Anthocyanins quantitative analysis

Keywords Anthocyanins, Quantitative Analysis, Analytical techniques... 

Jackman, R.L., Yada, R.Y., and Tung, M.A., Review separation and chemical properties of anthocyanins used for their qualitative and quantitative analysis, J. Food Biochem., 11, 279, 1987. 

Total flavonoid content. Quantitative analysis of flavonoids depends on the objective of the study. Colorimetric estimation of total flavonoid content is measured by the aluminum chloride colorimetric assay (Jia and others 1999 Chang and others 2002). The total flavonoid content measured in this way is normally expressed in equivalent values of a standard flavonoid, often catechin or quercetin equivalents. Not all subgroups of flavonoids can be quantified by colorimetric methods however, total anthocyanin content is determined using the pH-differentiation method (Boyles and others 1993). 

Numerous CE separations have been published for synthetic colours, sweeteners and preservatives (Frazier et al., 2000a Sadecka and Polonsky, 2000 Frazier et al., 2000b). A rapid CZE separation with diode array detection for six common synthetic food dyes in beverages, jellies and symps was described by Perez-Urquiza and Beltran (2000). Kuo et al. (1998) separated eight colours within 10 minutes using a pH 9.5 borax-NaOH buffer containing 5 mM /3-cyclodextrin. This latter method was suitable for separation of synthetic food colours in ice-cream bars and fmit soda drinks with very limited sample preparation. However the procedure was not validated for quantitative analysis. A review of natural colours and pigments analysis was made by Watanabe and Terabe (2000). Da Costa et al. (2000) reviewed the analysis of anthocyanin colours by CE and HPLC but concluded that the latter technique is more robust and applicable to complex sample types. Caramel type IV in soft drinks was identified and quantified by CE (Royle et al., 1998). 

Anthocyanin Measurements. Quantitative analysis of concentration by direct colorimetry in the visible range is not practical with red wines because of substances (tannins in particular) which absorb at the maximum for anthocyanins (from 500 to 550 nm). Such a simple method may be used if need be for plant parts especially rich in anthocyanins, such as grape skins. 

Frequently, the qualitative and quantitative analysis of anthocyanins is complicated by the presence of other compounds that may interfere with the measurements. The solvent systems generally used for extraction purposes are by no means specific for anthocyanins (Markakis, 1974 Jackman et al 1987). Purification of anthocyanin-containing extracts is often necessary because considerable amounts of other compounds may also be extracted and concentrated. The variety and levels of other compounds will depend on the solvent and methodologies used. The presence of extraneous materials could influence the stability or analysis of anthocyanins. Therefore, the next step towards anthocyanin characterization is the prefractionation of those extracts. 

Niketic-Aleksic, G. and Hrazdina, G. 1972. Quantitative analysis of the anthocyanin content in grape juices and wines. Lebensm. Wiss. U. Tech-nol. 5 163-165. 

Two predominant phenolic compounds (neochlorogenic and chlorogenic acids) in prunes and prune juice can be analyzed by reversed-phase HPLC with diode array detection along with other phenolic compounds (65). Phenolic compounds were extracted from prunes with methanol and aqueous 80% methanol and analyzed by HPLC. Ternary-gradient elution (a) 50 mM NaH4H2P04, pH 2.6, (b) 80% acetonitrile/20% (a), and (c) 200 mM phosphoric acid, pH 1.5, was employed for an 80-min run time. Four wavelengths were monitored for quantitation 280 nm for catechins and benzoic acids, 316 nm for hydroxycinnamates, 365 nm for flavonols, and 520 nm for anthocyanins. Phenolic analysis of pitted prune extract is presented in an HPLC chromatogram in Fig. 9, which is based on work done by Donovan and Waterhouse (65). 

It is critical to recognize the high reactivity and low stability of anthocyanins in the selection of methods of extraction and quantitation in the analysis of anthocyanins. An analysis of processed samples containing anthocyanins for polymeric color as well as monomeric anthocyanins is recommended in nutrition studies until we better understand any health effects (positive or negative) of the compounds that are part of the mix measured as polymeric color. ... 

