Validation of pH Differential Method for the Determination of Total Anthocyanin Content in Black Rice and Black Soybean with Interlaboratory Comparison

Abstract

An interlaboratory study was performed to find a method for the determination of total anthocyanin content in black rice and black soybean, based on the pH differential method. The extraction procedure of anthocyanin in milled materials of black rice and black soybean was optimized ; the recoveries determined for milled materials of rice and soybean spiked with cyanidin-3-glucoside were 98.9% and 92.1%, respectively. Duplicate determinations of total anthocyanin content performed by two technicians on five different test materials on three different days resulted in relative standard deviations of 1.6 to 2.7%. Seven collaborators in six laboratories participated, and measured the total anthocyanin content in five test materials as blind duplicates by the proposed method. A statistical treatment revealed that the relative standard deviation of the repeatability (RSDr) and the relative standard deviation of the reproducibility (RSDR) for the determination of total anthocyanin content were 0.9% to 4.0% and 1.5% to 6.1%, respectively. The HorRat values (RSDR/predicted RSDR) were 0.25 to 1.24. The present study demonstrated that the proposed method for the determination of total anthocyanin content in black rice and black soybean showed good interlaboratory reproducibility in the range from 0.50 to 2.71 mg/g. The expanded uncertainty, obtained by multiplying the standard deviation of the reproducibility (sR) by the coverage factor (k = 2), ranged from 0.021 to 0.326 mg/g.