Riboflavin fluorescence determination

Important organic applications are to the determination of quinine and the vitamins riboflavin (vitamin B2) and thiamine (vitamin Bj). Riboflavin fluoresces in aqueous solution thiamine must first be oxidised with alkaline hexacyanoferrate(III) solution to thiochrome, which gives a blue fluorescence in butanol solution. Under standard conditions, the net fluorescence of the thiochrome produced by oxidation of the vitamin Bj is directly proportional to its concentration over a given range. The fluorescence can be measured either by reference to a standard quinine solution in a null-point instrument or directly in a spectrofluorimeter.27... 

Fluorimetric determination After extraction and cieanup riboflavin fluorescence is measured employing 400 to 420 nm as the excitation wavelength and 550 to 570 nm as the emission wavelength. Despite its good sensitivity, this method has the disadvantage of interference from other fluorescent... 

Samples of urine are analyzed for riboflavin before and after taking a vitamin tablet containing riboflavin. Concentrations are determined using external standards or by the method of standard additions, fluorescence is monitored at 525 nm using an excitation wavelength of 280 nm. 

Riboflavin is heat-stable in the absence of light, but extremely photosensitive. It has a high degree of natural fluorescence when excited by UV light. This property can be used for detection and determination. Two coenzymes (Fig. 2), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are derived from riboflavin. 

C. Andres-Lacueva, F. Mattivi and D. Tonon, Determination of riboflavin, flavin mononucleotide and flavin-adenin dinucleotide in wine and other beverages by high-performance liquid chromatography with fluorescence detection. J. Chromatogr.A 823 (1998) 355-363. 

The movement of cutaneous interstitial fluids varies from a 3- to 4-fold range in normal individuals as determined by injecting riboflavin intradermally and recording the time required for one-half its fluorescence to disappear.31 When repeated tests were made on three individuals it was found that one gave relatively highly variable results. (S. D. 48.4), and the others gave relatively constant results (S.D. 9.5 and 5.8). 

Solid-phase extraction is routinely used to clean up extracts prior to quantitation (19,42,70, 80-82). Alternatively, endogenous fluorescent artifacts in food samples can be eliminated by oxidation with potassium permanganate/hydrogen peroxide/sodium metabisulphite. Benzyl alcohol has been used to extract riboflavin selectively without the coenzymes, permitting the determination of free riboflavin. 

In aqueous solution, riboflavin has absorption at ca 220—225, 226, 371, 444 and 475 nm. Neutral aqueous solutions of riboflavin have a greenish yellow color and an intense yellowish green fluorescence with a maximum at ca 530 nm and a quantum yield of = 0.25 at pH 2.6 (10). Fluorescence disappears upon the addition of acid or alkali. The fluorescence is used in quantitative determinations. The optical activity of riboflavin in neutral and acid solutions is [a]=-H56.5-59.5 (0.5%, dilHCl). In an alkaline solution, it depends upon the concentration, eg, [a] =—112-122 (50 mg in 2 mL 0.1 iV alcohoHc NaOH diluted to 10 mL with water). Borate-containing solutions are strongly dextrorotatory, because borate complexes with the ribitji side chain of riboflavin [ct] ° = +340° (pH 12). 

Riboflavin can be assayed by chemical, enzymatic, and microbiological methods. The most commonly used chemical method is fluorometry, which involves the measurement of iatense yeUow-green fluorescence with a maximum at 565 nm in. neutral aqueous solutions. The fluorometric determinations of flavins can be carried out by measuring the intensity of either the natural fluorescence of flavins or the fluorescence of lumiflavin formed by the kradiation of flavin in alkaline solution (68). The later development of a laser—fluorescence technique has extended the limits of detection for riboflavin by two orders of magnitude (69,70). 

Perez-Ruiz T, Martinez-Lozano C, Sanz A, Bravo E. Determination of riboflavin, flavin mononucletide and flavin adenine dinucleotide in biological tissues by capillary zone electrophoresis and laser-induced fluorescence detection. Electrophoresis 2001 22 1170-4. 

