Anthocyanins: analysis

As we have seen above, anthocyanins comprise an aglycone fraction commonly known as anthocyanidin and a frequently acylated osidic substituent. This characteristic leads to two different approaches for the analysis of these pigments (1) a direct anthocyanin analysis without a hydrolysis stage requiring identification of a number of molecules (several hundreds in the plant kingdom) or (2) an analysis of the anthocyanidin fraction only after hydrolysis of the anthocyanins present in the medium. 

The overall anthocyanin analysis is generally conducted using the Giusti and Wrolstad method based on the differences in absorbance of anthocyanins at pH 1 and pH 4.5. Then the pigment content is determined using the coefficient of molar extinction of the predominant anthocyanin. It should be noted that this technique only allows dosing of anthocyanins with a color difference between the two pH values (due to transition to the flavylium cation form). A more global analysis of total anthocyanin content may be conducted by direct spectrophotometry of the... 

Just as with anthocyanin analysis, the advent of HPLC/mass spectrometer coupling made it possible to avoid acid hydrolysis for flavonoid identification. Maata... 

CE was recently used for anthocyanin analysis because of its excellent resolution. This technique has different modes capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), micellar electrokinetic chromatography (MEKC), capillary electrochromatography (CEC), capillary isoelectric focusing (CIEE), and capillary isotachophoresis (CITP)."° CZE is the most popular method for anthocyanin... 

About 15 anthocyanins in bilberries (Vaccinium myrtillus L.) were reported by Ichiyanagi et al. using CZE separation and MS-NMR identification.Anthocyanin analysis of strawberry and elderberry extracts was performed by reverse HPLC at pH 1.8 and CZE using a standard silica capillary and pH 8.0 running buffer. Under these conditions, HPLC had more advantages than CZE in terms of anthocyanin separation in these extracts." ... 

Different types of mass analyzers have been used for anthocyanin analysis single or triple quadrupole mass analyzers, TOP mass analyzer,ion trap mass analyzers,and the combination of analyzers cited above. " ... 

This method is used for anthocyanin samples of lesser complexity that do not contain acylated pigments. It is the most common method used for anthocyanin analysis and is effective for most commodities the major exceptions being red grape and cabbage containing products. This protocol describes the dilution, filtration, and reversed-phase HPLC analysis of these samples. 

Individual anthocyanin composition is distinctive for any given plant, so anthocyanin analysis is very useful in distinguishing between species. Chemotaxonomic differentiation is commonly based on qualitative differences (163), furthermore within one cultivar (e.g., grapes) even varieties can be discriminated by quantitative differences (164). The anthocyanic profiles of 11 different grape varieties obtained with RP-HPLC are shown in Fig. 15 (165). The characteristic differences in anthocyanin patterns have also been successfully applied to the detection of adulterations in products of cranberries (166), black currants (166), blackberries (167), and grapes (168). 

Since the work of Manley and Shubiak (182), who were the first to apply HPLC to anthocyanin analysis, numerous HPLC techniques have been developed for the separation and quantification of anthocyanins and anthocyanidins. Nowadays HPLC has become the method of choice, because it offers the advantage that it is a rapid, sensitive, and quantitative method. For the peak identification and quantitative evaluation of chromatograms, the use of pure anthocyanin standards is recommended however, only a limited, but constantly increasing, number of substances is avail-... 

During the last two decades, RP-HPLC analysis has been successfully used to study anthocyanins in numerous foodstuffs. Because of the great number of publications, only a short excerpt from those many applications and corresponding HPLC-methods can be given in Table 6. To demonstrate the potential of this technique, an RP-HPLC separation of black currant juice is shown in Fig. 16. Despite the high costs of acquisition and maintenance, RP-HPLC will likely remain the best-qualified method for anthocyanin analysis in the coming years, because it offers,... 

Table 6 Some RP-HPLC Methods and Their Applications in Anthocyanin Analysis... 

Most anthocyanin analysis today is carried out using reverse-phase HPLC, often coupled with diode array detection, as this offers the best approach to assess these compounds. The use of reverse-phase HPLC means that predictions can be made about die elution order of compounds from the column, which is based on their polarity and makes the interpretation of the anthocyanin profiles easier. A couple of useful chapters that discuss the analysis of anthocyanin pigments more fully are those of Lea (1988) and Wrolstad et al. (1995). Both of these describe more fully the types of methods used and give examples of profiles found in different fruit types. The method used by Wrolstad et al., is similar to the procedure that is routinely used at RSSL for this type of analysis. 

