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Riboflavin: Properties and Determination
Riboflavin: Properties and Determination I Bitsch, Justus-Liebig-Universita¨t Giessen, Giessen, Germany R Bitsch, Friedrich–Schiller-Universita¨t Jena, Jena, Germany ã 2016 Elsevier Ltd. All rights reserved.
Physicochemical Characteristics Riboflavin, the prescribed name for vitamin B2 by the IUPAC– IUB, is the biologically active component of the prosthetic group of flavoproteins. Older designations are lactoflavin, ovoflavin, and uroflavin, which originated from the first isolation. It comprises a tricyclic, nitrogen-containing ring system, chemically defined as a substituted isoalloxazine with a ribitol side chain at N10. With the systematic nomenclature, the molecule is named as 7.8-dimethyl-10-(1-D-ribityl)-benzo[g]pteridin-2,4dion. The empirical formula is given as C17H20N4O6 with an MW of 376.36 (Figure 1). The substance is a yellow to orangeyellow powder and crystallizes in sharp, optically active needles, melting under decomposition at 275–282 C. The optical rotation is ½a25 D ¼ 112 C to 122 C (c ¼ 50 mg in 10 ml 0.02 N alcoholic NaOH). Riboflavin is sparingly soluble in water with a yellow-green fluorescence, very sparingly soluble in ethanol and insoluble in other organic solvents. It is readily soluble in dilute alkali, but rapidly deteriorated, being accelerated by light. In neutral or acid solutions, it is more stable even against heat and oxygen. Nevertheless, all operations with riboflavin solutions should be handled in the dark or at red light. Light sensitivity of the flavins has also consequences for the stability of vitamin B2-containing foods. In milk stored under day light, the riboflavin content is dropping off by more than 80% within hours. Processing losses in foods are given with 20%. Light exposure of alkaline solutions decomposes riboflavin to lumiflavin (7,8,10-trimethylisoalloxazine); in neutral or acidic solutions, the blue fluorescent lumichrome (7,8dimelthylalloxazine) is formed (Figure 2). Riboflavin is found in free-form only in the retina of the eye, in whey, and in urine. In other biological systems and tissues, the predominant forms are flavin mononucleotide (FMN) or riboflavin phosphate and flavin adenine dinucleotide (FAD). Free riboflavin and FMN-monosodium salt are the most important commercial products. FMN and FAD are effective as coenzymes or prosthetic groups in more than 100 enzymes in the plant and animal kingdom. The accepted designation for both compounds is incorrect, because FMN is not truly a nucleotide and FAD not a dinucleotide. The designations arose from the discovery of riboflavin and have been adopted ever since. Pure riboflavin was first isolated from yeast, egg white, and whey in 1933 (Kuhn and Wagner-Jauregg). Simultaneously, the so-called yellow coenzymes were discovered in yeast (Warburg and Christian), which could be identified as FMN. FMN is considerably more soluble in water than riboflavin. Its solubility in organic solvents as well as its stability against heat and light exposure is comparable to riboflavin. The flavoproteins FMN and FAD belong to the enzymatic class of oxidoreductases. The conjugated structure of the pteridine moiety of the basic molecule determines its coloring and
Encyclopedia of Food and Health
light absorbance in the UV and visible spectrum. Absorbance maxima of the flavins are listed in Table 1. The catalytic hydrogen transfer in the flavin enzymes takes place at both conjugated bound N1 and N5 atoms of the isoalloxazine molecule. The oxidized form of the flavoproteins, the flavoquinone, is thereby reversibly reduced to the colorless flavohydroquinone or leucobase (see Figure 1). The absorbance is lost, because double bonds are dissolved. As an intermediate product during this reaction, a sequinone radical is formed. During the redox process, the flavin molecule changes its conformation, which is planar in the oxidized and folded in a butterfly configuration in the reduced state. The characteristic yellow-green fluorescence of riboflavin and FMN in aqueous solutions exhibits maximal intensity at pH 6–7. The fluorescence of FAD is about 10–20% of that of riboflavin with the same spectral distribution but exhibiting a maximal intensity at pH 3. The fluorescence of flavins is quenched when bound to proteins. The irradiation products of the flavins are also fluorescing, and their spectrum is shifted to the blue (lumichrome) or green (lumiflavin) spectral range. For quantification of riboflavin, particularly the conversion to lumiflavin, measurement of its fluorescence intensity in chloroform solutions is used (ex 270 nm, em 418 nm).
Content and Reactions in Food Content Riboflavin belongs to the group of water-soluble vitamins and is generally referred to as vitamin B2. Riboflavin is an important compound of a healthy diet and is widely distributed in animal and vegetable foods. Protein-rich foods of animal origin are as rule considerable sources of this vitamin with good bioavailability. Particularly rich in riboflavin are offal, such as the heart, liver, and kidney, and eggs and dairy products, mostly as FMN and FAD. Of special importance for the dietary habits in Western countries are milk, dairy products, and meat, despite of a medium content of riboflavin. If those are omitted from the diet, problems may arise to cover an adequate vitamin B2 intake for human beings (Table 2). Furthermore, riboflavin is used as a safe coloring agent for some processed foods and pharmaceuticals and is included in nutrient supplements. Meat, fish, and dark green vegetables contain also significant concentrations of riboflavin. Milk and cheese are in agreement with their content of riboflavin products for which lightinduced quality deterioration is now widely recognized to be the result of riboflavin photosensitizing.
Stability of Riboflavin Riboflavin and its physiological compounds are relatively stable during thermal and nonthermal food processing and
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Riboflavin: Properties and Determination
O
O H3C
N NH
H3C
N
N
+ 2 [H] O
− 2 [H]
H3C
H N
H3C
N
N H
Ribit
P
NH
CH2 NH2
H C OH N
H C OH
O
P
Adenin
N
H C OH N H2C
O
P
O
P
CH2
N
O
Riboflavin
FMN (H2) OH
OH
FAD (H2) Figure 1 Riboflavin and its coenzyme forms.
CH3 H3C
N
H3C
N
N
O
H3C
N
H3C
N
N
O
N
N H O Lumiflavin (alkaline solution)
H O
Lumichrome (acidic solution)
Figure 2 Photolysis products of riboflavin.
storage, in the absence of light even at high temperature. But they are very unstable to light even at room temperature. Particularly, aqueous solutions of these substances are sensitive to UV and visible light, and they are degraded through a variety of reactions resulting in a number of photoproducts under aerobic and anaerobic conditions. The major photoproducts obtained through normal photolysis of riboflavin in aqueous solvents include formylmethylflavin, lumichrome, lumiflavin, and 2,3-butanedione. With multicomponent spectrometric methods, several further photoproducts are to be identified. Lumichrome is the major product under neutral or acidic pH; lumiflavin is formed in basic pH; 2,3-butanedione is the major volatile compound with a typical buttery odor. Lumichrome and lumiflavin are formed from unstable diradical excited triplet riboflavin by dealkylation; 2,3-butanedione is produced from the reaction between electrophilic singlet oxygen and electron-rich riboflavin. The kinetics of riboflavin photolysis under light exposure and in the pH range of 1–12 indicate that riboflavin has maximum photostability around pH 5–6.
Mostly destructive to riboflavin is exposure to light with wavelength of its absorption maximum at 450 nm.
Reactions of Riboflavin as a Photosensitizer Riboflavin and other flavins are efficient photosensitizers, inducing oxidative damage to light-exposed food and several food compounds. When exposed to light of wavelength 420–560 nm, the absorbed energy does excite riboflavin to triplet state. Riboflavin can accept or donate two hydrogen atoms and acts together with lumiflavin as efficient photosensitizer. These so-called sensitized photoreactions are typical for foods and essentially involve two pathways. Direct interaction of the excited sensitizer with food compounds initiates radical processes through electron transfer or hydrogen abstraction and is known as a type 1 photo-oxidation. This mechanism is favored under low oxygen pressure and is, for example, responsible for light-struck flavor formation in beer. Under aerobic conditions, excitation energy is preferably
Riboflavin: Properties and Determination
Table 1
635
Physicochemical data of flavines
Property
Riboflavin
Riboflavin-5-phosphate-Na (FMN-Na)
Molecular weight Melting point Solubility
376.36 278–282 C (decomposition) Readily soluble in dilute alkalis (decomposition) Sparingly soluble in water (70–100 mg l1, 27 C) Very sparingly soluble in ethanol (45 mg l1, 27 C) Insoluble in acetone, chloroform, ether, benzene 223, 267, 374, 444 nm (0.1 N HCl)
478.34 280–290 C (decomposition) Soluble in water at pH 7 (30 g l1, 25 C)
Absorbance (UV/ vis) Fluorescence Stability
Flavinadenindinukleotide-Na2 (FAD-Na2) 829.6 Soluble in water
Very sparingly soluble in ethanol
Yellow-green fluorescence of aqueous solutions at pH 6–7 (ex. 444 nm, em. ex. 400–500 nm, em. 530 nm 530–565 nm) In neutral and acidic medium relatively stable against heat and oxygen. Rapid photolysis by UV- and visible light to blue fluorescent lumichrome (acidic and neutral solutions) or yellow-green fluorescent lumiflavin (alkaline solutions). After reduction by dithionite, Zn-HCl, sodium hydrosulfite, etc., reversible conversion to leucoflavin (dihydroflavin)
Source: The Merck index: an encyclopaedia of chemicals, drugs and biologicals (2014) (15th ed.). New Jersey: The Royal Society of Chemistry; Eitenmiller, R. R., Landen Jr, W. O. and Ye, L. (2007). Vitamin analysis for the health and food sciences (2nd ed.). Boca Raton, FL: CRC Press.