Riboflavin (vitamin B2) is determined in a cereal sample by measuring its fluorescence intensity in 5% acetic acid solution. A calibration curve was prepared by measuring the fluorescence inteiisities of a series of standards of increasing concentrations. The following data were obtained. Use the method of least squares to obtain the best straight line for the calibration curve and to calculate flie concentration of riboflavin in the sample solution. The sample fluorescence intensity was 15.4. 

Riboflavin is strongly fluorescent in 5% acetic acid solution. The excitation and fluorescence spectra are obtained to determine the wavelengths of excitation and emission to use, and an unknown is determined by comparison to standards. 

Riboflavin was first observed in 1879 by the English chemist Alexander Wynter Blyth (1844-1921) who noticed a compound in cow s milk that glowed with a yellow fluorescence when exposed to light. Blyth called the compound lachtochrome (lachto- = milk and -chrome = color), but was unable to determine its chemical composition or its chemical properties. In fact, it was not until the 1930s that the chemical nature of the compound was determined. The Swiss chemist Paul Karrer (1889-1971) and the Austrian-German chemist Richard Kuhn (1900-1967) independently determined the chemical structure of riboflavin and first... 

Then, the authors recorded the fluorescence emission of Idra flour samples enriched with riboflavin (1.47, 2.94, 6.25, 12.48 and 25 pg/g) (Fig. 10.6) then plotted the integral of the fluorescence spectra from 520 to 775 nm against the riboflavin concentration added to the flour. A quadratic function was obtained allowing the determination of the concentration of riboflavin in native Idra flour. This riboflavin concentration was found excitation wavelength dependent. For example, scattered light is very important from 480 to 500 nm excitation wavelengths. The most suitable excitation wavelength to determine correctly the riboflavin concentration without any interference was found equal to 470 nm (Fig. 10.7). 

Zandomeneghi M, Carbonaro L, Calucci L, Pinzino C, Galleschi L, Ghiringhelli S. 2003, Direct fluorometric determination of fluorescent substances in powders the case of riboflavin in cereal flours. Journal of Agricultural and Food Chemistry 51, 2888-2895. 

A recent development has been the use of two-dimensional fluorescence spectroscopy as a new method for on-Hne monitoring of bioprocesses [108]. As ergot alkaloids fluoresce, the formation of the product during cultivation can be observed by two-dimensional fluorescence spectroscopy. Substraction spectra offered on-line real time information about the productivity during the cultivation. It was possible to follow the biomass concentration on-line by monitoring the culture fluorescence intensity in the region of riboflavine and its derivatives. This is a powerful application of this new sensor since the on-Une determination of biomass is extremely complicated for this fimgus. 

Riboflavin is commonly determined fluorimetri-cally, for instance in milk, by its strong native fluorescence at pH 7, which arises from the extended conjugation and rigidity of the nonribose portion of the molecule. Another fluorimetric method involves conversion of riboflavin into its fluorescent derivative lumiflavin using ultraviolet (UV) irradiation. Mixtures of thiamin and riboflavin in foods such as cereal products have been resolved using LC with fluorimetric detection. 

Sample treatment prior to SPE was required to convert FMN and FAD to riboflavin using a hot acid extraction procedure followed by enzymatic digestion, as the HPLC method applied allows the determination of only riboflavin. In this case, the recovery of riboflavin from FAD in pork samples was 94-98% with 2-4 hours of incubation time prolonging the incubation time to 24 hours did not improve the recovery. Indeed, the SPE procedure allowed a concentration factor of two to four fold, with recoveries for samples in the range 96-108%. The method was applied to 21 food samples, providing comparable results to the AO AC method (modified) apart from the crispbread sample. This was justified by the presence of a fluorescent contaminant in this sample that caused an overestimation of the amount of riboflavin in the fluorimetric, non-separative method. 

Riboflavin crystallizes from a variet of solvents as fine orange needles. The decomposition point is about 280° but values found in the literature may differ several degrees from this. It is very soluble in alkali and in 36% hydrochloric acid in the cold, and in 18% hydrochloric acid when heated. The vitamin is unstable in alkali while relatively stable toward acid. The water solution is yellow in color and shows an intense yellowish-green fluorescence (maximum 565 m/r) which is useful for quantitative determination. 