With HPLC methods where individual anthocyanins are separated and the method of detection is DAD, individual anthocyanin standards are preferred for quantitation, but not always available. A standard mix of the glucoside form of the six aglycones of the anthocyanidins has been commercially available. However, obtaining a consistent purity has been a challenge at times. Because of the large numbers of anthocyanins present, it is not reasonable at this point in time to have a standard for every individual anthocyanin. Thus, some assumptions and compromises must be made in any quantitative analysis of anthocyanins. Calculation of the anthocyanins containing the same anthocyanidin based upon the glucoside equivalent has been a workable compromise. 

Paper chromatography may be used as one step for separation and/or isolation of anthocyanins from complex samples. Thin-layer chromatography, performed on cellulose or silica gel plates, was in the first period of use a convenient extension of paper chromatography. The introduction of new stationary phases and the development of instrumentation offering the possibility of quantitative analysis provided new possibilities for method developments. The advantages of TLC are the simplicity in terms of instrumentation, sample preparation and data evaluation. 

In the last years new developments in the field of plate chromatography have led to high performance thin layer chromatographic (HPTLC) systems. HPTLC offer simultaneous and cost-efficient screening of anthocyanin samples and provides the possibility for quantitative analysis. 

For analysis of the presence/absence of individual anthocyanins, HPLC is the method of choice. Coupled to UV-detection, HPLC is useful for qualitative fingerprint analysis or for quantitative analysis if the anthocyanins detected are known and authentic reference standards are available. If struetural elucidation is deemed neeessary the coupling of HPLC to mass spectrometry is inevitable. Mass speetrometry is the most powerful routine technique to explore the strueture of anthoeyanins on-line after separation by HPLC. 

Li X, Song H, Yao S, Jia C, Yang Y, Zhu W (2011) Quantitative analysis and recovery optimisation of flavonoids and anthocyanins in sugar-making process of sugarcane industry. Food Chem 125 150-157... 

Many techniques for the analysis of anthocyanins have been used for almost a century and are still of importance, along with considerable advances in technologies such as mass spectroscopy (MS) and nuclear magnetic resonance (NMR). This section summarizes the analytical procedures for quantitative and qualitative analyses of anthocyanins, including classical and modem techniques. 

HPLC has proved to be fast and sensitive for the analyses of phenolic plant constit-nents, and is especially useful for the analysis of anthocyanins. The first application of HPLC to anthocyanin analyses was in 1975 by Manley and Shubiak and it has now become the method of choice for the separation of mixtures of anthocyanins and anthocyanidins. HPLC is now used for anthocyanin qualitative, quantitative, and preparative work, offering improved resolution compared to chromatographic procedures previously employed. It also allows for simultaneous rapid monitoring of the eluting anthocyanins. ... 

A similar RP-HPLC method was developed and employed for the separation and quantitative determination of nine anthocyanins in red grape cultivars and red wines. Analysis... 

Because of their high separation capacity, short analysis time, low reagent consumption and simplicity, various electrophoretic methods have found application in the separation and quantitative determination of anthocyanins in various complex matrices [267].The different techniques used for the measurement of anthocyanins in beverages [268], the application of capillary electrophoresis (CE) for the analysis of natural food pigments [269], the use of CE for the determination of anthocyanins in foods [270] and in medicinal plants [271] have been previously reviewed. 

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-... 

The analysis of urine, plasma and tissue samples presents challenges because of the low concentrations of anthocyanins and the formation of metabolites and the lack of appropriate standards. There are few reports of the quantitative analyses of anthocyanins in dietary supplements and biological matrices (Cooke et ah, 2006 Tian et al., 2006 Giordano et al., 2007). 

Polyphenol Analyses. Skin and seed extracts were prepared as described elsewhere (4,15). Flavonol and anthocyanin composition of grape skin extracts and wines were determined by direct reversed-phase HPLC analysis with diode array detection. The chromatographic conditions were the same as described earlier (16) but the formic acid concentration in the elution solvents was raised to 5% to improve anthocyanin resolution. Quantitations were based on peak areas, using mdvidin-3-glucoside (at 530nm) and quercetin-3-glucoside (at 360 nm) response factors, respectively, for anthocyanins and flavonols. 

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