Anthocyanins identification

A systematic approach for optimizing the extraction and identification of anthocyanins from blueberries was explored using HPLC-UV and HPLC-ESI-IT-TOF-MS (Barnes et al., 2009). A method was developed for anthocyanin identification without the use of standards. Consideration was given to elution order by chromatographic separation with selective detection at 520 nm, high mass accuracy m/z values, tandem MS fragmentation, and previously published literature. Overall, 25 anthocyanins from a wild type highbush blueberry were identified and reported (Barnes et al., 2009). 

De la Cruz AA, Hilbert G, Riviere C, Mengin V, Ollat N, Bordenave L, Decroocq S, Delaunay J-C, Detot S, Merillon J-M, Monti J-P, Gomes E, Richard T (2012) Anthocyanin identification and composition of wild Vitis spp. accessions by using LC-MS and LC-NMR. Anal Chim Acta 732 145-152... 

Based on experimental results the method for identification of anthocyanin metallocomplexes was suggested. 

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. 

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

Longo, L. et al.. Identification of anthocyanins in Rhamnus alatemus L. berries, J. Agric. Food Chem., 53, 1723, 2005. 

The total number of different anthocyanins reported to be isolated from plants was 539. However, the number of anthocyanins found in foods is much smaller. Although a large number of papers were published regarding anthocyanin composition in several foods, investigators in most studies used only chromatographic and chemical behaviors as bases for identification. In this chapter, we considered only papers in which identification was based at least on mass spectrometry (MS). In fact, the use of only MS and UV-visible information can easily lead to misidentifi-cation as the following example shows. 

Fortunately, the appearance in the last years of new, more accurate methods and sophisticated equipments permitted the isolation of anthocyanins on a preparative scale and allowed the identification of extremely complex and stable anthocyanins. 

Longo, L. and Vasapollo, G., Extraction and identification of anthocyanins from Smilax aspera L. berries. Food Chem., 94, 226, 2006. 

Cooney, J.M., Jensen, D.J., and McGhie, T.K., LC-MS identification of anthocyanins in boysenberry extract and anthocyanin metabolites in human urine following dosing, J. Sci. Food Agric., 84, 237, 2004. 

Mozetic, B. and Trebse, P., Identification of sweet cherry anthocyanins and hydrox-ycinnamic acids using HPLC coupled with DAD and MS detector, Acta Chim. Slov., 51, 151, 2004. 

Revilla, 1. et al.. Identification of anthocyanin derivatives in grape skin extracts and red wines by liquid chromatography with diode array and mass spectrometric detection, J. Chromatogr. A, 847, 83, 1999. 

Purification of anthocyanin-containing extracts is often necessary for further structural identification. Since none of the solvents used for extraction is specific for anthocyanins, considerable amounts of other compounds may be also extracted and concentrated. The variety and concentration of other compounds will depend on the solvent and methodologies used. The presence of extraneous materials could affect the stability and/or analysis of anthocyanins. Therefore, the next step toward anthocyanin characterization is the purification of those extracts. 

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." ... 

Pati, S. et al.. Simultaneous separation and identification of oligomeric procyanidins and anthocyanin-derived pigments in raw red wine by HPLC-UV-ESI-MS, J. Mass Spectrom., 41, 861, 2006. 

Lopes-Da-Silva, E. et al.. Identification of anthocyanin pigments in strawherry (cv Camarosa) hy LC using DAD and ESI-MS detection, Eur. Food Res. Technol., 214, 248, 2002. 

The optimized RPLC UV-Vis ESI MS method for all typical blue colourants (indi-goids, hematein, tannins, anthocyanins and selected flavonoids) was used for the identification of dyes extracted from a thread taken from an Italian tapestry of unknown origin from the collection of the National Museum in Warsaw (Poland). It was found that to obtain the red-blue colour of the fibre a mixture of dyestuffs was probably used. The presence of indigotin, tannic and ellagic acid (at m/z 301, NI), as well as carminic acid, suggested the use of indigo and cochineal. Reseda luteola could also have been used due to the presence of luteolin and apigenin. 

Prior RL, Lazarus SA, Cao G, Muccitelli H and Hammerstone JF. 2001. Identification of procyanidins and anthocyanins in blueberries and cranberries (Vaccinium spp.) using high-performance liquid chromatog-raphy/mass spectrometry. J Agric Food Chem 49(3) 1270-1276. 

Hassimotto NMA, Genovese MI and Lajolo FM. 2007. Identification and characterisation of anthocyanins from wild mulberry (Moms nigra L.) growing in Brazil. Food Sci Technol Int 13(1) 17—25. 

Netzel M, Netzel G, Tian Q, Schwartz S and Konczak I. 2006. Sources of antioxidant activity in Australian native fruits. Identification and quantification of anthocyanins. J Agric Food Chem 54(26) 9820-9826. 

TOGURI, T., UMEMOTO, N., KOBAYASHI, O., OHTANI, T., Activation of anthocyanin synthesis genes by white light in eggplant hypocotyl tissues, and identification of an inducible P-450 cDNA, Plant. Mol. Biol., 1993, 23, 933-46. 

The separation and identification of flavanol-anthocyanin adducts in wine and in model solutions were performed with RP-HPLC coupled to DAD or ESI-MS. The investigation was motivated by the assumption that the formation of flavanol-anthocyanin complexes may influence the organoleptic characteristics of wine during ageing. Measurements were carried out in an ODS column (250 X 2 mm i.d. particle size 5 pm) at 30°C. The flow rate was 0.25 ml/min. Solvent A was water-formic acid (95 5), solvent B consisted of ACN— solvent A (80 20, v/v). The Gradient elution began with 3 per cent B for 7min to 20 per... 

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