Improving the Analysis of Anthocyanidins from Blueberries Using Response Surface Methodology

Abstract

Background: Recent interest in the health promoting potential of anthocyanins points to the need for robust and reliable analytical methods. It is essential to know that the health promoting chemicals are present in juices and other products processed from whole fruit. Many different methods have been published using a wide variety of conditions for the hydrolysis of anthocyanins to anthocyanidins. Objective: To investigate the factors influencing the hydrolytic conversion efficiency. The optimum set of conditions will maximize the recovery of anthocyanidins. Method: Extraction procedure (freeze drying vs. direct liquid extraction), heating method (reflux vs. sealed vial), nitrogen purging and acid type were investigated. Response surface methodology was then used to find the optimum combination of incubation time, acid concentration and incubation temperature. Results: Anthocyanidin recovery can be maximized using this procedure: Freeze-dry homogenized fruit and extract with methanol:water:TFA, place 1 mL extract or juice in a test tube and add 440 µL 37% HCl, purge the tube with N2, seal with a PTFE lined cap, vortex, then heat at 99°C for 6.4 minutes. Filter the hydrolysate into an autosampler vial and analyze by UPLC immediately. Conclusion: Maximizing the recovery of anthocyanidins (by manipulating conditions in order to maximize peak areas) leads to a more accurate measure of the anthocyanidins present in blueberries.