Table 2
Riboflavin content of foods
Content (mg/ 100) ingested food
Milk and milk products
>3.00 2.50–3.00
0.30–0.50
0.20–0.30
0.15–0.20
Cereals
Vegetables and legumes
Fruits and nuts
Fish
Pig’s liver Beef liver, calf liver Beef kidney, calf kidney Pig’s kidney Pig’s heart
2.00–2.50 1.50–2.00 1.00–1.50 0.70–1.00 0.50–0.70
Meat and meat products
Camembert type, cheddar type, Danish blue type, parmesan Cream brie (50%), edam type, fresh cheese (skim milk) Fresh cheese (50%), gouda cheese (45%), yogurt (low fat) Cottage cheese, cow’s milk (fresh)
Wheat germs Wheat bran
Eggs, lamb
Eel, mackerel
Goose, pork (lean) veal
Beef (lean), chicken, duck, turkey
Oat flakes, whole meal flower (wheat, rye)
0.10–0.15
Green cabbage, soybean meal
Cashew nuts, hazelnuts, sallow thorn
Broccoli (boiled), mangold, soybean sprouts, spinach (boiled) Asparagus (boiled), Brussels sprouts
Avocado
Flounder, herring, pilchard, plaice Haddock, salmon
Peanuts (roasted)
Source: Souci, S. W., Fachmann, W. and Kraut, H. (2008). Food composition and nutrition tables (7th ed.), compiled by Dr. Eva Kirchhoff. Stuttgart: Medpharm.
transferred to oxygen to generate singlet oxygen, the pivotal intermediate in a type II photo-oxidation. This reaction is responsible for oxygenation of unsaturated fatty acids in vegetal oils causing high peroxide values and eventually rancidity, while attack of amino acid residues in dairy proteins may give rise to sulfury and potato-like off-flavors. Both
photo-oxidation pathways induced by triplet-excited riboflavin lead to premature aging or spoiling of foods and beverages. Photosensitizing of riboflavin causes production of reactive oxygen species such as super oxide anion, singlet oxygen, hydroxyl radical, and hydrogen peroxide. Radicals and reactive oxygen species accelerate the decomposition of food compounds
636
Riboflavin: Properties and Determination
as amino acids, proteins, lipids, carbohydrates, and vitamins and cause significant alterations of nutritional value of foods. Carbohydrates are less sensitive to riboflavin-photosensitized oxidation than proteins, lipids, or vitamins. Exposed to visible light, riboflavin acts as a potent photosensitizer in food and beverages presenting a risk for their stability. Decomposition of the nutrients depends on the wavelength and energy of light, the concentration of oxygen and riboflavin, and the presence of activating or inactivating compounds. Riboflavin is an excellent photosensitizer for singlet oxygen formation and a superb reactant for singlet oxygen, with the reaction rate of 1.01 1010/M/s. The triplet-excited state of riboflavin is relatively long-living and as a biradical a very powerful oxidant compared to ground state riboflavin, leading to direct oxidation of most types of biomolecules.
Riboflavin Losses in Dairy Products Due to the relative heat stability, only minor vitamin B2 losses occur during processing. Likewise, the pasteurization of milk in the absence of light causes losses of < 10%. On the other hand, exposure of dairy products to day light may result in a remarkable decrease of the riboflavin content being dependent on the surface exposed. In milk stored under daylight, the riboflavin content is dropping off by more than 80% within hours with both lipids and proteins being oxidized under photosensitized conditions. The photodegradation leads to the formation of carbonyl compounds such as pentanal, hexanal, heptanal, 2-nonenal, 2,4-nonadienal, and 2,4-decadienal all attributed to lipid photo-oxidation. The chemical reaction between methionine and singlet oxygen, which has been formed by riboflavin photosensitization, produces dimethyl-disulfide, which is responsible for the sunlight flavor of milk. In an actual study, it could be demonstrated that photodegradation of riboflavin in milk may be considerably reduced by the addition of small amounts of gum arabic microcapsules, avoiding large losses in the nutritional value of milk.
Riboflavin Losses in Beer and Cereals Light exposure of beer generates an obnoxious off-flavor after a complex interplay of light, excited riboflavin, isohumulones and sulfur-containing amino acids, peptides, and proteins. The radical chemical degradation path of these compounds results in 3-methylbut-2-ene-1-thiol, the main off-flavor compound that characterizes light-exposed beers with the trivial name of skunky thiol. Vitamin losses occur also during the milling process of cereals. White flour with a low extraction rate contains about one-third of riboflavin of the whole grain flour.
Photodegradation of Amino Acids and Vitamins The deactivation of triplet-excited state riboflavin by several food components leading to photodegradation of nutrients in food, beverages, and model solutions is described in actual studies. For instance, the amino acids cysteine, histidine, methionine, tyrosine, and tryptophan are rapidly photodegraded in aqueous solutions in the presence of triplet exited riboflavin. These amino acids are the primary targets of photochemical oxidation in proteins.
Both fat-soluble and water-soluble vitamins are sensitive to photo-oxidation as sensitized by riboflavin. The vitamins ascorbate, folate, thiamine, and pyridoxal phosphate deactivate triplet-excited riboflavin and are photodegraded as well as vitamins A and D3 and tocopherols in aqueous solutions. A good photostability in the presence of riboflavin was demonstrated for the vitamins biotin and niacin. Carotenoids seem to reduce the formation of triplet-excited riboflavin through an inner filter, which protects these substances against the riboflavin-induced photosensitizing damage.
How to Prevent Reactions Polyphenolic compounds are efficient antioxidants with strong free radical scavenging ability. It was demonstrated that the plant polyphenols rutin and catechin, several flavonoids, and phenolic compounds present in beverages quench the tripletexcited state of riboflavin and protect the beverages that contain these compounds from light-induced off-flavor formation.
Methods of Determination In earliest reports, the most frequently applied assays of flavins as vitamin B2 active components made use of the direct fluorimetric assay of the intact flavins or after hydrolysis to FMN or free riboflavin. Alternatively, the lumiflavin method based upon the conversion of the chloroform-insoluble riboflavin into chloroform-soluble lumiflavin after photodegradation in alkaline solution was used, despite a transformation rate of only 60–70%. Currently, microbiological assays and highperformance liquid chromatography (HPLC) procedures with enhanced sensitivity are widespread utilized (see also Table 3). In principle, riboflavin is extracted from dry materials by autoclaving in 0.1 N HCl for 30 min or on water bath for 1–2 h. By using acid extraction, flavins are released from their protein binding and FAD and FMN are converted to free riboflavin. In protein-rich materials, the acidic extraction may be followed by enzymatic hydrolysis for complete liberation of the flavins. It is of importance to carry out all operations relating to the extraction and quantitation of flavins and riboflavin under subdued light and in brown or dark vessels. An optimal extraction procedure applicable for microbiological or HPLC determination of thiamine and riboflavin has been worked out by the European Measurement and Testing Program. The steps of the extraction are detailed in autoclaving, incubation with takadiastase, and filtration. For biological samples, such as plasma/serum, the preferred extraction medium is 5–10% trichloroacetic acid, which is suitable for denaturing the relatively weak protein binding while keeping the phosphorylated forms intact.
