**6. Identification and quantification: a primer**

The identification of anthocyanins has a critical role in taxonomic [214] and adulteration [215] studies, besides anthocyanins might replay synthetic days. HPLC, especially in the reversed phase, is the most widely used separation technique. Due to pH-dependent interconversions among various molecular forms of the anthocyanins, a highly acidic mobile phase (pH < 2) is required to ensure that they are maintained predominantly in the flavylium cationic form for maximum chromatographic efficiency. However, even at low pH [31, 32, 35, 110] some interconversion between the anthocyanin flavylium cationic and carbinol pseudobasic forms occurs.

Regarding chromatographic detection techniques for the study of anthocyanins we have [168, 180, 216, 217] diode array detection (DAD) and MS or tandem mass spectrometry (MS/MS) among the most widely used. Spectroscopy is the main technique used due to its simplicity and low cost providing very useful qualitative and quantitative information (anthocyanins have a specific and intense absorbance band in the range of 520–560 nm) [218–220], however the difficulty in obtaining reference compounds and the spectral similarities of the anthocyanins represent important drawbacks.

Various MS instruments, as well as the advances in nuclear magnetic resonance (NMR) have given a fresh impetus to anthocyanin analysis [78, 83, 221]. MS/MS is particularly suited for structure elucidation and compound identification [217, 222, 223] since information pertaining to the aglycone moiety, type and number of sugars and other substituents can be obtained and many of the previously proposed reaction mechanisms for the formation of polymeric anthocyanins and other new pigments have been verified. NMR identification of anthocyanin compounds [78] offers new promising approaches for analysis of complex phenolic mixtures. NMR is based primarily on the analysis of <sup>1</sup> H NMR spectra but important structural information can also be provided by 13C NMR [170] and, especially for compounds that have many quaternary carbons, by combining homo and heteronuclear 2D and 3D techniques. However, the relatively high capital costs are still an impediment [218] to their routine use in enforcement laboratories, a fact that must be taken into consideration.

The almost universal distribution of anthocyanins in flowering plants makes them also suitable for chemotaxonomic considerations [224] both at the family and genus level. Differential anthocyanins profiles may be used [164] for the detection and adulteration in specific commodities of berry fruit products. In the last few years, special attention has been paid [225] to the isolation and characterization of compounds that may delay the onset of aging, as occurs with some berry phenolics. The extremely low levels of anthocyanins usually present in biological samples [57, 168, 181, 186] (blood plasma and body tissues) possess further challenges to their identification and quantification, together with the lack of commercially available anthocyanin standards.
