mobile phase and octadecyl-silica columns. An ultrasound extraction procedure was optimized by a 3(2) factorial design (%HCl in methanol, temperature, and time) and response surface methodology. Method validation was performed according to the following parameters: linearity (R(2)>0.99, p-value<10(-4), F>725), LOD (3-7 μmol L(-1)) and LOQ (9-22 μmol L(-1)), selectivity/specificity (baseline separation of all analytes and peak purity), instrumental precision (<6.4%CV), repeatability (<6.3%CV) and intermediate precision (<9.9%CV), recovery (83-99%), robustness (mobile phase pH, column temperature and flow rate) and stability (high temperatures and storage; 1st order kinetics). The antioxidant power of anthocyanins was measured on-line (ABTS(+) reaction; Trolox as reference). Ten strawberry extracts were quantified (average values: 24.2 μg/g for cyanidin-3-glucoside and 49.1 μg/g for pelargonidin-3-glucoside).