Direct Fluorimetry Direct measurement of the native fluorescence of riboflavin may be applicable for pharmaceutical preparations or food samples with a relatively high vitamin content. After acid extraction, the remaining proteins are removed by precipitation at pH 4.5. Interfering substances are subsequently oxidized with potassium permanganate and the excess oxidant
Riboflavin: Properties and Determination
Table 3
637
Methods of riboflavin determination
Method
Principles
Specials
Detection limits
Direct fluorimetry Lumiflavin method Microbiological assay HPLC analysis
Fluorescence measurement of riboflavin at 440 (ex)/565 (em) nm Fluorescence measurement after irradiation at pH 10–12 (ex 450, em 513 nm) Turbidimetric measurement of bacterial growth response dependent on the B2 content Separation of flavins on RP-C18 materials with aqueous/ organic solvents and fluorimetric/UV detection
Conversion of FAD and FMN to riboflavin by acidic hydrolysis Lumiflavin is separated by chloroform extraction Interference of growth response with nutrients/food components Differentiated analysis of flavins, combined analysis with other B vitamins
0.1 mg B2/g sample
destroyed with hydrogen peroxide or sodium bisulfite. The fluorescence of the clear sample solution, commonly spiked with riboflavin as an internal standard, is registered by difference measurement before and after reduction of riboflavin with sodium bisulfite to the leuco form for correction by unspecific fluorescences (ex ¼ 440 nm; em ¼ 565 nm). The detection limit of this method is about 0.1 mg riboflavin per gram sample. The direct fluorimetry of flavins had been adopted by the US Pharmacopoeia for the riboflavin analysis in tablets and injections and by the AOAC for the riboflavin determination in foods, as in milk-based formulas.
Lumiflavin Method By this method, riboflavin is photodegraded in alkaline solution at pH 10–12 to the stronger fluorescent lumiflavin (ex ¼ 450 nm; em ¼ 513 nm). This reaction is specific for riboflavin since other flavins interfering with riboflavin fluorescence are not converted into lumiflavin. Lumiflavin is separated from riboflavin and other fluorescent substances by extraction with chloroform. The transformation into lumiflavin is not quantitative and ranges between 60% and 70%. Reliable results can actually only be obtained by comparison with a riboflavin standard under identical irradiation conditions for sample and standard solution. While the original lumiflavin method is sparsely used, the principal methodology was applied to HPLC analyses of the riboflavin content in foods with detection limits of 0.02 ng riboflavin per injection.
Microbiological Assay Besides bioassays, as chicken and curative rat growth tests, microbial assays belong to the first widely used test methods measuring the biological activity of preparations containing total vitamin B2. The most frequently used test organism is Lactobacillus casei subsp. rhamnosus (ATCC No. 7469). The sensitivity limit is indicated to be 0.5 ng riboflavin absolutely. Occasionally, other test organisms are proposed such as Enterococcus faecalis (ATCC No. 10100) and Leuconostoc mesenteroides (ATCC No. 9135) with enhanced sensitivity of 0.1 ng ml1 riboflavin. The microbiological assay with L. casei is approved
0.02 ng B2 0.1–0.5 ng B2 0.21 ng riboflavin 0.89 ng FMN 11.15 ng FAD 0.02 ng B2 (precolumn irradiation to lumiflavin)
by the AOAC International Methods for the riboflavin determination in vitamin preparations. Growth response of bacteria is proportional to the riboflavin content of the medium and measured turbidimetrically. Earlier techniques determined metabolically formed lactic acid by titration. However, comparisons of turbidimetry after 16 h incubation obtained results that agreed with those obtained by titration of lactic acid after 72 h. Thus, the turbidimetric measurement is at present preferred in view of its shorter incubation time and easier handling. The growth response of lactobacilli differs significantly between free riboflavin and FMN and FAD. Acidic or enzymatic treatment is therefore needed for releasing and converting the flavins into riboflavin as described under methods of determination, followed by adjustment of the extract to pH 4.5 before incubation. In case of enzymatic release using takadiastase or clarase, it has to be considered that some enzyme preparations contain variable traces of riboflavin. Starch, glycogen, free fatty acids, and other lipids and protein degradation products can interfere with the test by either stimulating or inhibiting the bacterial growth. Lipids in a relevant content can be removed either by filtration or by ether or petroleum ether extraction before hydrolysis. Proteins are precipitated at pH 4.5 and starch is split by the acidic or enzymatic hydrolysis step. As with the other analytic procedures, the microbiological test has also to be done under subdued light. Of importance is that the used glass vessels do not release any traces of alkali and should thus be cleaned with acid before use, because the flavins are alkali-sensitive. Hence, it is advisable to dissolve riboflavin standards in 0.02 M acetic acid. An optimal extraction procedure applicable for microbiological or HPLC determination of thiamin and riboflavin has been worked out by the European Measurement and Testing Program. The steps are detailed in autoclaving in 0.1 N HCl at 121 C for 30 min and adjusting thereafter to pH 4.0 with 4 M sodium acetate buffer (pH 6.1), addition of takadiastase (0.1 g g1 sample), incubation at 37–45 C (18 h), filtration, or centrifugation after cooling.
HPLC Procedures HPLC methods enable separate quantification of individual flavins or simultaneous analysis of riboflavin including FMN
638
Riboflavin: Properties and Determination
and FAD. A broad variety of HPLC techniques are described, varying the column material, mobile phase, separation mode, and detection system as well. Of the numerous analytic and technical variations communicated during the last 20 years, the separation techniques can be categorized as follows: Reversed phase C18 materials are utilized as stationary phases in most cases, combined with aqueous/organic mobile phases on the basis of water, methanol, acetonitrile with or without phosphate, or acetate buffer in an isocratic mode. Rarely, ion pair chromatography with sodium salts of hexane or heptanesulfonic acid in the mobile phase (ion interaction chromatography) was utilized; in some cases, the separation of riboflavin and flavins was accomplished with gradient elution technique. Ion-pair HPLC seems more favorable only when riboflavin is determined simultaneously with other B vitamins (e.g., B1, B6, niacin, folic acid, and B12), because this technique results in better peak shapes. Retention times of vitamin B2 active compounds are between 5 (FAD) and 20 (riboflavin) minutes. Fluorescence monitoring is preferred for detection; UV or visible absorbance detection at 270 or 446 nm is restricted to early HPLC analyses or measurements of pharmaceuticals and enriched foods. Limits of fluorescence detection at 450/520 nm (ex./em.) for the native flavins were given to 0.55 pmol (0.21 ng) riboflavin, 1.96 pmol (0.89 ng) FMN, and 14.19 pmol (11.15 ng) FAD. In an earlier HPLC method, the detection limit could be increased to 0.02 ng riboflavin/injection by irradiation of the sample extract and conversion of the extracted riboflavin to lumiflavin prior to HPLC separation. A specific and sensitive UHPLC/LC-MS method was developed for the simultaneous determination of riboflavin and pyridoxine in infant food. In principle, extraction techniques and sample cleanup suitable for HPLC analysis resemble those used for quantitation by the other methods discussed. Mostly, vitamin B2 active substances are released from the food matrix by autoclaving with 0.1 N mineral acid (HCl or H2SO4) followed by enzymatic digestion with papain, takadiastase, or claradiastase. Solid-phase cleanup procedures on C18 materials or Florisil are often used for support prior to injection. The extraction procedure is guided by the analytic aim, whether the total riboflavin content, that is, the sum of FAD, FMN, and free riboflavin, shall be determined or the differentiated quantitation of FAD and FMN in addition to free riboflavin is strived for. Treatment with potassium permanganate can be omitted, because possibly remaining extraneous fluorescent substances are chromatographically separated. The combined acid and enzymatic hydrolysis is advantageous particularly for foods with high starch or protein content to liberate the bound flavins. Occasionally, autoclaving with dilute mineral acid is incomplete, particularly for the conversion of FMN to riboflavin, as indicated by the appearance of an FMN peak in the following chromatogram. Additionally, FMN may be partially converted during acid hydrolysis to biologically active isomeric riboflavin phosphates, which are separated from FMN by HPLC analysis and remain neglected for the calculation of total riboflavin. The combination of acidic and enzymatic digestion is thus advisable prior to HPLC analysis for the determination of total riboflavin. When using this extraction procedure and
following HPLC analysis, the riboflavin content of various foods such as breakfast cereals, porridges, milk, and milk products correlated well with the microbiological assay using Lactobacillus casei. The simultaneous determination of FAD and FMN besides free riboflavin needs nondegradative techniques, such as extraction of flavins by methanol/dichloromethane followed by partitioning with citrate buffer (pH 5.5) or extraction with 6% formic acid containing 2 M urea. The HPLC separation and quantitation of the flavins was accomplished by using internal standards (7-ethyl-8-methyl-riboflavin, sorboflavin isoriboflavin, nicotinamide, and others). This analytic device enabled the flavin analysis (FAD, FMN, and riboflavin) of milk and dairy products, fruits and vegetables, meats, and cereal products. In many cases, HPLC methods were developed for the simultaneous or sequential determination of riboflavin and thiamin in foods. Both vitamins are extracted by a common procedure using acidic and enzymatic digestion. For biological samples, as blood or plasma/serum, the preferred extraction medium is 5–10% trichloroacetic acid, which is suitable for denaturing the relatively weak protein binding while keeping the phosphorylated forms intact. In this way, flavins in whole blood can be analyzed by isocratic RP-HPLC against an external standard. A compilation of usual HPLC procedures is listed in Table 4. An overview on methods of riboflavin determination can be taken from Table 3.