Reversed-phase high-performance liquid chromatography (RP-HPLC) is the usual method of choice for the separation of anthocyanins combined with an ultraviolet-visible (UV-Vis) or diode-array detector (DAD)(Hebrero et al., 1988 Hong et ah, 1990). With reversed-phase columns the elution pattern of anthocyanins is mainly dependent on the partition coefficients between the mobile phase and the Cjg stationary phase, and on the polarity of the analytes. The mobile phase consists normally of an aqueous solvent (water/carboxylic acid) and an organic solvent (methanol or acetonitrile/carboxylic acid). Typically the amount of carboxylic acid has been up to 10%, but with the addition of a mass spectrometer as a detector, the amount of acid has been decreased to as low as 1 % with a shift from trifluoroacetic acid to formic acid to prevent quenching of the ionization process that may occur with trifluoroacetic acid. The acidic media allows for the complete displacement of the equilibrium to the fiavylium cation, resulting in better resolution and a characteristic absorbance between 515 and 540 nm. HPLC separation methods, combined with electrochemical or DAD, are effective tools for anthocyanin analysis. The weakness of these detection methods is a lack of structural information and some nonspecificity leading to misattribution of peaks, particularly with electrochemical... 

Wu, X. Prior, R.E. 2008. Anthocyanins Analysis and distribution in selected medicinal plants. Asian Chem. Lett. 12 9-22. 

Wightman, J.D. and Wrolstad, R.E. 1995. Anthocyanin analysis as a measure of glycosidase activity in enzymes for juice processing. J. Food Sci. 60(4) 862—867. 

Consequently, detailed knowledge of the chemical properties of anthocyanins occurring in berries and of analytical methods used are important to judge the quality of anthocyanin extracts from berries. The following overview introduces the chemistry of berry anthocyanins and their occurrence in individual berries, addresses all relevant topics of anthocyanin analysis, substantiate the need to replace simple by sophisticated methodology and proposes a sequence analytical methods for quantitative and qualitative analysis under routine conditions. 

HPLC is the method of choice to confirm the distribution/occiurence of individual anthocyanins hence HPLC is most often conclusive for the evaluation of source material and product quality. HPLC is mandatory for berry anthocyanin analysis especially for extracts claimed to stem from a specific berry source. 

A series of HPLC methods for quantitative anthocyanin analysis have been published in the last years (Table 11). Most methods are comprised of reversed-phase chromatography (Cl8 columns, standard dimensions) at 0.5-2.0 mL/min and 25-45 °C for native anthocyanin separation with subsequent UV-detection at 520-530 nm and quantification against external anthocyanin standards. 

Table 14. Overview of CZE methods used for anthocyanin analysis...

On basis of the published literature presented in this chapter and with our own practical experience gathered, we developed following approach for accurate and reliable anthocyanin analysis in berries, berry extracts and products containing berry anthocyanins. 

If those tests provide inconclusive results, the next step of analysis is quantitative HPLC coupled to UV-detection with external cahbration for anthocyanin analysis. HPLC/UV analysis will reveal blending of berries due to the distribution/relative content of anthoeyanins and adulteration due to the low absolute content of anthoeyanins. 

Stoj, A., Targonski, Z., Malik, A. Use of anthocyanin analysis for detection of berry juice adulterations. Acta Sci. Pol. Technol. Aliment 5 (1), 73-85,2006. 

Anthocyanin analysis generally involves extraction with weakly acidified alcoholic solvent, followed by concentration under vacuum and purification and separation of the pigments, while the whole extract is used for coloring foodstuffs. 

Analytical methods for the isolation, separation, and characterization of anthocyanins have been described (Markham, 1982 Jackman et al., 1987 Harbome, 1998 Rivas-Gonzalo, 2003 Andersen and Francis, 2004). A comprehensive and highly recommended source for anyone involved in anthocyanin analysis is that of Strack and Wray (1989). Further details of flavonoid chemistry can be found in volumes of The Flavonoids series (Harbome et al., 1975 Harbome and Mabry, 1982 Harbome, 1988, 1994). Recent advances in flavonoid research are thoroughly described in the book Flavonoids Chemistry, Biochemistry and Applications, edited by Andersen and Markham (2006). Extensive information on the occurrence of anthocyanins in various natural products reported after 1992 was presented by Andersen and Jordheim (2006). One of the most useful sources of current protocols of anthocyanin analysis is the Handbook of Food Analytical Chemistry, Pigments, Colorants, Flavors, Texture, and Bioactive Food Components, edited by Wrolstad et al. (2004). This book is a practical how to manual that contains detailed in-stmctions on the following topics (1) extraction, isolation, and purification of anthocyanins, (2) characterization and measurement of anthocyanins by UV-Vis... 

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