**7. Final comments**

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

134 Phenolic Compounds - Natural Sources, Importance and Applications

binol pseudobasic forms occurs.

macroporus polymeric adsorbent [198].

nins represent important drawbacks.

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 car-

**Figure 6.** Schematic representation of a highly separation and purification of anthocyanins from bilberry based on

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 anthocya-

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 analy-

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

H NMR

sis of complex phenolic mixtures. NMR is based primarily on the analysis of <sup>1</sup>

In last decades, polyphenol chemistry has experienced an explosion of knowledge, being anthocyanins one of the most widely studied groups [27, 226, 227], due to its great potential for practical applications in various fields, contributing in addition this to obtain a better understanding of the chemistry of life.

Anthocyanins occur in all plant tissues including leaves, stems, roots, flowers and fruits imparting color. Anthocyanins are responsible for the red, purple and dark blue colors of many fruits and berries [68, 228–230]. Anthocyanins have antioxidant activity [55, 129, 231– 233] preventing radical formation. These nontoxic natural pigments have received considerable attention from such as food, pharmaceutical and nutritional industries due to their potential applications in color-processed food and medicines [31, 33, 147, 234] which may replace synthetic dyes.

It was only a few decades ago that anthocyanins were regarded as highly degradable compounds and the research studies mainly were focused on their chemical structures, color stability, use as food constituents and changes in foods during storage. Anthocyanins are now recognized as food constituents with potential health benefits [102, 131] and research related to these properties has markedly progressed at the molecular level. Anthocyanins will continue to attract researchers across various disciplines, including those involved in the creation of new flower varieties with novel colors. Research on the health benefits of anthocyanins will provide information [172] on underlying molecular mechanisms and absorption and metabolism. Moreover, once these benefits are proven in humans, development of foods and dietary supplements in a capsule form [164, 235, 236] can be accelerated to promote the proven functions, i.e. berry extracts are being commercialized as nutraceuticals and as dietary supplements to fulfill consumer demands.

The development of analytical techniques to determine the identity and quantities of anthocyanins in natural products, as well as their effects *in vivo* and *in vitro*, is challenging. Up to date, there is no universal extraction procedure suitable for extraction of all plant phenolics. The choice of an extraction method should maximize pigment recovery [237] with a minimal amount of adjuncts and minimal degradation or alteration of the natural state.

Solvent extraction involving the use of acidic solvents has been the most commonly used method [36, 79] for the recovery of diverse compounds found in flavonoids, including anthocyanins. The traditional solid-liquid or liquid-liquid extraction offers good recovery. Nevertheless, they are often described as laborious, time and solvent consuming and prone to errors. However, in recent years there are trends toward other environmentally and economically friendlier extraction techniques [38, 39, 110, 167, 184, 206, 238] using a smaller amount of (nontoxic) solvents and sample sizes, reducing working time and increasing selectivity, specificity, recovery and potential of automation. MAE, SFE, PLE, or PHWE are among the greener techniques that have experienced a large increase in recent years to extract anthocyanins from plant material and other samples.