European Standard Method The European Technical Committee for Standardization (CEN/TC 275) specifies a method with the title “Foodstuffs: Determination of vitamin B2 by high performance liquid chromatography and fluorescence detection.” The document number is DIN EN 14152:2014, the publication date is 2014-08, the original language is German, and the responsible standards committee for food and agricultural products is NA 057-01-13 AA: Vitamins and Carotenoids. The ‘Deutsches Institut fu¨r Normung e.V. (DIN)’ is the responsible committee for the standardization in the field of food as well as in the agricultural sector. This method has been validated in two interlaboratory studies. The first study was for the analysis of samples of milk powder and pig’s liver ranging from 1.45 to 10.68 mg/100 g. The second study was for the analysis of samples of tube feeding solution, baby food, powdered milk, meal with fruits, yeast, cereal, and chocolate powder ranging from 0.2 to 87.1 mg/100 g. Vitamin B2 is the mass fraction of total riboflavin including its phosphorylated derivates. In this European norm, riboflavin is extracted from foods after acidification, followed by an enzymatic dephosphorylation. The quantification takes place with subsequent HPLC separation, fluorimetric detection, and extern standards.
See also: Chromatography: High-Performance Liquid Chromatography; Enzymes: Functions and Characteristics.
Riboflavin: Properties and Determination
Table 4
639
HPLC analysis of flavins
Food sample/matrix (compounds determined)
Detection (nm)
Detection limit
H2O/CH3OH/acetic acid ¼ 65/35/1 0.3 M KH2PO4/ CH3OH ¼ 83.3/16.7 (pH 2.9)
UV 270
10 ng
Fluorescence 470/525 ex. em.
m Bondapak C18
CH3OH/0.005 M phosphate buffer pH 7.0 ¼ 35/65
Fluorescence 440/520 ex. em.
FMN 15 nM FAD 20 nM Riboflavin 10 nM 80–96% recovery
Spherisorb ODS 2.5 mm
H2O/CH3OH 0.65/35
Fluorescence 445/525 ex. em.
20 pg
LC-18, 3 mm (Supelco)
14% acetonitrile in 0.1 M KH2PO4, pH 2.9
Fluorescence 450/530 ex. em.
FMN 2.5 nM FAD 3 nM Riboflavin 2.5 nM
2PLRP-S, 5 mm column
temperature 40 C
Acetonitrile/ 0.1% sodium azide in 0.01 M citratephosphate buffer, pH 5.5 gradient elution
Altex
Ultrasphere ODS 5 mm
H2O/
Extraction procedure
Column
Mobile phase
Dairy products (total riboflavin) Blood (FAD, FMN, riboflavin)
Disperse in water, clean up on C18 cartridge 10% TCA at 4 C (30 min), adding NaOAc-buffer, centrifugation
Biosil ODS5S C18 Hypersil ODS 5 mm
Cereals, various foods (thiamin and total riboflavin)
0.1 N HCl, autoclave (15 min) at 125 C, adjust to pH 4.0–4.5 (2 N NaOAc), incubation with claradiastase at 50 C (3 h), 50% TCA and heating at 90 C (15 min), adjust to pH 3.5 (2 N NaOAc), filtration 0.05 M (0.1 N) H2SO4, autoclave (20 min) at 121 C, adjust to pH 4.5 (2.5 M acetate buffer), incubation with claradiastase at 45 C (overnight); filtrated sample passed through C18 Sep-Pak, elution with 40–70% CH3 OH Homogenize with 6% formic acid, containing 2 M urea pass through C18 solid phase, elute with 10% formic acid/CH3OH ¼ 4/1 Homogenize with CH3OH/CH2Cl2 ¼ 9/ 10 mix with citrate-phosphate buffer, pH 5.5 (containing 0.1% sodium azide), centrifugation
Dairy þ meat products, fruits, vegetables, flour, baked products, beer, coffee (total riboflavin)
Dairy products (FAD, FMN, riboflavin) sorboflavin as internal standard Dairy products, raw and cooked meats, cereals (FAD, FMN, riboflavin) 7-ethyl-8methyl-riboflavin as internal standard
Fluorescence 450/522 ex. em. Fruits, vegetables (total riboflavin) CH3OH ¼ 60/40 þ 5 mM heptanesulfonic acid, pH 4.5 Potatoes, vegetables (total riboflavin) Serum, urine (riboflavin) isoriboflavin as internal standard
96–113% recovery 0.1 N HCl (30 min) at 100 C, incubation with amylase (38 C) Fluorescence 450/530 ex. em. 0.1 N HCl (30 min) at 121 C, incubation with takadiastase (45–50 C) Homogenize/mix with TCA (100 g l1), centrifugation, pass through SepPak C18 (serum)
0.2 ng/ inject. m-Bondapak C18 ROSIL C18 HL 5 mm
CH3OH/H2O ¼ 30/70
CH3OH/H2O/ CH3COOH ¼ 36.7/ 63.7/0.1
Fluorescence 450/510 ex. em. Fluorescence 450/530 ex. em.
0.1 ng/ inject. 10 mg l1
Source: De Leenheer, A. P., Lambert, W. E. and van Bocxlaer, J. F. (2000). Modern chromatographic analysis of vitamins (3rd ed.). New York: Marcel Dekker, Inc.; Ball, G. F. M. (2013). Water-soluble vitamin assays in human nutrition. New York: Springer; Eitenmiller, R. R., Landen, Jr. W. O. and Ye, L. (2007). Vitamin analysis for the health and food sciences (2nd ed.). Boca Raton, FL: CRC Press.
Further Reading Ball GFM (2005) Vitamins in foods: analysis, bioavailability and stability. Boca Raton, FL: CRC Press. Ball GFM (2013) Water-soluble vitamin assays in human nutrition. New York: Springer. Bitsch R (2003) Riboflavin – properties and determination. In: Caballero B, Trugo L, and Finglas PM (eds.) Encyclopaedia of food sciences and nutrition, 2nd ed., pp. 4983–4989. London: Academic Press.
Bitsch R and Bitsch I (2011) HPLC determination of riboflavin in fortified foods. In: Rychlik M (ed.) Fortified foods with vitamins, analytical concepts to assure better and safer products, pp. 103–110. Weinheim: Wiley-VHC. Boiero ML, Mandrioli M, Vanden Braber N, Rodriguez-Estrada MT, Garcia NA, Borsarelli CD, and Montenegro MA (2014) Gum arabic microcapsules as protectors of the photoinduced degradation of riboflavin in whole milk. Journal of Dairy Science 97: 5328–5336. http://dx.doi.org/10.3168/ jds2013-7886.
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Riboflavin: Properties and Determination
Cardoso DR, Libardi SH, and Skibsted LH (2012) Riboflavin as a photosensitizer, effects on human health and food quality. Food & Function 3: 487–502. Choe E, Huang R, and Min DB (2005) Chemical reactions and stability of riboflavin in foods. Journal of Food Science 70: R28–R36. De Leenheer AP, Lambert WE, and van Bocxlaer JF (2000) Modern chromatographic analysis of vitamins, 3rd ed. New York: Marcel Dekker, Inc. Eitenmiller RR, Landen Jr. WO Jr., and Ye L (2007) Vitamin analysis for the health and food sciences, 2nd ed. Boca Raton, FL: CRC Press. Friedrich W (1988) Vitamin B2: riboflavin and its bioactive variants. In: Vitamins. New York: Walter de Gruyter Chapter 7. Huvaere K and Skibsted LH (2014) Flavonoids protecting food and beverages against light. Journal of the Science of Food and Agriculture 95: 20–35. http://dx.doi.org/ 10.1002/jsfa.6796. Sheraz MA, Kazi SH, Ahmed S, Quadeer K, Khan F, and Ahmed S (2013) Multicomponent spectrometric analysis of riboflavin and photoproducts and their kinetic applications. Central European Journal of Chemistry 12: 635–642.
Souci SW, Fachmann W, and Kraut H (2008) Food composition and nutrition tables, 7th ed. Stuttgart: Medpharm Compiled by Dr. Eva Kirchhoff. The Merck Index: an encyclopaedia of chemicals, drugs and biologicals, 15th ed. New Jersey: The Royal Society of Chemistry. De Horwitz W and Latimer GW (eds.) The Official Methods of Analysis of AOAC International, 19th ed. Rockville, MD: AOAC International vol. ll. Zand N, Chowdhry BZ, Pullen FS, Snowden MJ, and Tetteh J (2012) Simultaneous determination of riboflavin and pyridoxine by UHPLC/LC-MS. Food Chemistry 135: 2743–2749.
