Flavonoids fluorescence detection

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. 

Sample extraction and hydrolysis details e.g., solvent extraction after freeze drying, with optimized acid or enzymatic hydrolysis Preparation of flavonoid standards and use of internal standards Chromatographic separation and detection method used, ideally RP-HPLC with UV or fluorescent detection Outline of quality assurance procedures employed... 

E. de Rijke, H. Zappey, F. Ariese, C. Gooijer, U.A.Th. Brinkman, Flavonoids in Leguminosae Analysis of extracts ofT. pratense L T. dubium L T. repens L., and L. comiculatus L. leaves using LC with UV, MS and fluorescence detection. Anal. 

As regards the sensitivity of the MS detection in HPLC analysis of flavonoids, this technique has proved to be the most sensitive as compared to UV and fluorescence detection. A very comprehensive comparison of the four detection systems—UV, fluorescence, and two MS systems [atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI)]—for the determination of the previously identified 3, 4, 5 -trimethoxyflavone is presented in Table 1. Fluorescence detection is 10 times more sensitive than UV detection, whereas MS detection is 50 times more sensitive than UV detection and 5 times more sensitive than fluorescence detection. 

Capillary electrophoresis has been applied to the analysis of enzymically glucosylated flavonoids and of monosaccharides from glycosaminoglycans, using UV detection and indirect laser-induced fluorescence detection, respectively, and the separation of the cyanogenic glycosides amygdalin and prunasin from their isomers has been achieved by a micellar capillary electrophoresis method. ... 

Fluorescence detection in conjtmction with HPLC post-colunui treatment is commonly used to fulfill the requirements of sensitivity and specificity needed for the study of flavonoids in body fluid. The number of flavonoids that exhibit native fluorescence is limited, and derivatization of flavonoids with reagents such as Al " [63] and Tb [64] is needed before detection. If a hydroxyl group is replaced by a methoxy group, fluorescence becomes considerably more intense as demonstrated by a study using luteofin flavones [65]. 

The presence of at least one aromatic ring in the flavonoid chemical structure ensures the absorption of UV radiation as mentioned above, a first maximum occurs in the range of 240-285 nm, and a second one in the 300-550 nm range. Thus, UV detection is a satisfactory tool for use in screening and/or quantification studies. In contrast, the use of fluorescence detection is rarer since only few flavonoids exhibit native fluorescence, mostly isoflavones [61] and some flavones [62]. In other cases, derivatization processes, through the reaction between the flavonoid and metal cations, have been carried out for... 

Although the number of flavonoids that are naturally fluorescent is quite limited, fluorescence detection can provide better selectivity and sensitivity than UV detection, permitting selective detection of flavonoids in complex mixtures. Thus, fluorescence detection was reported to be tenfold more sensitive than UV detection. A fluorescence detector, mainly set at 280-270 and 310 mn for excitation and emission wavelengths, respectively, connected in series with a UV detector allows a more complete characterization of fluorescent and nonfluorescent compounds. 

Hollman, P.C.H., van Trijp, J.M.P., and Buysman, M.N.C.P., Fluorescence detection of flavonoids in HPLC by postcolumn chelation with aluminium. Anal. Chem., 68, 3511,... 

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]. 

Fluorescence detectors have also been employed for flavonoid determination, offering higher sensitivity and selectivity, as suggested by the results in the analysis of 3, 4, 5 -trimethoxyflavone, which has been determined by excitation at 330 nm and by detection at 440 nm. - ... 

Microspectrofluorometry was employed for mapping the location of phenolic substances in maize kernels. Autofluorescence due to phenolic acids was detected mainly in the embryo, aleurone and pericarp of maize kernel cross sections. Boric acid (H3BO3) reagent enhanced the fluorescence due to flavonoids in the aleurone layer. The amides of phenolic acids required derivatization with Ehrlich s reagent (168) to reveal fluorescence in the embryo and aleurone. The localization of phenolic amines was conflrmed by HPLC analysis. Phenolic compounds are important in the resistance of maize kernels to pests. Resistant maize types showed higher intensities of phenolic fluorescence but no unusual distributions of these compounds. ... 

Kappenberg [68] derivatized sample solutions of inflorescenses of hawthorn and lime (Tilia sp.), among other species, with dansylchloride (5-dimethylamino-l-naphthalinsulfonyl chloride) prior to thin layer chromatography on silica. Detection is based on the measurement of fluorescence at 365 nm. Under the applied experimental conditions phenolic acids and flavonoid glycosides remained at the start, whereas derivatives of B-type procyanidin dimers were developed as a single spot. The sensitivity was reported to be ten times higher compared to the vanillin condensation products. Drawbacks of this method are that it is sensitive to light and that so far, no studies on reaction kinetics of individual procyanidins have been performed. 

Separation of Marrubii herba extract (3) In solvent C and detection with NP/PEG reagent reveals six blue fluorescent zones (e.g. caffeic acid derivatives) between A, 0.15 and i , — 0.8 and two weak green-yellow flavonoid glycosides at R 0.5-0.65. 

C The separation in solvent system C and detection with NP/PEG reagent reveals three yellow-orange fluorescent flavonoids at Rf 0.2-0.45, with quercetin-3-O-rhainnosyl-arabinoside and kaempferol-3-0-robmosyl-7-rhamiioside as major zones. 

The flavonoid content of the methanolic extract of Convallariae herba (2a) is low. Undefined yellow-green flavonoid glyco.side zones in the R, range 0.25-0.45, bine fluorescent phenol carboxylic acids and red chlorophyll zones (front) are detectable. 

Fig. 6A Detection with NP reagent only Arnicae flos (A. montana, A. chamissonis 5-8) Astragalin is found as a bright green fluorescent zone at Rf 0.8 (T5), while the other flavonoid glycoside,s below only appear pale orange-brown (see fig. 5). Blue fluorescent chlorogenic acid at R, 0.45 and caffeic acid at Rp 0.9 are detectable.

Fig. I2C Methanoltc extracts of Farfarae folium (4) and the adulterant Petasitidis folium (5-7) have a very similar pattern of blue fluorescent phenol carboxylic acids. Flavonoid monoglycosides (R, range 0.5-0.65) are present in varying concentrations in Petasites species, while rutin is not detectable in the samples 5,6 and there are only traces in sample 7.

In UV-365 nm six yellow, orange-red or green fluorescent flavonoid aglycones are detectable eriodictyol (R, 0.3/T3) followed by a yellow-green zone of chrysoeriodictyol, an orange-red zone of xanthoeriodictyol and the green zone of homoeriodictyol (Rf — 0.5.5/T4). 

Vitis idaeae folium (1) is characterized by six yellow-orange fluorescent flavonoid glycosides in the R, range 0.35-0.8. The major zones are found at R, 0.8 and in the R, range of the hyperoside test (T2). As a minor zone rutin is detected at R,- — 0.35 (T2). 

Detection of flavonoids, aioin. Intense fluorescence is produced in UV-365 nm. PEG increases the sensitivity (from lOpg to 2,5 pg). The fluorescence behaviour is structure dependent. 

Detection reagents can also be applied to the layer as a vapor, as mentioned above for iodine. Other reagents delivered to the layer by vapor exposure include t-butyl hypochlorite and HCl, both of which form fluorescent derivatives with a variety of compounds. The Analtech vapor-phase fluorescence (VPF) visualization chamber provides detection of compounds such as sugars, lipids, steroids, flavonoids, and antibiotics by induced fluorescence after heating the sealed chamber, containing the plate and ammonium bicarbonate crystals, on a hotplate to a temperature that decomposes the salt to ammonia. 

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