**5. Sample preparation and extraction of anthocyanins**

The presence of phytochemical bioactive compounds in food and dietary supplements poses difficult problems in connection with the optimization of their extraction process and determination. Aspects related to Ref. [165] the complexity of sample matrices, the presence of varying forms of bioactive substances and interaction with other components need to be solved. A number of factors including pH, metal ions, complex formation, light, temperature, enzymes, sugars, oxygen and ascorbic acid exert influence [166] on the stability of anthocyanins. The role of analytical chemistry is vital in this context, e.g., [34, 76, 91, 167–171] promoting advances in separation science.

Most phenolic compounds are made up of only C, H and O, differing in some cases even by only one atom and in many others by constitutional or stereochemical isomers. The identification of species proves thus be difficult because of subtle structural changes, being necessary for this purpose to apply [38] complementary techniques. Anthocyanins may be forming part of complexes, may be present in matrices of a complex nature and may appear in distinct equilibrium forms [30, 32, 34], depending of the pH of the medium. Acid dissociation, rate constant and tautomerization constants are of great importance in the analysis of bioactive compounds and in the interpretation of their mechanisms of action.

Time-consuming processes are involved [172] in the isolation, purification and determination of the structures of anthocyanins, which must be accomplished with care. However, there is no universal sample pretreatment technique applicable to all kind of samples. The primary steps required, sampling, sample preservation and sample preparation, are not always properly documented [173] in the analytical literature.