Relevant Websites www.ncbi.nlm.nih.gov/Pubchem.Compound/Riboflavin. www.ncbi.nlm.nih.gov/Pubchem.Substance/Riboflavin.
Physicochemical Characteristics Riboflavin, the prescribed name for vitamin B2 by the IUPAC– IUB, is the biologically active component of the prosthetic group of flavoproteins. Older designations are lactoflavin, ovoflavin, and uroflavin, which originated from the first isolation. It comprises a tricyclic, nitrogen-containing ring system, chemically defined as a substituted isoalloxazine with a ribitol side chain at N10. With the systematic nomenclature, the molecule is named as 7.8-dimethyl-10-(1-D-ribityl)-benzo[g]pteridin-2,4dion. The empirical formula is given as C17H20N4O6 with an MW of 376.36 (Figure 1). The substance is a yellow to orangeyellow powder and crystallizes in sharp, optically active needles, melting under decomposition at 275–282 C. The optical rotation is ½a25 D ¼ 112 C to 122 C (c ¼ 50 mg in 10 ml 0.02 N alcoholic NaOH). Riboflavin is sparingly soluble in water with a yellow-green fluorescence, very sparingly soluble in ethanol and insoluble in other organic solvents. It is readily soluble in dilute alkali, but rapidly deteriorated, being accelerated by light. In neutral or acid solutions, it is more stable even against heat and oxygen. Nevertheless, all operations with riboflavin solutions should be handled in the dark or at red light. Light sensitivity of the flavins has also consequences for the stability of vitamin B2-containing foods. In milk stored under day light, the riboflavin content is dropping off by more than 80% within hours. Processing losses in foods are given with 20%. Light exposure of alkaline solutions decomposes riboflavin to lumiflavin (7,8,10-trimethylisoalloxazine); in neutral or acidic solutions, the blue fluorescent lumichrome (7,8dimelthylalloxazine) is formed (Figure 2). Riboflavin is found in free-form only in the retina of the eye, in whey, and in urine. In other biological systems and tissues, the predominant forms are flavin mononucleotide (FMN) or riboflavin phosphate and flavin adenine dinucleotide (FAD). Free riboflavin and FMN-monosodium salt are the most important commercial products. FMN and FAD are effective as coenzymes or prosthetic groups in more than 100 enzymes in the plant and animal kingdom. The accepted designation for both compounds is incorrect, because FMN is not truly a nucleotide and FAD not a dinucleotide. The designations arose from the discovery of riboflavin and have been adopted ever since. Pure riboflavin was first isolated from yeast, egg white, and whey in 1933 (Kuhn and Wagner-Jauregg). Simultaneously, the so-called yellow coenzymes were discovered in yeast (Warburg and Christian), which could be identified as FMN. FMN is considerably more soluble in water than riboflavin. Its solubility in organic solvents as well as its stability against heat and light exposure is comparable to riboflavin. The flavoproteins FMN and FAD belong to the enzymatic class of oxidoreductases. The conjugated structure of the pteridine moiety of the basic molecule determines its coloring and
Encyclopedia of Food and Health
light absorbance in the UV and visible spectrum. Absorbance maxima of the flavins are listed in Table 1. The catalytic hydrogen transfer in the flavin enzymes takes place at both conjugated bound N1 and N5 atoms of the isoalloxazine molecule. The oxidized form of the flavoproteins, the flavoquinone, is thereby reversibly reduced to the colorless flavohydroquinone or leucobase (see Figure 1). The absorbance is lost, because double bonds are dissolved. As an intermediate product during this reaction, a sequinone radical is formed. During the redox process, the flavin molecule changes its conformation, which is planar in the oxidized and folded in a butterfly configuration in the reduced state. The characteristic yellow-green fluorescence of riboflavin and FMN in aqueous solutions exhibits maximal intensity at pH 6–7. The fluorescence of FAD is about 10–20% of that of riboflavin with the same spectral distribution but exhibiting a maximal intensity at pH 3. The fluorescence of flavins is quenched when bound to proteins. The irradiation products of the flavins are also fluorescing, and their spectrum is shifted to the blue (lumichrome) or green (lumiflavin) spectral range. For quantification of riboflavin, particularly the conversion to lumiflavin, measurement of its fluorescence intensity in chloroform solutions is used (ex 270 nm, em 418 nm).
Content and Reactions in Food Content Riboflavin belongs to the group of water-soluble vitamins and is generally referred to as vitamin B2. Riboflavin is an important compound of a healthy diet and is widely distributed in animal and vegetable foods. Protein-rich foods of animal origin are as rule considerable sources of this vitamin with good bioavailability. Particularly rich in riboflavin are offal, such as the heart, liver, and kidney, and eggs and dairy products, mostly as FMN and FAD. Of special importance for the dietary habits in Western countries are milk, dairy products, and meat, despite of a medium content of riboflavin. If those are omitted from the diet, problems may arise to cover an adequate vitamin B2 intake for human beings (Table 2). Furthermore, riboflavin is used as a safe coloring agent for some processed foods and pharmaceuticals and is included in nutrient supplements. Meat, fish, and dark green vegetables contain also significant concentrations of riboflavin. Milk and cheese are in agreement with their content of riboflavin products for which lightinduced quality deterioration is now widely recognized to be the result of riboflavin photosensitizing.
Stability of Riboflavin Riboflavin and its physiological compounds are relatively stable during thermal and nonthermal food processing and
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633
634
Riboflavin: Properties and Determination
O
O H3C
N NH
H3C
N
N
+ 2 [H] O
− 2 [H]
H3C
H N
H3C
N
N H
Ribit
P
NH
CH2 NH2
H C OH N
H C OH
O
P
Adenin
N
H C OH N H2C
O
P
O
P
CH2
N
O
Riboflavin
FMN (H2) OH
OH
FAD (H2) Figure 1 Riboflavin and its coenzyme forms.
CH3 H3C
N
H3C
N
N
O
H3C
N
H3C
N
N
O
N
N H O Lumiflavin (alkaline solution)
H O
Lumichrome (acidic solution)
Figure 2 Photolysis products of riboflavin.
storage, in the absence of light even at high temperature. But they are very unstable to light even at room temperature. Particularly, aqueous solutions of these substances are sensitive to UV and visible light, and they are degraded through a variety of reactions resulting in a number of photoproducts under aerobic and anaerobic conditions. The major photoproducts obtained through normal photolysis of riboflavin in aqueous solvents include formylmethylflavin, lumichrome, lumiflavin, and 2,3-butanedione. With multicomponent spectrometric methods, several further photoproducts are to be identified. Lumichrome is the major product under neutral or acidic pH; lumiflavin is formed in basic pH; 2,3-butanedione is the major volatile compound with a typical buttery odor. Lumichrome and lumiflavin are formed from unstable diradical excited triplet riboflavin by dealkylation; 2,3-butanedione is produced from the reaction between electrophilic singlet oxygen and electron-rich riboflavin. The kinetics of riboflavin photolysis under light exposure and in the pH range of 1–12 indicate that riboflavin has maximum photostability around pH 5–6.
Mostly destructive to riboflavin is exposure to light with wavelength of its absorption maximum at 450 nm.
Reactions of Riboflavin as a Photosensitizer Riboflavin and other flavins are efficient photosensitizers, inducing oxidative damage to light-exposed food and several food compounds. When exposed to light of wavelength 420–560 nm, the absorbed energy does excite riboflavin to triplet state. Riboflavin can accept or donate two hydrogen atoms and acts together with lumiflavin as efficient photosensitizer. These so-called sensitized photoreactions are typical for foods and essentially involve two pathways. Direct interaction of the excited sensitizer with food compounds initiates radical processes through electron transfer or hydrogen abstraction and is known as a type 1 photo-oxidation. This mechanism is favored under low oxygen pressure and is, for example, responsible for light-struck flavor formation in beer. Under aerobic conditions, excitation energy is preferably
Riboflavin: Properties and Determination
Table 1
635
Physicochemical data of flavines
Property
Riboflavin
Riboflavin-5-phosphate-Na (FMN-Na)
Molecular weight Melting point Solubility
376.36 278–282 C (decomposition) Readily soluble in dilute alkalis (decomposition) Sparingly soluble in water (70–100 mg l1, 27 C) Very sparingly soluble in ethanol (45 mg l1, 27 C) Insoluble in acetone, chloroform, ether, benzene 223, 267, 374, 444 nm (0.1 N HCl)
478.34 280–290 C (decomposition) Soluble in water at pH 7 (30 g l1, 25 C)
Absorbance (UV/ vis) Fluorescence Stability
Flavinadenindinukleotide-Na2 (FAD-Na2) 829.6 Soluble in water
Very sparingly soluble in ethanol
Yellow-green fluorescence of aqueous solutions at pH 6–7 (ex. 444 nm, em. ex. 400–500 nm, em. 530 nm 530–565 nm) In neutral and acidic medium relatively stable against heat and oxygen. Rapid photolysis by UV- and visible light to blue fluorescent lumichrome (acidic and neutral solutions) or yellow-green fluorescent lumiflavin (alkaline solutions). After reduction by dithionite, Zn-HCl, sodium hydrosulfite, etc., reversible conversion to leucoflavin (dihydroflavin)
Source: The Merck index: an encyclopaedia of chemicals, drugs and biologicals (2014) (15th ed.). New Jersey: The Royal Society of Chemistry; Eitenmiller, R. R., Landen Jr, W. O. and Ye, L. (2007). Vitamin analysis for the health and food sciences (2nd ed.). Boca Raton, FL: CRC Press.
