Flavonoids, detection

CHARRIER, B., LEROUX, C., KONDOROSI, A., RATET, P., The expression of alfalfa flavanone 3-hydroxylase promoter-gus fusion in Nicotiana benthamiana correlates with the presence of flavonoids detected in situ. Plant Mol. Biol., 1996, 30, 1153-1168. 

FIGURE 18.1 Chemical structure of various flavonoids detected in pistachios. 

Improvements in the instrumentation, ionization sources, high-resolution mass analyzers, and detectors [67-69], in recent years have taken mass spectrometry to a different level of HPLC-MS for natural product analysis. Mass spectrometry detection offers excellent sensitivity and selectivity, combined with the ability to elucidate or confirm chemical structures of flavonoids [70-72]. Both atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) are most commonly used as ionization sources for flavonoid detection [73-76]. Both negative and positive ionization sources are applied. These sources do not produce many fragments, and the subsequent collision-induced dissociation energy can be applied to detect more fragments. Tandem mass spectrometry (MS , n> 2) provides information about the relationship of parent and daughter ions, which enables the confirmation of proposed reaction pathways for firagment ions and is key to identify types of flavonoids (e.g., flavones, flavonols, flavanones, or chalcones) [77-80]. 

Heartwoods of 23 taxa of Rhus were screened and 14 flavonoids detected. All uniformly contained eight deoxyflavonoids sulphuretin (29) and its 6,3 -digluco-side, fisetin (16), butein (27), 3,7,4 -trihydroxyflavone (88), 3, 4 -dihydroxyflavone (91), fustin (93), and garbanzol (92). Among the new compounds reported is... 

More recently, the flavonoid pattern found in the Hepaticae and the Musci has been discussed by Zinsmeister and Mues (348), Chromatographic characteristics of the flavonoids detected in the Hepaticae and a chemosystematic study of the Marchantiales by use of flavonoids have been reported in a recent review (199),... 

Figure 7.25 Chlorogenic acids and flavonoids detected in peaches and nectarines.

After the dipped or sprayed chromatogram has been dried in a stream of cold air long-wave UV light (2 = 365 nm) reveals fluorescent yellow zones (flavonoids). Sterigmatocystine, which can be detected without derivatization on account of its red intrinsic fluorescence (detection limit 0.5 pg), also fluoresces pale yellow after being heated to 80°C [9] or 100°C [13] for 10 min on the other hand, citrinine, zearalenone and vomitoxin fluoresce blue. 

Note The natural fluorescence colors of some flavonoids [7, 9] and anthracene derivatives [16] are altered by the ammonia treatment. This makes possible differentiation on the basis of color. Detection limits per chromatogram zone have been reported of 2 ng for morphine and heroin [2], 6 ng for ochratoxin A [5] and 1 pg for penicillic acid [13]. 

To detect glycosides heat the chromatograms to 130—150°C for 15 min. Blue-grey zones are produced (detection limit prunasin 0.3—0.5 pg [18]). Flavonoids are better detected with a modified reagent of the following composition phosphoric acid (85%) — acetic acid — aniline — diphenylamine (20 ml - -100 ml + 5 ml + 5 g). 

The visual detection limits for polyphenols and flavonoids are 0.2 to 1 pg substance per chromatogram zone [1]. The photometric detection limits are appreciably lower (see Procedure Tested ). 

The detection limits for phenols, phenol carboxylic acids, cumarins and flavonoids are 0.1-1 substance per chromatogram zone [1, 3, 7]. ... 

The detection limits for flavonoid substances are 5-10 ng substance per chromatogram zone [4]. 

The reaction of eq. 16.9 will regenerate the antioxidant Arj-OH at the expense of the antioxidant At2-OH. Despite the fact that such regeneration reactions are not simple electron transfer reactions, the rate of reactions like that of eq. 16.9 has been correlated with the E values for the respective Ar-0. Thermodynamic and kinetic effects have not been clearly separated for such hierarchies, but for a number of flavonoids the following pecking order was established in dimethyl formamid (DMF) by a combination of electrolysis for generating the a-tocopherol and the flavonoid phenoxyl radicals and electron spin resonance (ESR) spectroscopy for detection of these radicals (Jorgensen et al, 1999) ... 

Knowledge of the identity of phenolic compounds in food facilitates the analysis and discussion of potential antioxidant effects. Thus studies of phenolic compounds as antioxidants in food should usually by accompanied by the identification and quantification of the phenols. Reversed-phase HPLC combined with UV-VIS or electrochemical detection is the most common method for quantification of individual flavonoids and phenolic acids in foods (Merken and Beecher, 2000 Mattila and Kumpulainen, 2002), whereas HPLC combined with mass spectrometry has been used for identification of phenolic compounds (Justesen et al, 1998). Normal-phase HPLC combined with mass spectrometry has been used to identify monomeric and dimeric proanthocyanidins (Lazarus et al, 1999). Flavonoids are usually quantified as aglycones by HPLC, and samples containing flavonoid glycosides are therefore hydrolysed before analysis (Nuutila et al, 2002). 

In the intermolecular reactions between anthocyaifins and flavonoids mediated by acetaldehyde, new compounds linked by an ethyl bridge are formed. Three new compounds were detected by the reaction of malvidin 3-glucoside and proanthocy-... 

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