More than 100 years ago a fluorescent compound was isolated first fi om whey, and later from different biological materials. When it Ijecame clear that the isolated yellow pigments, named lactochrome, ovoflavin, or lactoflavin, had a common structure, the new compound was named riboflavin (vitamin B2) (for historical review see 2). In the years between 1933 and 1935 the structure and the main chemical reactions of riboflavin were studied and the chemical synthesis was performed. Soon afterward, the coenzyme forms, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), were isolated in pure form, and the structures were determined. In the last 50 years many flavoproteins were isolated and their physicochemical properties were studied. Succinate dehydrogenase was the first enzyme found with the prosthetic group (FAD) covalently bound to the protein. About 20 flavoproteins are now known to contain covalently bound coenzyme (mainly via carbon atom 8a) (3). In mammalian tissue, the number of covalently bound flavoproteins appears to be limited. 

The quantitation of riboflavin together with thiamine and niacin using HPTLC silica gel plates and methanol/water (70 30, v/v) as mobile phase was described by Diaz et al. (30). For riboflavin the native fluorescence was used and a preplate derivatization was applied for the other two vitamins (addition of a fluorescent tracer to label nicotinic acid conversion of thiamine into thio-chrome). The developed plates were scanned by a commercially available bifurcated flber-optic-based instrument that transferred the excitation and emission energies between the plate and the fluorescence spectrometer. Calibration curves for the determination of riboflavin 48 to 320 ng, thiamine 300 to 750 ng, and niacin 10 to 100 ng were established. The advantages of this method are that no elimination of excess oxidation reagent is necessary and that the simultaneous determination of vitamins with only one detector is possible. 

A simple determination of riboflavin from urine and plasma based on a fluorometric titration was described by Kodentsova et al. (36). This method is based on the fact that the formation of riboflavin-apoprotein complex is accompanied by a complete loss of fluorescence peculiar to free riboflavin. A plasma sample of 1 mL was acidified by the addition of trichloroacetic acid. After centrifugation and neutralization, the fluorescence intensity was measured in a 3-mL cuvette (ex. 465 nm em. 525 nm). The measurement was repeated after addition of riboflavin standard and riboflavin-binding apoprotein, respectively. An amount of 1.5 ng riboflavin/mL plasma can be detected using this method. 

Some methods have been applied only to selected foods and their general validity has not been demonstrated, whereas other methods were successfully used with a wide variety of different food matrices (58-61,63). For simultaneous determination of riboflavin and thiamine in foods, various workers have proposed HPLC methods, which analyze riboflavin either as riboflavin or lumiflavin and thiamine as thiochrome by fluorescence detection. The thiochrome can be formed either before or after column separation, while lumiflavin was formed precolumn. A strong argument against the precolumn oxidation of thiamine to thiochrome is the fact that some of the riboflavin may be destroyed during the oxidation step in alkaline solution. Therefore, a state-of-the-art procedure for this special separation is the postcolumn derivatization of thiamine (see chapter on vitamin Bi). 

The majority of breakfast cereals in the United States are fortified with PN, and additional PN is also added to infant formula products to ensure adequate vitamin Be supply to the infant. Gregory (100) reported an isocratic HPLC method for the determination of PN in breakfast cereals (Table 5). Other investigators attempted simultaneous determination of PN and other vitamins used in food fortification. Wehling and Wetzel used ion pair HPLC to separate pyridoxine, riboflavin and thiamine from each other after acid extraction of the vitamins from cereals (101). Using a dual fluorescence detector setup, pyridoxine and riboflavin were monitored by the first detector. After the column eluate had passed the first detector, an alkaline ferricyanide solution was introduced, resulting in the formation of a fluorescent thiochrome derivative of thiamine, which was detected by the second fluorescence detector. A similar method for simultaneous determination of pyridoxine and riboflavin in infant formula products has also been described (102). 

Although the measurement of the fluorescence of riboflavine solutions at known pH values has been widely used for assay and forms the basis of the U.S.P. method, riboflavine is best determined in simple formulations by extraction and measurement of the extinction at 444 m. ... 

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