Table 2
Riboflavin content of foods
Content (mg/ 100) ingested food
Milk and milk products
>3.00 2.50–3.00
0.30–0.50
0.20–0.30
0.15–0.20
Cereals
Vegetables and legumes
Fruits and nuts
Fish
Pig’s liver Beef liver, calf liver Beef kidney, calf kidney Pig’s kidney Pig’s heart
2.00–2.50 1.50–2.00 1.00–1.50 0.70–1.00 0.50–0.70
Meat and meat products
Camembert type, cheddar type, Danish blue type, parmesan Cream brie (50%), edam type, fresh cheese (skim milk) Fresh cheese (50%), gouda cheese (45%), yogurt (low fat) Cottage cheese, cow’s milk (fresh)
Wheat germs Wheat bran
Eggs, lamb
Eel, mackerel
Goose, pork (lean) veal
Beef (lean), chicken, duck, turkey
Oat flakes, whole meal flower (wheat, rye)
0.10–0.15
Green cabbage, soybean meal
Cashew nuts, hazelnuts, sallow thorn
Broccoli (boiled), mangold, soybean sprouts, spinach (boiled) Asparagus (boiled), Brussels sprouts
Avocado
Flounder, herring, pilchard, plaice Haddock, salmon
Peanuts (roasted)
Source: Souci, S. W., Fachmann, W. and Kraut, H. (2008). Food composition and nutrition tables (7th ed.), compiled by Dr. Eva Kirchhoff. Stuttgart: Medpharm.
transferred to oxygen to generate singlet oxygen, the pivotal intermediate in a type II photo-oxidation. This reaction is responsible for oxygenation of unsaturated fatty acids in vegetal oils causing high peroxide values and eventually rancidity, while attack of amino acid residues in dairy proteins may give rise to sulfury and potato-like off-flavors. Both
photo-oxidation pathways induced by triplet-excited riboflavin lead to premature aging or spoiling of foods and beverages. Photosensitizing of riboflavin causes production of reactive oxygen species such as super oxide anion, singlet oxygen, hydroxyl radical, and hydrogen peroxide. Radicals and reactive oxygen species accelerate the decomposition of food compounds
636
Riboflavin: Properties and Determination
as amino acids, proteins, lipids, carbohydrates, and vitamins and cause significant alterations of nutritional value of foods. Carbohydrates are less sensitive to riboflavin-photosensitized oxidation than proteins, lipids, or vitamins. Exposed to visible light, riboflavin acts as a potent photosensitizer in food and beverages presenting a risk for their stability. Decomposition of the nutrients depends on the wavelength and energy of light, the concentration of oxygen and riboflavin, and the presence of activating or inactivating compounds. Riboflavin is an excellent photosensitizer for singlet oxygen formation and a superb reactant for singlet oxygen, with the reaction rate of 1.01 1010/M/s. The triplet-excited state of riboflavin is relatively long-living and as a biradical a very powerful oxidant compared to ground state riboflavin, leading to direct oxidation of most types of biomolecules.
Riboflavin Losses in Dairy Products Due to the relative heat stability, only minor vitamin B2 losses occur during processing. Likewise, the pasteurization of milk in the absence of light causes losses of < 10%. On the other hand, exposure of dairy products to day light may result in a remarkable decrease of the riboflavin content being dependent on the surface exposed. In milk stored under daylight, the riboflavin content is dropping off by more than 80% within hours with both lipids and proteins being oxidized under photosensitized conditions. The photodegradation leads to the formation of carbonyl compounds such as pentanal, hexanal, heptanal, 2-nonenal, 2,4-nonadienal, and 2,4-decadienal all attributed to lipid photo-oxidation. The chemical reaction between methionine and singlet oxygen, which has been formed by riboflavin photosensitization, produces dimethyl-disulfide, which is responsible for the sunlight flavor of milk. In an actual study, it could be demonstrated that photodegradation of riboflavin in milk may be considerably reduced by the addition of small amounts of gum arabic microcapsules, avoiding large losses in the nutritional value of milk.
Riboflavin Losses in Beer and Cereals Light exposure of beer generates an obnoxious off-flavor after a complex interplay of light, excited riboflavin, isohumulones and sulfur-containing amino acids, peptides, and proteins. The radical chemical degradation path of these compounds results in 3-methylbut-2-ene-1-thiol, the main off-flavor compound that characterizes light-exposed beers with the trivial name of skunky thiol. Vitamin losses occur also during the milling process of cereals. White flour with a low extraction rate contains about one-third of riboflavin of the whole grain flour.
Photodegradation of Amino Acids and Vitamins The deactivation of triplet-excited state riboflavin by several food components leading to photodegradation of nutrients in food, beverages, and model solutions is described in actual studies. For instance, the amino acids cysteine, histidine, methionine, tyrosine, and tryptophan are rapidly photodegraded in aqueous solutions in the presence of triplet exited riboflavin. These amino acids are the primary targets of photochemical oxidation in proteins.
Both fat-soluble and water-soluble vitamins are sensitive to photo-oxidation as sensitized by riboflavin. The vitamins ascorbate, folate, thiamine, and pyridoxal phosphate deactivate triplet-excited riboflavin and are photodegraded as well as vitamins A and D3 and tocopherols in aqueous solutions. A good photostability in the presence of riboflavin was demonstrated for the vitamins biotin and niacin. Carotenoids seem to reduce the formation of triplet-excited riboflavin through an inner filter, which protects these substances against the riboflavin-induced photosensitizing damage.
How to Prevent Reactions Polyphenolic compounds are efficient antioxidants with strong free radical scavenging ability. It was demonstrated that the plant polyphenols rutin and catechin, several flavonoids, and phenolic compounds present in beverages quench the tripletexcited state of riboflavin and protect the beverages that contain these compounds from light-induced off-flavor formation.
Methods of Determination In earliest reports, the most frequently applied assays of flavins as vitamin B2 active components made use of the direct fluorimetric assay of the intact flavins or after hydrolysis to FMN or free riboflavin. Alternatively, the lumiflavin method based upon the conversion of the chloroform-insoluble riboflavin into chloroform-soluble lumiflavin after photodegradation in alkaline solution was used, despite a transformation rate of only 60–70%. Currently, microbiological assays and highperformance liquid chromatography (HPLC) procedures with enhanced sensitivity are widespread utilized (see also Table 3). In principle, riboflavin is extracted from dry materials by autoclaving in 0.1 N HCl for 30 min or on water bath for 1–2 h. By using acid extraction, flavins are released from their protein binding and FAD and FMN are converted to free riboflavin. In protein-rich materials, the acidic extraction may be followed by enzymatic hydrolysis for complete liberation of the flavins. It is of importance to carry out all operations relating to the extraction and quantitation of flavins and riboflavin under subdued light and in brown or dark vessels. An optimal extraction procedure applicable for microbiological or HPLC determination of thiamine and riboflavin has been worked out by the European Measurement and Testing Program. The steps of the extraction are detailed in autoclaving, incubation with takadiastase, and filtration. For biological samples, such as plasma/serum, the preferred extraction medium is 5–10% trichloroacetic acid, which is suitable for denaturing the relatively weak protein binding while keeping the phosphorylated forms intact.
