This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/bmc.4150 This article is protected by copyright. All rights reserved. Statistical optimization of an RP-HPLC method for the determination of selected flavonoids in berry juices and evaluation of their antioxidant activities Andrija Ciric a,* , Milena Jelikic-Stankov b , Milica Cvijovic c , Predrag Djurdjevic a a University of Kragujevac, Faculty of Science, P.O. Box 60, 34000 Kragujevac, Serbia b University of Belgrade, Faculty of Pharmacy, V. Stepe 450, Belgrade, Serbia c University of Kragujevac, Faculty of Agriculture, Cara Dusana 34, 32000 Cacak, Serbia Abstract 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 berries 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, pH of 2.8 (adjusted with formic acid), flow rate 0.5 mL/min and column temperature of 35 °C. The obtained levels of bioflavonoids (mg/100 mL 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. This is considerably lower than what is found 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 14.05 mg L -1 . These values are approximately 2.5 to 3 times lower than quercetin, ascorbic acid and Trolox®, but compared to rutin and butylhydroxytoluene, berries show similar or better antioxidant activity by both the DPPH and ABTS methods.