Determination of DPPH Free Radical Scavenging Activity by RP-HPLC, Rapid Sensitive Method for the Screening of Berry Fruit Juice Freeze Dried Extract

Abstract

USP official UV-VIS spectrophotometric method for the evaluation of antioxidant activity by scavenging the DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical was not suitable to determine the antioxidant activity of Berry fruit juice freeze dried extract due to interferences from the matrix. Interference was also observed from the color pigments of the Berry fruit juice freeze dried extract. This has leads to the development of a specific analytical method for evaluation of the DPPH free radical scavenging activity of Berry fruit juice freeze dried extract. RP-HPLC method was developed on a Phenomenex Luna-C18 Column (250 mm × 4 mm, 5 μM) with mobile phase methanol and water mixed online in the ratio of 80:20 (v/v), pumped at a flow rate of 1 mL/min. DPPH peaks were quantified at wavelength 517 nm. Method was standardized using the anti-oxidant ascorbic acid. The 50% free radical scavenging activity (IC50) determined by the novel HPLC method was correlating with the results obtained by USP UV-VIS spectrophotometric method. While the USP UV-VIS spectrophotometric method failed to estimate the free radical scavenging activity of Berry fruit juice freeze dried extract, whereas the developed HPLC method was specific, sensitive, robust, accurate and precise and rapid in the determination.