Direct Fluorimetry Direct measurement of the native fluorescence of riboflavin may be applicable for pharmaceutical preparations or food samples with a relatively high vitamin content. After acid extraction, the remaining proteins are removed by precipitation at pH 4.5. Interfering substances are subsequently oxidized with potassium permanganate and the excess oxidant
Riboflavin: Properties and Determination
Table 3
637
Methods of riboflavin determination
Method
Principles
Specials
Detection limits
Direct fluorimetry Lumiflavin method Microbiological assay HPLC analysis
Fluorescence measurement of riboflavin at 440 (ex)/565 (em) nm Fluorescence measurement after irradiation at pH 10–12 (ex 450, em 513 nm) Turbidimetric measurement of bacterial growth response dependent on the B2 content Separation of flavins on RP-C18 materials with aqueous/ organic solvents and fluorimetric/UV detection
Conversion of FAD and FMN to riboflavin by acidic hydrolysis Lumiflavin is separated by chloroform extraction Interference of growth response with nutrients/food components Differentiated analysis of flavins, combined analysis with other B vitamins
0.1 mg B2/g sample
destroyed with hydrogen peroxide or sodium bisulfite. The fluorescence of the clear sample solution, commonly spiked with riboflavin as an internal standard, is registered by difference measurement before and after reduction of riboflavin with sodium bisulfite to the leuco form for correction by unspecific fluorescences (ex ¼ 440 nm; em ¼ 565 nm). The detection limit of this method is about 0.1 mg riboflavin per gram sample. The direct fluorimetry of flavins had been adopted by the US Pharmacopoeia for the riboflavin analysis in tablets and injections and by the AOAC for the riboflavin determination in foods, as in milk-based formulas.
Lumiflavin Method By this method, riboflavin is photodegraded in alkaline solution at pH 10–12 to the stronger fluorescent lumiflavin (ex ¼ 450 nm; em ¼ 513 nm). This reaction is specific for riboflavin since other flavins interfering with riboflavin fluorescence are not converted into lumiflavin. Lumiflavin is separated from riboflavin and other fluorescent substances by extraction with chloroform. The transformation into lumiflavin is not quantitative and ranges between 60% and 70%. Reliable results can actually only be obtained by comparison with a riboflavin standard under identical irradiation conditions for sample and standard solution. While the original lumiflavin method is sparsely used, the principal methodology was applied to HPLC analyses of the riboflavin content in foods with detection limits of 0.02 ng riboflavin per injection.
Microbiological Assay Besides bioassays, as chicken and curative rat growth tests, microbial assays belong to the first widely used test methods measuring the biological activity of preparations containing total vitamin B2. The most frequently used test organism is Lactobacillus casei subsp. rhamnosus (ATCC No. 7469). The sensitivity limit is indicated to be 0.5 ng riboflavin absolutely. Occasionally, other test organisms are proposed such as Enterococcus faecalis (ATCC No. 10100) and Leuconostoc mesenteroides (ATCC No. 9135) with enhanced sensitivity of 0.1 ng ml1 riboflavin. The microbiological assay with L. casei is approved
0.02 ng B2 0.1–0.5 ng B2 0.21 ng riboflavin 0.89 ng FMN 11.15 ng FAD 0.02 ng B2 (precolumn irradiation to lumiflavin)
by the AOAC International Methods for the riboflavin determination in vitamin preparations. Growth response of bacteria is proportional to the riboflavin content of the medium and measured turbidimetrically. Earlier techniques determined metabolically formed lactic acid by titration. However, comparisons of turbidimetry after 16 h incubation obtained results that agreed with those obtained by titration of lactic acid after 72 h. Thus, the turbidimetric measurement is at present preferred in view of its shorter incubation time and easier handling. The growth response of lactobacilli differs significantly between free riboflavin and FMN and FAD. Acidic or enzymatic treatment is therefore needed for releasing and converting the flavins into riboflavin as described under methods of determination, followed by adjustment of the extract to pH 4.5 before incubation. In case of enzymatic release using takadiastase or clarase, it has to be considered that some enzyme preparations contain variable traces of riboflavin. Starch, glycogen, free fatty acids, and other lipids and protein degradation products can interfere with the test by either stimulating or inhibiting the bacterial growth. Lipids in a relevant content can be removed either by filtration or by ether or petroleum ether extraction before hydrolysis. Proteins are precipitated at pH 4.5 and starch is split by the acidic or enzymatic hydrolysis step. As with the other analytic procedures, the microbiological test has also to be done under subdued light. Of importance is that the used glass vessels do not release any traces of alkali and should thus be cleaned with acid before use, because the flavins are alkali-sensitive. Hence, it is advisable to dissolve riboflavin standards in 0.02 M acetic acid. An optimal extraction procedure applicable for microbiological or HPLC determination of thiamin and riboflavin has been worked out by the European Measurement and Testing Program. The steps are detailed in autoclaving in 0.1 N HCl at 121 C for 30 min and adjusting thereafter to pH 4.0 with 4 M sodium acetate buffer (pH 6.1), addition of takadiastase (0.1 g g1 sample), incubation at 37–45 C (18 h), filtration, or centrifugation after cooling.
HPLC Procedures HPLC methods enable separate quantification of individual flavins or simultaneous analysis of riboflavin including FMN
638
Riboflavin: Properties and Determination
and FAD. A broad variety of HPLC techniques are described, varying the column material, mobile phase, separation mode, and detection system as well. Of the numerous analytic and technical variations communicated during the last 20 years, the separation techniques can be categorized as follows: Reversed phase C18 materials are utilized as stationary phases in most cases, combined with aqueous/organic mobile phases on the basis of water, methanol, acetonitrile with or without phosphate, or acetate buffer in an isocratic mode. Rarely, ion pair chromatography with sodium salts of hexane or heptanesulfonic acid in the mobile phase (ion interaction chromatography) was utilized; in some cases, the separation of riboflavin and flavins was accomplished with gradient elution technique. Ion-pair HPLC seems more favorable only when riboflavin is determined simultaneously with other B vitamins (e.g., B1, B6, niacin, folic acid, and B12), because this technique results in better peak shapes. Retention times of vitamin B2 active compounds are between 5 (FAD) and 20 (riboflavin) minutes. Fluorescence monitoring is preferred for detection; UV or visible absorbance detection at 270 or 446 nm is restricted to early HPLC analyses or measurements of pharmaceuticals and enriched foods. Limits of fluorescence detection at 450/520 nm (ex./em.) for the native flavins were given to 0.55 pmol (0.21 ng) riboflavin, 1.96 pmol (0.89 ng) FMN, and 14.19 pmol (11.15 ng) FAD. In an earlier HPLC method, the detection limit could be increased to 0.02 ng riboflavin/injection by irradiation of the sample extract and conversion of the extracted riboflavin to lumiflavin prior to HPLC separation. A specific and sensitive UHPLC/LC-MS method was developed for the simultaneous determination of riboflavin and pyridoxine in infant food. In principle, extraction techniques and sample cleanup suitable for HPLC analysis resemble those used for quantitation by the other methods discussed. Mostly, vitamin B2 active substances are released from the food matrix by autoclaving with 0.1 N mineral acid (HCl or H2SO4) followed by enzymatic digestion with papain, takadiastase, or claradiastase. Solid-phase cleanup procedures on C18 materials or Florisil are often used for support prior to injection. The extraction procedure is guided by the analytic aim, whether the total riboflavin content, that is, the sum of FAD, FMN, and free riboflavin, shall be determined or the differentiated quantitation of FAD and FMN in addition to free riboflavin is strived for. Treatment with potassium permanganate can be omitted, because possibly remaining extraneous fluorescent substances are chromatographically separated. The combined acid and enzymatic hydrolysis is advantageous particularly for foods with high starch or protein content to liberate the bound flavins. Occasionally, autoclaving with dilute mineral acid is incomplete, particularly for the conversion of FMN to riboflavin, as indicated by the appearance of an FMN peak in the following chromatogram. Additionally, FMN may be partially converted during acid hydrolysis to biologically active isomeric riboflavin phosphates, which are separated from FMN by HPLC analysis and remain neglected for the calculation of total riboflavin. The combination of acidic and enzymatic digestion is thus advisable prior to HPLC analysis for the determination of total riboflavin. When using this extraction procedure and
following HPLC analysis, the riboflavin content of various foods such as breakfast cereals, porridges, milk, and milk products correlated well with the microbiological assay using Lactobacillus casei. The simultaneous determination of FAD and FMN besides free riboflavin needs nondegradative techniques, such as extraction of flavins by methanol/dichloromethane followed by partitioning with citrate buffer (pH 5.5) or extraction with 6% formic acid containing 2 M urea. The HPLC separation and quantitation of the flavins was accomplished by using internal standards (7-ethyl-8-methyl-riboflavin, sorboflavin isoriboflavin, nicotinamide, and others). This analytic device enabled the flavin analysis (FAD, FMN, and riboflavin) of milk and dairy products, fruits and vegetables, meats, and cereal products. In many cases, HPLC methods were developed for the simultaneous or sequential determination of riboflavin and thiamin in foods. Both vitamins are extracted by a common procedure using acidic and enzymatic digestion. For biological samples, as blood or plasma/serum, the preferred extraction medium is 5–10% trichloroacetic acid, which is suitable for denaturing the relatively weak protein binding while keeping the phosphorylated forms intact. In this way, flavins in whole blood can be analyzed by isocratic RP-HPLC against an external standard. A compilation of usual HPLC procedures is listed in Table 4. An overview on methods of riboflavin determination can be taken from Table 3.
