Vitamin , natural fluorescence

Fluorimetric methods of analysis make use of the natural fluorescence of the analyte, the formation of a fluorescent derivative or the quenching of the fluorescence of a suitable compound by the analyte. Fluorescence cannot occur unless there is light absorption, so that all fluorescent molecules absorb, but the reverse is not true only a small fraction of all absorbing compounds exhibits fluorescence. The types of molecule most likely to show useful fluorescence are those with delocalised ji-orbital systems. Often, the more rigid the molecule the stronger the fluorescence intensity. Naturally fluorescent compounds include Vitamin A, E (tocopherol). 

Some analytes, such as riboflavin (vitamin B2)16 and polycyclic aromatic compounds (an important class of carcinogens), are naturally fluorescent and can be analyzed directly. Most compounds are not luminescent. However, coupling to a fluorescent moiety provides a route to sensitive analyses. Fluorescein is a strongly fluorescent compound that can be coupled to many molecules for analytical purposes. Fluorescent labeling of fingerprints is a powerful tool in forensic analysis.17 Sensor molecules whose luminescence responds selectively to a variety of simple cations and anions are available.18 Ca2+ can be measured from the fluorescence of a complex it forms with a derivative of fluorescein called calcein. 

Riboflavin (vitamin B2) Riboflavin exists primarily as the coenzyme forms flavin mononucleotide and flavin adenine dinucleotide. Not commonly measured, both can be measured by their natural fluorescence at 530 nm following reversed-phase... 

Several vitamins are naturally fluorescent, a physical characteristic that can be used in their sensitive and selective detection in LC. The detection limit for riboflavin by fluorescence detection is less than 1 pmol, whereas in UV detection it is 30 times higher. The detection of an LC assay for thiamin and its phosphate esters using the fluorimetric... 

Whereas the measurement of B vitamin status has, in recent years, tended to focus on blood analysis, perhaps mainly because of the convenience of sample collection, the development of blood-based status analysis for niacin has lagged behind that of the other components of the B complex. Some studies have indeed suggested that the erythrocyte concentration of the niacin-derived coenzyme NAD may provide useful information about the niacin status of human subjects that a reduction in the ratio of NAD to NADP to below 1.0 in red cells may provide evidence of niacin deficiency and that a decline in plasma tryptophan levels may indicate a more severe deficiency than a decline in red cell NAD levels. These claims now need to be tested in naturally deficient human populations. The niacin coenzymes can be quantitated either by enzyme-linked reactions or by making use of their natural fluorescence in alkaline solution. 

Because of the time and expense involved, biological assays are used primarily for research purposes. The first chemical method for assaying L-ascorbic acid was the titration with 2,6-dichlorophenolindophenol solution (76). This method is not appHcable in the presence of a variety of interfering substances, eg, reduced metal ions, sulfites, tannins, or colored dyes. This 2,6-dichlorophenolindophenol method and other chemical and physiochemical methods are based on the reducing character of L-ascorbic acid (77). Colorimetric reactions with metal ions as weU as other redox systems, eg, potassium hexacyanoferrate(III), methylene blue, chloramine, etc, have been used for the assay, but they are unspecific because of interferences from a large number of reducing substances contained in foods and natural products (78). These methods have been used extensively in fish research (79). A specific photometric method for the assay of vitamin C in biological samples is based on the oxidation of ascorbic acid to dehydroascorbic acid with 2,4-dinitrophenylhydrazine (80). In the microfluorometric method, ascorbic acid is oxidized to dehydroascorbic acid in the presence of charcoal. The oxidized form is reacted with o-phenylenediamine to produce a fluorescent compound that is detected with an excitation maximum of ca 350 nm and an emission maximum of ca 430 nm (81). 

More than one century ago a yellow, fluorescent pigment was isolated from whey by Blyth In the subsequent years yellow pigments were extracted from various biological materials. Depending either on the source of isolation or the physical appearance, these natural products were named e.g. lactochrome , lycochrome , ovoflavin , lactoflavin , hepatoflavin , or verdoflavin . Later, it became evident that all these compounds are riboflavin (vitamin B2). 

The most obvious fluorescent compound in milk is riboflavin, which absorbs strongly at 440-500 nm and emits fluorescent radiation with a maximum at 530 nm. Riboflavin in whey is measured easily by fluorescence (Amer. Assoc. Vitamin Chemists 1951). Proteins also fluoresce because of their content of aromatic amino acids. Part of the ultraviolet radiation absorbed at 280 nm is emitted at longer wavelengths as fluorescent radiation. A prominent maximum near 340 nm is attributable to tryptophan residues in the protein. Use of fluorescence for quantitation of milk proteins was proposed by Konev and Kozunin (1961), and the technique has been modified and evaluated by several groups (Bakalor 1965 Fox et al. 1963 Koops and Wijnand 1961 Porter 1965). It seems to be somewhat less accurate than desired because of difficulties in disaggregating the caseinate particles and in standardizing instruments. It also involves a basic uncertainty due to natural variations in the proportions of individual proteins which differ in tryptophan content. 

