**5.1. Sample preparation**

A number of strategies are used [37, 174, 175] for the characterization of phenolic samples in plant materials. In any case extraction techniques and semipreparative isolation methods are usually applied prior to the separation and quantification steps. After the first operations of drying and of powdering the plant material, it follows [176]: (i) a previous extraction step of the plant materials as well as a preliminary consequent purification step; (ii) fractionation of the mixture in order to isolate pure pigments and (iii) the final characterization and identification of pure anthocyanins compounds (**Table 3**).

populations, may also explain the wide range of anthocyanin consumption estimated by different authors. Anthocyanin intake in the German young shows differences between girls and

Up to now, anthocyanins have not been detected in processed food such as canned food, bread, or cereals. Also, although prepared baby food containing blueberries, rich in anthocyanins, are expected to find these compounds, analyses have hardly detected them [162]. In

Due to antioxidant and other potential beneficial properties, grapes, various berries, red cabbage and other anthocyanin-rich foods are becoming more popular. Berry extracts are also being commercialized as nutraceuticals and dietary supplements [164] to meet consumer demands.

Currently, there is no recommended intake level of anthocyanins for optimal health or to avoid adverse effects; however, future research and continued consumer interest will undoubtedly

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] promot-

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

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 prop-

A number of strategies are used [37, 174, 175] for the characterization of phenolic samples in plant materials. In any case extraction techniques and semipreparative isolation methods are

boys [161], decreasing from young childhood to adolescence.

128 Phenolic Compounds - Natural Sources, Importance and Applications

young infants [163] the anthocyanidin intake was found to be zero.

present opportunities for pursing dietary guidance recommendations.

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

compounds and in the interpretation of their mechanisms of action.

erly documented [173] in the analytical literature.

**5.1. Sample preparation**

ing advances in separation science.

In order to avoid sample oxidation, thermal degradation, chemical and biochemical changes under mild extraction conditions [177] are recommended and drying, lyophilized, or frozen samples should be used. The deterioration processes of the compounds may be avoided by the


Nuclear magnetic resonance (NMR) Capillary electrophoresis (CE) Thin layer chromatography (TLC) Voltammetry Others Hyphenated techniques GC-MS, LC-MS, LC-DAD-ES-MS/MS, CE-MS, LC-NMR, others

**Table 3.** Strategies for preparation and characterization of anthocyanin samples from plant materials [38].

addition of antioxidants compounds, by using inert atmospheres or working in the absence of light. However, no definitive procedure for storage and collection has been established.

To determine either the target analytes (in their various conjugated forms) or the aglycones is an important question [175, 178] to answer. When dealing with plant, food products and biological matrices, the instant conjugates are usually search, whereas in the other instances it is necessary to carry out a preliminary hydrolysis, e.g., an enzymatic or chemical (acidic or alkaline) treatment. Intentional hydrolysis for obtaining the aglycones of some flavonoid or derivatization of some fatty acids to esters is sometimes intentionally incorporated [79] to the extraction process.

Extraction represents an important phase [34, 38, 39, 79, 169] in the isolation, identification and utilization of anthocyanins. Anthocyanins are usually recovered by mean of a solvent extraction procedure. Parameters such as solvent-extraction kind and its composition, liquid-tosolid ratio, extraction time and temperature require [179] proper optimization. The flavylium cation form of anthocyanins is red and stable in a highly acidic medium. Thus, the extraction solution should be enough (slightly) acid to maintain the flavylium cation form [180], but not so much as to cause partial hydrolysis of the acyl moieties in acylated anthocyanins. Protocols of extraction and analysis of plant materials and biological fluids are, however, difficult to accomplish because of [181] the structural diversity of anthocyanins and their susceptibility to heat, pH, metal complexes and copigmentation.

In the same way as flavonoids, in general, anthocyanins possess aromatic rings that contains polar substituent groups (─OH, ─C═O, or ─OCH3 ) and glucosyl residues, which altogether [28, 29] constitute a polar molecule. Flavonoid glycosides are of a more polar nature, whereas aglicones are extracted either with alcohols or alcohol-water mixtures. Cold acidified solvents (polar organic solvents, water) under mild conditions [167, 182] are used for the extraction of anthocyanins. The organic solvent usually used is methanol. However, solvents such as acetone, ethanol, or acetonitrile may be used. These solvents system denature the cell membranes [93] also dissolving and stabilizing the anthocyanins. Acetic acid at about 7% or trifluoroacetic acid at about 3% are usually used; the organic solvent content [183] varying from 50 to 100% in the mixture. When a mineral acid is used it may assist [174] to the loss of the attached acyl group. Sulfurous water also allows [181] the reduction of organic solvent and cost extraction.

Phytochemical recovery of a good antioxidant from various sources may be achieved by using solvent extraction. The conventional solvent extraction procedure suffers from the drawback of requiring subsequent extraction and cleanup prior analysis. In addition, health and safety risks are associated with the use of large amounts of organic solvents, being on the other hand environmentally unfriendly. A modern trend toward [184]: (1) the use of samples smaller in size, volume, or organic solvent content; (2) an extraction with increasing selectivity or specificity; (3) improved recoveries and reproducibility; (4) greater automation facilities. A variety of modern techniques have been developed for this purpose, including solid phase extraction (SPE), countercurrent chromatography (CCC), microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), pressurized hot water extraction (PHWE) and high hydrostatic pressure extraction (HHP), among others. Selected applications of sample preparation techniques on anthocyanin compounds are listed in **Table 4** [185–213], an extension of applications previously published by the authors in [39]. The applications of other novel nonthermal techniques will be the subject of further study. **Figure 6** shows a schematic representation of a highly separation and purification methodology based on a macroporus polymeric adsorbent for the determination of anthocyanins in bilberry [198].

addition of antioxidants compounds, by using inert atmospheres or working in the absence of light. However, no definitive procedure for storage and collection has been established.

