W19 - Flow Injection Determination of the Antioxidant Class Anthocyanin in Fruit and Beverages

In fifteen words or less, explain the significance of this contribution (Novel Aspect).: No previously reported flow injection method for anthocyanins.

Abstract Text:

Anthocyanins represent an important type of polyphenolic compounds found in fruit. The color of anthocyanins is pH dependent. At pH 4.5, anthocyanin is colorless, with a hemiketal structure, but at pH 1 the conjugated flavylium cation forms, causing a reddish color. The difference in absorbance at 520  nm of these forms of anthocyanin is a measure of total anthocyanin in a fruit sample. We have developed a quality control method for anthocyanins in various fruit beverages using flow injection analysis. The homebuilt instrument is composed of a four solvent line HPLC pump, a HPLC injection valve, and a HPLC UV-VIS detector with peak height readout. Method 1 involved the sequential injection of all the samples into the acetate pH 4.5 buffer mobile phase, then re-equilibrating the instrument with a sulfuric acid pH 1 buffer, and finally re-injecting all the samples again. Method 2 involved injection of each sample at pH 4.5 and then that same sample at pH 1, taking advantage of the multi-solvent lines of the pump. Solvent line A was equilibrated with pH 4.5 buffer and solvent line B with pH 1 buffer. With solvent line A set at 100% buffer, the sample was injected in triplicate and then switched to 100% B. After a 90 second wait to ensure the instrument was completely equilibrated with pH 1 buffer, the samples were injected again in triplicate. Depending on flowrate and measurement of replicate peaks, sample analysis time can range from about 3-7 min. Reproducibility of triplicate injections was generally 1-3% RSD. A comparison of Method 1 and Method 2, done several months apart giving a correlation coefficient of 0.974, was based on the analysis of the following juice samples: blueberry, pomegranate, grape, cherry, prune, antioxidant blend, and blueberry vitamin water. Anthocyanins in blueberry and pomegranate were about four times higher as compared to that for grape which was twice that of cherry which was twice that of the other drinks. A 50 % water-50% methanol extraction of ground pomegranate was found to give the best anthocyanin signal.