Methods of analysis for anthocyanins in plants and biological fluids

Anthocyanins are the largest group of water-soluble pigments in the plant kingdom. They are responsible for most of the red, blue, and purple colors of fruits, vegetables, flowers, and other plant tissues or products. The analysis of anthocyanins is complex as a result of their ability to undergo structural transformations and complexation reactions. In addition, they are difficult to measure independently of other flavonoids, as they have similar structural and reactivity characteristics. Anthocyanins are generally extracted with weakly acidified alcohol-based solvents, followed by concentration (under vacuum), and purification of the pigments. Paper and/or thin-layer chromatography and UV-Vis spectroscopy have traditionally been used for the identification of anthocyanins. Capillary zone electrophoresis, a hybrid of chromatography and electrophoresis, is gaining popularity for the analysis of anthocyanins; however, liquid chromatography (LC) has become the standard method for identification and separation in most laboratories and may be used for both preparative and quantitative analysis. LC with mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are possibly the most powerful methods for the structural elucidation of anthocyanins available, to date. At present, the most satisfactory method for mixture analysis is the multistep method of separation, isolation, and quantification by LC with peak identification by MS and high-field NMR.