**Table 3.** Strategies for preparation and characterization of anthocyanin samples from plant materials [38].

Voltammetry Others

Hyphenated techniques

CE-MS, LC-NMR, others

Nuclear magnetic resonance (NMR) Capillary electrophoresis (CE) Thin layer chromatography (TLC)

GC-MS, LC-MS, LC-DAD-ES-MS/MS,

To determine either the target analytes (in their various conjugated forms) or the aglycones is an important question [175, 178] to answer. When dealing with plant, food products and biological matrices, the instant conjugates are usually search, whereas in the other instances it is necessary to carry out a preliminary hydrolysis, e.g., an enzymatic or chemical (acidic or alkaline) treatment. Intentional hydrolysis for obtaining the aglycones of some flavonoid or derivatization of some fatty acids to esters is sometimes intentionally incorporated [79] to the

Extraction represents an important phase [34, 38, 39, 79, 169] in the isolation, identification and utilization of anthocyanins. Anthocyanins are usually recovered by mean of a solvent extraction procedure. Parameters such as solvent-extraction kind and its composition, liquid-tosolid ratio, extraction time and temperature require [179] proper optimization. The flavylium cation form of anthocyanins is red and stable in a highly acidic medium. Thus, the extraction solution should be enough (slightly) acid to maintain the flavylium cation form [180], but not so much as to cause partial hydrolysis of the acyl moieties in acylated anthocyanins. Protocols of extraction and analysis of plant materials and biological fluids are, however, difficult to accomplish because of [181] the structural diversity of anthocyanins and their susceptibility

In the same way as flavonoids, in general, anthocyanins possess aromatic rings that contains

[28, 29] constitute a polar molecule. Flavonoid glycosides are of a more polar nature, whereas aglicones are extracted either with alcohols or alcohol-water mixtures. Cold acidified solvents (polar organic solvents, water) under mild conditions [167, 182] are used for the extraction of anthocyanins. The organic solvent usually used is methanol. However, solvents such as acetone, ethanol, or acetonitrile may be used. These solvents system denature the cell membranes [93] also dissolving and stabilizing the anthocyanins. Acetic acid at about 7% or trifluoroacetic acid at about 3% are usually used; the organic solvent content [183] varying from 50 to 100%

) and glucosyl residues, which altogether

extraction process.

to heat, pH, metal complexes and copigmentation.

130 Phenolic Compounds - Natural Sources, Importance and Applications

polar substituent groups (─OH, ─C═O, or ─OCH3





**Countercurrent chromatographic methods**

Mulberry fruit/anthocyanins MTBE, 1-butanol,

MTBE/n-butanol/

n-butanol/MTBE/

v/v)

132 Phenolic Compounds - Natural Sources, Importance and Applications

HSCCC

HSCCC

acetonitrile/0.01% TFA (1:3:1:5,

MTBE/n-butanol/acetonitrile/ water/TFA (2:2:1:5:0.01, v/v).

acetonitrile, water and TFA (10:30:10:50:0.05; %, v/v)

acetonitrile/0.1% aqueous TFA (0.715:1.0:0.134:1.592, v/v/v/v).

**Solution (target compounds) Adsorbent Eluting and/or regenerating** 

Blackberries/anthocyanins Polyamide resin Deionized water and ethanol

divinylbenzene and ethylene glycol dimethyl acrylate

Cross-linked acrylonitrile-codivinylbenzene (AN/DVB)

Black carrot/anthocyanins T-10-coded 1% acetic acid solution and

Crude mulberry/ cyanidin-3-glucoside and cyanidin-3-rutinoside

Blue honeysuckle fruits/ cyanidin 3-glucoside

Petals of *Chaenomeles sinensis*/

**Adsorbents and eluting agents**

Blueberries/anthocyanins and

Jamun (*Syzygium cumini* L.)/

Aronia melanocarpa berries/ antioxidant phenolics

Strawberry/aroma compounds

Muscadine (*Vitis rotundifolia*) juice pomace/anthocyanins

polyphenols

anthocyanins

and Anthocyanins

Bilberry based/anthocyanins Copolymerization of

Purple-fleshed potato/ anthocyanins

Anthocyanidins

**Type of matrix/analyte Solvent system Elution mode/comment References**

The upper phase was used as the stationary phase and the lower phase as

Head–tail elution mode with the upper organic phase as stationary phase

Stationary phase: upper organic phase. Mobile phase: lower aqueous phase. The elution was in "head to tail" mode

The lower phase in the solvent separator was pumped into the mobile phase bottle, the pumps for pumping MTBE, n-butanol, acetonitrile and 0.1% TFA aqueous were set to a certain flow rate, the solvents were mixed and separated into two layers; and after rinsing the solvent separator, the upper phase flowed into the stationary [192]

[193]

[194]

[195]

**References**

[196]

[197]

[200]

[203]

[204]

the mobile phase

phase bottle

**agent**

FPX66 resin 3 bed volumes of 95% ethanol [199]

XAD 7HP resin Ethanol-water mixtures [201]

(above 40%, v/v)

ethanol (70%)

methanol

Amberlite XAD7HP Aqueous acidified ethanol

FPX-66 resin Three bed volumes of aqueous

acid) aqueous ethanol

(0.1% v/v HCl, pH = 3)

Ethanol [198]

Methanol [202]

Amberlite XAD-7HP 75 vol.% acidic (7 vol.% acetic


**Table 4.** Selected applications of extraction techniques applied to anthocyanins.

**Figure 6.** Schematic representation of a highly separation and purification of anthocyanins from bilberry based on macroporus polymeric adsorbent [198].
