Anthocyanins HPLC

Anthocyanin HPLC Peak ESMS Mass/Charge Ratio m/z... 

The anthocyanin HPLC chromatograms corresponding to Graciano, Tempranillo and Cabernet Sauvignon wines after 12 months in bottle, are illustrated in Figure 7 (a, b and c, respectively). 

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. 

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

Wang, J. et al.. Comparison between HPLC and MALDI-TOE-MS analysis of anthocyanins in high bush blueberries, J. Agric. Food Chem., 48, 3330, 2000. 

Hong, V. and Wrolstad, R.E., Use of HPLC separation/photodiode array detection for characterization of anthocyanins, J. Agric. Food Chem., 38, 708, 1990. Osmianski, J. and Lee, C.Y., Isolation and HPLC determination of phenolic compounds in red grapes. Am. J. Enol. Vitic., 41, 204, 1990. 

Betalains are vacuolar plant pigments. Hence their hydrophilic nature is comprehensible. Although they are slightly soluble in ethanol and methanol, water is the best snited solvent both for stability and solnbility reasons. In contrast to the antho-cyanins, the betalains are even more polar as can be demonstrated by shorter retention times in RP-HPLC and lower solubilities in alcoholic solutions. The varying polarities may also be beneficially used to separate anthocyanins from betalains on an RP-18 solid-phase extraction cartridge (Stintzing, unpublished data). 

Stintzing, F.C., Trichterborn, J., and Carle, R., Characterisation of anthocyanin-beta-lain mixtures for food colouring by chromatic and HPLC-DAD-MS analyses. Food Chem., 94, 296, 2006. 

Based on peak size of HPLC chromatogram or percent above 10. Only anthocyanins exceeding 1% listed. 

Studies of the stability and stabilization of anthocyanins are still required, based on the extreme importance of those pigments for food colors. Modem HPLC-MS equipment also allows us to easily follow the copigmentation reactions in detail, calculate their kinetic and thermodynamic parameters, identify the products formed during the reactions, and thus shed new light on the stability and stabilization of these pigments. Since anthocyanins play important roles as natural colorants for... 

Wu, X. and Prior, R.L., Systematic identification and characterization of anthocyanins by HPLC-ESl-MS/MS in common foods in the United States fruits and berries, J. Agric. Food Chem., 53, 2589, 2005. 

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. 

Chandra, A., Rana, J., and Li, Y., Separation, identification, quantification, and method validation of anthocyanins in botanical supplement raw materials by HPLC and HPLC-MS, J. Agric. Food Chem., 49, 3515, 2001. 

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. ... 

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." ... 

The availability of HPLC systems coupled to photodiode array detectors allows for online spectral characterization of anthocyanins. 

The use of mass spectroscopic analyses for characterization of anthocyanins has increased dramatically over the past decade. Most reports cite the use of HPLC coupled to MS detectors or isolating individual pigments prior to the mass spectroscopic analysis. - - " ... 

Hale, M.L., Erancis, E.J., and Eagerson, I.S., Detection of enocyanin in cranherry juice cocktail hy HPLC anthocyanin profile, J. Food Set, 51, 1511, 1986. 

Hong, V. and Wrolstad, R.E., Use of HPLC separation/photodiode array detection for characterization of anthocyanins, J. Agric. Food Chem., 38, 708, 1990. 

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. 

Stintzing, unpublished observations). Analogous aspects were recently addressed in an extended study of anthocyanin quantification. Spectrophotometric determination is still the most preferred method that has been shown to compare well with HPLC measurements." Hence, only in rare cases have purified standards from a particular food commodity been used for calibration by HPLC-DAD. " ... 

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). 

Fig. 2.71. HPLC chromatogram of the neutral (a) and acidic fractions (b) and the acid-catalysed hydrolysed product of freshly squeezed cranberry juice (c) at 280 nnm. Peaks in a 1 = ( + )-cate-chin 2 = myicetin 3 = quercetin (added as internal standard). Peaks in b 1 = anthocyanin derivative I 2 = benzoic acid 3 = p-anisic acid 4 = quercetin (added as internal standard). Peaks in c 1 = ( + )-catechin 2 = anthocyanin derivative I 3 = anthocyanin derivative II 4 = benzoic acid 5 = anthocyanin derivative III 6 = p-anisic acid 7 = myricetin 8 = quercetin. Reprinted with permission from H. Chen et al. [188].

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... 

TLC and HPLC have been applied for the separation of the flower pigments of some Delphinium species. Anthocyanins were extracted with 70 per cent aqueous ACN containing... 

RELATIVE ANTHOCYANIN COMPOSITION OF PETALS OF ELPHINIUM CULTIVARS. INTERSPECIFIC HYBRIDS AND THEIR PARENTS BY HPLC ANALYSIS. RELATIVE LEVEL OF ANTHOCYANIN (AREA PERCENTAGE AT 510 NM) ARE REPORTED (VALUES UNDER 2PER CENT ARE EXCLUDED)... 

---