Development and validation of green chromatography for the determination of anthocyanins in haskap berry, mulberry and blackberry

Abstract

This study was aimed at developing and validating a green HPLC-DAD method to determine anthocyanins using ethanol and an alpha-hydroxy acid aqueous solution as a mobile phase. The effects of alpha-hydroxy acids on the chromatographic behaviors of cyanidin-3-O-glucoside, the most predominant anthocyanin in nature, in a C18 column were investigated. Among seven selected alpha-hydroxy acids, tartaric acid exhibited potential for improving the chromatographic separation of cyanidin-3-O-glucoside. Cyanidin-3-O-glucoside was the main anthocyanin in haskap berry, mulberry, and blackberry, and the three berries were selected for further method development and validation. The optimized mobile phase was a mixture of ethanol and a 0.25 mol L⁻¹ tartaric acid aqueous solution at the 20 : 80 (v/v) ratio. The retention time of cyanidin-3-O-glucoside was less than 1.8 min, and an optimum separation of anthocyanins was achieved in 4 min. The developed method was validated in terms of linearity, limits of detection and quantification, precision, repeatability, stability, and recovery. The calibration curve revealed good linearity (R² = 0.9999), and the limits of detection and quantification were 0.038 mg L⁻¹ and 0.125 mg L⁻¹, respectively. The relative standard deviations of intra- and inter-day precision, repeatability, and stability were less than 5%. The recoveries of cyanidin-3-O-glucoside from the berry samples were between 99% and 101%, a satisfactory level. Additionally, the optimized mobile phase had a negligible effect on the C18 column stability. Therefore, the developed method was safe, rapid, and valid for anthocyanin analysis.