Riboflavin fluorescence

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... 

As the measurement of riboflavin fluorescence in the urine after oral dosage is very easy to perform, riboflavin is given in some studies to test the compliance of other oral drugs in patients (25). 

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. 

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 alkah. The fluorescence is used in quantitative deterrninations. The optical activity of riboflavin in neutral and acid solutions is [a]=+56.5-59.5° (0.5%, dil HCl). In an alkaline solution, it depends upon the concentration, eg, [a] J =—112-122° (50 mg in 2 mL 0.1 Ai alcohohc NaOH diluted to 10 mL with water). Borate-containing solutions are strongly dextrorotatory, because borate complexes with the ribityl side chain of riboflavin = +340° (pH 12). 

Riboflavin can be assayed by chemical, en2ymatic, and microbiological methods. The most commonly used chemical method is fluorometry, which involves the measurement of intense yeUow-green fluorescence with a maximum at 565 nm in neutral aqueous solutions. The fluorometric deterrninations 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 irradiation 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). 

Two types of fluorescent proteins have been isolated from luminous bacteria and studied in detail. The first of them are the blue fluorescent lumazine proteins (LumPs) containing lumazine as their chromophores, which were isolated from P. phosphoreum and P. fischeri (Gast and Lee, 1978 Koda and Lee, 1979 O Kane et al.y 1985). The second are the yellow fluorescent proteins (YFPs) containing a chromophore of FMN or riboflavin, isolated from P. fischeri strain Y-l (Daubner et al., 1987 Macheroux et ai, 1987 ... 

Fig. 2.4 The spectrum of bacterial luminescence measured with B. harveyi luciferase, FMN, tetradecanal and NADH, in 50 mM phosphate buffer, pH 7.0, at 0°C (dashed line from Matheson et al., 1981) and the absorption and fluorescence emission spectra of LumP (solid lines) and Rf-LumP (dotted lines) obtained from P. leiog-natbi, in 25 mM phosphate buffer, pH 7.0, containing 1 mM EDTA and 10 mM 2-mercaptoethanol, at room temperature (from Petushkov et al, 2000, with permission from Elsevier). LumP is a lumazine protein, and Rf-LumP contains riboflavin instead of lumazine in the lumazine protein. Fluorescence emission curves are at the right side of the absorption curves.

Petushkov, V. N., Gibson, B. G., and Lee, J. (1995). The yellow bioluminescence bacterium, Vibrio fischeri Yl, contains a bioluminescence active riboflavin protein in addition to the yellow fluorescence FMN protein. Biochem. Biophys. Res. Commun. 211 774-779. 

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. 

Fig. 1. a) UV-Vis absorption and fluorescence emission spectra of riboflavin (RF, 20 pM) and Gum Arabic aqueous solutions at pH 7 (phosphate buffer 100 mM). b) Transient absorption spectra of RF (35 pM) in N2-saturated MeOH-Water (1 1) solution. The insets show the transient decay at 720 nm for the RF species and the Stern-Volmer plot for the quenching of 3RF by GA, eqn 11. 

The excitation and emission wavelengths used for fluorescence detection in HPLC analysis are 350 and 450 nm for isoxanthopterin and 340 and 450 nm for 2,4,7-trioxopteridine, respectively." The absorption spectra of riboflavin present... 

The types of compounds that can be analyzed by fluorometry are rather limited. Benzene ring systems, such as the vitamins riboflavin (Figure 8.13) and thiamine, are especially highly fluorescent compounds and are analyzed in foods and pharmaceutical preparations by fluorometry. Metals can be analyzed by fluorometry if they are able to form complex ions by reaction with a ligand having a benzene ring system. 

Obtain fluorescence measurements (F) on all of your standards, the unknown, and control. Use 5% acetic acid for a blank. Your instructor will demonstrate the use of the instrument, and explain how it was set up to measure riboflavin. 

HPLC with fluorescence detection was employed for the analysis of riboflavin (RF), flavin mononucleotide (FMN) and flavin-adenin dinucleotide (FAD) in beer, wine and other beverages. The investigation was motivated by the finding that these compounds are responsible for the so-called taste of light which develops in beverages exposed to light. Samples were filtered and injected in to the analytical column without any other pretreatment. Separations were carried out in an ODS column (200 X 2.1mm i.d. particle size 5 pm). Solvents A and B were 0.05 M phosphate buffer (pH 3) and ACN, respectively. The... 

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). 

We should note that there are other intrinsic fluoro-phores that can be used in binding studies. NADH and NADPH are both highly fluorescent coenzymes. So too are riboflavin and FAD, but flavoproteins are not typi-... 

The riboflavin vitamers have UV absorbance and fluorescence, even if EAD s fluorescence is 10 times lower than riboflavin and FMN. 

Formation of a chemical complex with other molecules in solution can change fluorescence behaviour, e.g. the presence of caffeine in solution reduces the fluorescence of riboflavin. This alteration of fluorescence upon binding is used to advantage when examining binding of fluorescent molecules to proteins or other constituents of cells. 

Riboflavin (vitamin B2 6.18) consists of an isoalloxazine ring linked to an alcohol derived from ribose. The ribose side chain of riboflavin can be modified by the formation of a phosphoester (forming flavin mononucleotide, FMN, 6.19). FMN can be joined to adenine monophosphate to form flavin adenine dinucleotide (FAD, 6.20). FMN and FAD act as co-enzymes by accepting or donating two hydrogen atoms and thus are involved in redox reactions. Flavoprotein enzymes are involved in many metabolic pathways. Riboflavin is a yellow-green fluorescent compound and, in addition to its role as a vitamin, it is responsible for the colour of milk serum (Chapter 11). 

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). 

---