Optimization and application of HPLC for simultaneous separation of six well-known major anthocyanins in blueberry

Anthocyanins have attracted great attention because of their potential therapeutic benefit. However, the effective technique for simultaneous separation and preparation multiple anthocyanin monomers with high purity and high yield is still deficient. In this study, the chromatographic conditions of HPLC were optimized to investigate six well-known major anthocyanins (delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, pelargonidin-3-O-glucoside, peonidin-3-O-glucoside and malvidin-3-O-glucoside) in blueberry. The separation conditions were optimized in analytical HPLC and further applied in semi-preparative HPLC to prepare anthocyanin monomers. The results showed that six well-known major anthocyanins were well separated under the condition of using acetonitrile-water (contained 0.3% phosphoric acid) as a mobile phase with gradient elution at a detection wavelength of 520 nm. The method showed good linear correlations between the concentrations and peak areas of the six components with correlation coefficients greater than 0.9994, and the detection limits of the six anthocyanins were 0.010-0.035 μg/mL, and the quantification limits were 0.033-0.117 μg/mL, which was suitable for the determination of anthocyanins in products. In the same experimental conditions, six well-known major anthocyanins were simultaneously prepared by semi-preparative HPLC with high purity to 99% and high yield to 22.5%. This study provides a practical and valuable method for simultaneous determination and preparation of six well-known major anthocyanins.