European Standard Method The European Technical Committee for Standardization (CEN/TC 275) specifies a method with the title “Foodstuffs: Determination of vitamin B2 by high performance liquid chromatography and fluorescence detection.” The document number is DIN EN 14152:2014, the publication date is 2014-08, the original language is German, and the responsible standards committee for food and agricultural products is NA 057-01-13 AA: Vitamins and Carotenoids. The ‘Deutsches Institut fu¨r Normung e.V. (DIN)’ is the responsible committee for the standardization in the field of food as well as in the agricultural sector. This method has been validated in two interlaboratory studies. The first study was for the analysis of samples of milk powder and pig’s liver ranging from 1.45 to 10.68 mg/100 g. The second study was for the analysis of samples of tube feeding solution, baby food, powdered milk, meal with fruits, yeast, cereal, and chocolate powder ranging from 0.2 to 87.1 mg/100 g. Vitamin B2 is the mass fraction of total riboflavin including its phosphorylated derivates. In this European norm, riboflavin is extracted from foods after acidification, followed by an enzymatic dephosphorylation. The quantification takes place with subsequent HPLC separation, fluorimetric detection, and extern standards.
See also: Chromatography: High-Performance Liquid Chromatography; Enzymes: Functions and Characteristics.
Riboflavin: Properties and Determination
Table 4
639
HPLC analysis of flavins
Food sample/matrix (compounds determined)
Detection (nm)
Detection limit
H2O/CH3OH/acetic acid ¼ 65/35/1 0.3 M KH2PO4/ CH3OH ¼ 83.3/16.7 (pH 2.9)
UV 270
10 ng
Fluorescence 470/525 ex. em.
m Bondapak C18
CH3OH/0.005 M phosphate buffer pH 7.0 ¼ 35/65
Fluorescence 440/520 ex. em.
FMN 15 nM FAD 20 nM Riboflavin 10 nM 80–96% recovery
Spherisorb ODS 2.5 mm
H2O/CH3OH 0.65/35
Fluorescence 445/525 ex. em.
20 pg
LC-18, 3 mm (Supelco)
14% acetonitrile in 0.1 M KH2PO4, pH 2.9
Fluorescence 450/530 ex. em.
FMN 2.5 nM FAD 3 nM Riboflavin 2.5 nM
2PLRP-S, 5 mm column
temperature 40 C
Acetonitrile/ 0.1% sodium azide in 0.01 M citratephosphate buffer, pH 5.5 gradient elution
Altex
Ultrasphere ODS 5 mm
H2O/
Extraction procedure
Column
Mobile phase
Dairy products (total riboflavin) Blood (FAD, FMN, riboflavin)
Disperse in water, clean up on C18 cartridge 10% TCA at 4 C (30 min), adding NaOAc-buffer, centrifugation
Biosil ODS5S C18 Hypersil ODS 5 mm
Cereals, various foods (thiamin and total riboflavin)
0.1 N HCl, autoclave (15 min) at 125 C, adjust to pH 4.0–4.5 (2 N NaOAc), incubation with claradiastase at 50 C (3 h), 50% TCA and heating at 90 C (15 min), adjust to pH 3.5 (2 N NaOAc), filtration 0.05 M (0.1 N) H2SO4, autoclave (20 min) at 121 C, adjust to pH 4.5 (2.5 M acetate buffer), incubation with claradiastase at 45 C (overnight); filtrated sample passed through C18 Sep-Pak, elution with 40–70% CH3 OH Homogenize with 6% formic acid, containing 2 M urea pass through C18 solid phase, elute with 10% formic acid/CH3OH ¼ 4/1 Homogenize with CH3OH/CH2Cl2 ¼ 9/ 10 mix with citrate-phosphate buffer, pH 5.5 (containing 0.1% sodium azide), centrifugation
Dairy þ meat products, fruits, vegetables, flour, baked products, beer, coffee (total riboflavin)
Dairy products (FAD, FMN, riboflavin) sorboflavin as internal standard Dairy products, raw and cooked meats, cereals (FAD, FMN, riboflavin) 7-ethyl-8methyl-riboflavin as internal standard
Fluorescence 450/522 ex. em. Fruits, vegetables (total riboflavin) CH3OH ¼ 60/40 þ 5 mM heptanesulfonic acid, pH 4.5 Potatoes, vegetables (total riboflavin) Serum, urine (riboflavin) isoriboflavin as internal standard
96–113% recovery 0.1 N HCl (30 min) at 100 C, incubation with amylase (38 C) Fluorescence 450/530 ex. em. 0.1 N HCl (30 min) at 121 C, incubation with takadiastase (45–50 C) Homogenize/mix with TCA (100 g l1), centrifugation, pass through SepPak C18 (serum)
0.2 ng/ inject. m-Bondapak C18 ROSIL C18 HL 5 mm
CH3OH/H2O ¼ 30/70
CH3OH/H2O/ CH3COOH ¼ 36.7/ 63.7/0.1
Fluorescence 450/510 ex. em. Fluorescence 450/530 ex. em.
0.1 ng/ inject. 10 mg l1
Source: De Leenheer, A. P., Lambert, W. E. and van Bocxlaer, J. F. (2000). Modern chromatographic analysis of vitamins (3rd ed.). New York: Marcel Dekker, Inc.; Ball, G. F. M. (2013). Water-soluble vitamin assays in human nutrition. New York: Springer; Eitenmiller, R. R., Landen, Jr. W. O. and Ye, L. (2007). Vitamin analysis for the health and food sciences (2nd ed.). Boca Raton, FL: CRC Press.
Further Reading Ball GFM (2005) Vitamins in foods: analysis, bioavailability and stability. Boca Raton, FL: CRC Press. Ball GFM (2013) Water-soluble vitamin assays in human nutrition. New York: Springer. Bitsch R (2003) Riboflavin – properties and determination. In: Caballero B, Trugo L, and Finglas PM (eds.) Encyclopaedia of food sciences and nutrition, 2nd ed., pp. 4983–4989. London: Academic Press.
Bitsch R and Bitsch I (2011) HPLC determination of riboflavin in fortified foods. In: Rychlik M (ed.) Fortified foods with vitamins, analytical concepts to assure better and safer products, pp. 103–110. Weinheim: Wiley-VHC. Boiero ML, Mandrioli M, Vanden Braber N, Rodriguez-Estrada MT, Garcia NA, Borsarelli CD, and Montenegro MA (2014) Gum arabic microcapsules as protectors of the photoinduced degradation of riboflavin in whole milk. Journal of Dairy Science 97: 5328–5336. http://dx.doi.org/10.3168/ jds2013-7886.
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Riboflavin: Properties and Determination
Cardoso DR, Libardi SH, and Skibsted LH (2012) Riboflavin as a photosensitizer, effects on human health and food quality. Food & Function 3: 487–502. Choe E, Huang R, and Min DB (2005) Chemical reactions and stability of riboflavin in foods. Journal of Food Science 70: R28–R36. De Leenheer AP, Lambert WE, and van Bocxlaer JF (2000) Modern chromatographic analysis of vitamins, 3rd ed. New York: Marcel Dekker, Inc. Eitenmiller RR, Landen Jr. WO Jr., and Ye L (2007) Vitamin analysis for the health and food sciences, 2nd ed. Boca Raton, FL: CRC Press. Friedrich W (1988) Vitamin B2: riboflavin and its bioactive variants. In: Vitamins. New York: Walter de Gruyter Chapter 7. Huvaere K and Skibsted LH (2014) Flavonoids protecting food and beverages against light. Journal of the Science of Food and Agriculture 95: 20–35. http://dx.doi.org/ 10.1002/jsfa.6796. Sheraz MA, Kazi SH, Ahmed S, Quadeer K, Khan F, and Ahmed S (2013) Multicomponent spectrometric analysis of riboflavin and photoproducts and their kinetic applications. Central European Journal of Chemistry 12: 635–642.
Souci SW, Fachmann W, and Kraut H (2008) Food composition and nutrition tables, 7th ed. Stuttgart: Medpharm Compiled by Dr. Eva Kirchhoff. The Merck Index: an encyclopaedia of chemicals, drugs and biologicals, 15th ed. New Jersey: The Royal Society of Chemistry. De Horwitz W and Latimer GW (eds.) The Official Methods of Analysis of AOAC International, 19th ed. Rockville, MD: AOAC International vol. ll. Zand N, Chowdhry BZ, Pullen FS, Snowden MJ, and Tetteh J (2012) Simultaneous determination of riboflavin and pyridoxine by UHPLC/LC-MS. Food Chemistry 135: 2743–2749.
Relevant Websites www.ncbi.nlm.nih.gov/Pubchem.Compound/Riboflavin. www.ncbi.nlm.nih.gov/Pubchem.Substance/Riboflavin.