The detection of the individual C vitamers is complicated by their distinctly different properties. Although AA and DHAA are both ultraviolet (UV) absorbers, the absorbance maximum of DHAA is between 210 and 230 nm (15,18,42,43). For practical detection purposes, this makes DHAA particularly susceptible to interferences from a number of naturally occurring food constituents and limits the choice of reagents and solvents. In contrast, AA exhibits a pH-dependent absorbance maximum of 245-265 nm, which makes UV absorbance an ideal choice for detection. On the strength of its reducing capacity, AA can be detected electrochemically, but DHAA is electrochemically inactive. Neither AA nor DHAA fluoresce naturally. However, DHAA readily forms a fluorescent quinoxaline derivative upon reaction with o-phenylenediamine. As a result, chemical derivatization is often used to achieve the sensitivity needed to detect the naturally occurring vitamin C in food. 

One major reason for nutrient loss and off-flavor development today is due to extended exposure to fluorescent light in food retail display cases. Many foods and beverages are susceptible to light-induced reactions, especially those with photo-sensitizers. Natural pigments found in foods that commonly act as photochemical initiators are flavonoids, riboflavin (vitamin B2), chlorophyll, heme, and vitamin K. 

Additional support for the nonequivalence of the two subunits has been claimed by Everse 360) from fluorescence quenching studies of the stoichiometry of vitamin A acid binding. The nonlinear nature of such quenching Section II,F) might, however, require more direct binding studies for confirmation, especially since the results were obtained in the absence of coenzyme. 

Reports on the applications of h.p.l.c. to specific problems include an efficient separation of the reduction products of progesterone, of the conjugates of natural bile acids, and of 2-hydroxy- and 2-methoxy-oestrogens ( catechol oestrogens). Fluorescence detection is reported to be some 500 times more sensitive than u.v. absorption for h.p.l.c. of oestriol. The h.p.l.c. behaviour of compounds in the vitamin D series appears to be correlated with the degree of molecular planarity. " A first report on the use of cholesteric liquid crystals as stationary phases for h.p.l.c. shows promise. Various cholesteryl esters coated on or bonded to Corasil II showed increased capacity factors (k ) when steroids were chromatographed, and permitted some useful separations. 

C20H30O, Mr 286.46, yellow prisms, mp. 63 -64°C, bp. 120-125°C (0.6 Pa), exhibits a characteristic green fluorescence in solution. The action of uv light converts R. to products without any vitamin A activity. R. is very sensitive to oxygen and heavy metal ions (Cu, Co) singlet oxygen can completely destroy R. by formation of peroxides. R. forms esters with acids, the esters are more stable towards autoxidation. Occurrence Of the possible stereoisomers of R. only few occur in nature (e.g., 11-cis- and 13-cis-R.). R. is very rare in the plant kingdom but its provitamin p-carotene is widely distributed. The body s require-... 

Vitamin C Vitamin C activity resides in two naturally occurring compounds ascorbic acid and its oxidation product, dehydroascorbic acid. In human tissues ascorbic acid predominates. Ascorbic acid is labile in most samples, oxidizing to dehydroascorbic acid and then degrading to 2,3-diketogluconic acid. Various reagents can be used to prevent this oxidation in plasma or whole blood samples. Extraction with 5% metaphosphoric or trichloroacetic acid is the usual initial preparation. Only ascorbic acid may be detected by UV spectrophotometry at 245-265 nm, the absorption maxima of dehydroascorbic acid being 210 nm. A similar problem exists with electrochemical detection where ascorbic acid oxidizes at +0.7V with carbon electrodes. Fluorescent derivatives may be formed with 2-4-din-itrophenylhydrazine or o-phenyldiamine. These derivatives can be assayed by reversed-phase HPLC. 

Tocopherols and tocotrienols appear to unite all necessary physicochemical properties to make them the ideal analytes for a liquid chromatographic separation and quantitation. They are nonpolar, nonvolatile, unstable, and easily detectable owing to their favorable UV, fluorescent, and electrochemical characteristics. Their nonpolar nature together with the absence of silanol sensitive functional groups minimizes unwanted chromatographic phenomena such as peak tailing and low efficiency. Unlike GC, LC of vitamin E proceeds at room temperature and does not require derivatization to improve its chromatographic properties or... 

Several LC modes have been used for the separation of fat-soluble vitamins. The choice depends on the vitamin forms to be determined, the nature of the food matrix, and the sample treatment. Adsorption chromatography has two main advantages (a) geometric and positional isomers are generally resolved on silica stationary phases [86,87] (b) relatively high loads of lipoidal material can be tolerated by this type of column. The latter feature allows the direct injection of extracts obtained by means of direct liquid extraction [88] or sample dissolution in hexane (e.g., vitamin E from oils) [89], when the recourse to saponification is not essential for the analyte isolation. In these cases, fluorescence detection is... 

Several comprehensive reviews have been published on the existing chromatographic methods for the analysis of lipophilic antioxidants (tocopherols, tocotrienols, and carotenoids) in various sample matrices (Abidi, 2000 Aust et al., 2001) on electrochemical approaches in the sensing of natural or biological antioxidants and antioxidant capacity (mainly polyphenols and vitamins C and E) using cyclic voltammetry on flow injection analysis (FIA) with amperometric detection in food and biological samples (Blasco et al., 2007) and on chemiluminescence (CL) and fluorescence (FL) methods for the analysis of oxidative stability, antioxidant activity, and lipid hydroperoxide content in edible oils (Christodouleas et al., 2012). 

Chen (1965) examined several fluorescent dyes for the detection of vitamin D on thin-layer chromatograms. When the fluorescence was visualized under light having wavelengths of 365 and 254 mji, significant differences were observed in the nature of the fluorescence for a number of sterols. This method afforded a good qualitative differentia-... 

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