Statistical optimization of an RP-HPLC method for the determination of selected flavonoids in berry juices and evaluation of their antioxidant activities

An isocratic RP-HPLC method for the separation and identification of selected flavonoids (quercetin, rutin, luteolin-7-O-glucoside, kaempferol and kaempferol-3-O-glucoside) in commercial berry juices (blackcurrant, blueberry, red raspberry and cherry) was developed with the aid of central composite design and response surface methodology. The optimal separation conditions were a mobile phase of 85:15 (% v/v) water-acetonitrile, pH2.8 (adjusted with formic acid), flow rate 0.5mL min(-1) and column temperature 35 degrees C. The obtained levels of bioflavonoids (mg per 100mL of juice) were as follows: for quercetin, ca. 0.21-5.12; for kaempferol, ca. 0.05-1.2; for rutin, ca. 0.4-6.5; for luteolin-7-O-glucoside, ca. 5.6-10.2; and for kaempferol-3-O-glucoside, ca. 0.02-0.12. These are considerably lower than the values in fresh fruits. Total phenolic, flavonoid and anthocyanin contents were determined spectrophotometrically. Total flavonoid content varied as follows: blackcurrant > blueberry > red raspberry > cherry. The antioxidant activity of juice extracts (DPPH and ABTS methods) expressed as IC50 values varied from 8.56 to 14.05mg L-1. These values are similar to 2.5-3 times lower than quercetin, ascorbic acid and Trolox (R), but compared with rutin and butylhydroxytoluene, berries show similar or better antioxidant activity by both the DPPH and ABTS methods.