Abstract: In this study, the influence of different molar extinction coefficients and reference substances on the determination of total anthocyanins in black chokeberry by the pH differential method and high performance liquid chromatography (HPLC), respectively was investigated. The results obtained by the optimized pH differential method were compared with those obtained by the HPLC method using t-test. Results showed that the selection of molar extinction coefficient and reference substance had a significant impact on the results of the two methods. Moreover, the pH differential method with cyanidin-3-O-galactoside, which is abundant in black chokeberry as the reference substance, and HPLC with a mixed reference standard were superior to the traditional method with cyanidin-3-O-glucoside as the reference substance (P < 0.05). There was no significant difference between the results of HPLC with cyanidin-3-O-galactoside as the reference substance and those with the mixed reference standard (P > 0.05). The results of HPLC were higher than those of the pH differential method. Collectively, despite its low cost, HPLC with the mixed reference standard was the most accurate method for the determination of total anthocyanins in black chokeberry. HPLC with cyanidin-3-O-galactoside as the reference substance was simple, accurate and economical and also suitable for this purpose.

Key words: black chokeberry; anthocyanins; high performance liquid chromatography; pH differential method