This method is specified for the determination of vitamin B2 content in all foods by fluorometric method.
2. SAFETY
When using a hazardous reagent such as concentrated acid or alkali, gloves should be worn, eye protected, and the work done in a fume hood.
3. REFERENCES
3.1 Horwitz, W. (editor) Official Methods of Analysis of AOAC International, 17th ed., Vol. 2., Riboflavin Fluorometric Methods 970.65 Ch. 45 pp. 9-10, 2000.
3.2 Shah, J.J. Riboflavin. In: Methods of vitamin assay 4th ed. Edited by Jory Augustin et al. A Wiley Interscience publication, USA. 1985: 365 - 381.
4. DEFINITION
Riboflavin is 7,8-dimethyl-10-(1’-D-ribityl) isoalloxazine, a yellow-green, fluorescence and has the empirical formula C12H17N4O2. Riboflavin is the official name. The name vitamin B2 is frequently used, and vitamin G, lyochrome, ovoflavin, lactoflavin, uroflavin and hepatoflavin are historical names. Riboflavin can occur in food as free riboflavin and as two of its derivatives, flavin mononucleotide (FMN, riboflavin 5’-monophosphate) and flavin adenine dinucleotide (FAD). The richest natural sources of vitamin B2 are yeast, milk and milk products, meat, egg, legumes, fruits, fresh vegetables, and cereal grains.
5. PRINCIPLE
Since riboflavin may occur combined with proteins, the foodstuff has to be treated with enzyme or dilute acid to liberate the vitamin. Protein and other interfering substances are then precipitated at a pH of 6.0. After filtering, any protein remaining in solution may be further precipitated at a pH of 4.5.
Potassium permanganate is then added to the extract to remove interfering fluorescent substances, followed by the addition of hydrogen peroxide to remove excess permanganate. Fluorescence of the extract may then be measured in a fluorometer. A little sodium hydrosulphite is added to quench the fluorescence due to riboflavin and the fluorescence reading again taken. The remaining fluorescence is due to other interfering substances and should be subtracted from the initial fluorescent reading.
6. REAGENTS
6.1 Hydrochloric acid, 0.1N
Add 8.3 mL concentrated hydrochloric acid to 500 mL of deionized water and dilute to 1L with deionized water.
6.2 0.02 N CH3COOH
Add 1.14 mL glacial acetic acid to 500 mL of deionized water and dilute to 1 L with deionized water.
6.3 1 N sodium hydroxide 6.4 Glacial acetic acid
6.5 Potassium permanganate solution, 4%
6.6 Hydrogen peroxide solution, 3%
Dilute 30% H202 1:10 with water. Prepare just before use.
6.7 Sodium hydrosulphite (sodium dithionite).
Check suitability for use as follows:
a. To each of two tubes, add 10 ml water and 1 ml standard riboflavin solution containing 20 μg per ml.
b. Add 1 ml of glacial acetic acid to each tube and mix.
c. Add 0.5 ml of 4% potassium permanganate, mix, and allow to stand for 2 minutes. D
d. Add 0.5 ml 3% hydrogen peroxide; mix thoroughly. Red colour should disappear within 10 seconds.
e. Measure fluorescence in a fluorometer.
f. Add 8 mg of sodium hydrosulphite and mix.
g. Read solution again in fluorometer; riboflavin should be completely reduced in 5 seconds.
6.8 Riboflavin standard solutions.
a. Stock solution: dissolve, with heating, 100 mg pure riboflavin in 1 litre of 0.02N acetic acid. Store in amber bottle under toluene in refrigerator (5-10oC). Solution contains 100 μg riboflavin per ml.
b. Intermediate solution: dilute 10 ml stock solution to 100 ml with 0.02N acetic acid to give a concentration of 10 μg per ml. Store in amber bottle under toluene in refrigerator (5-10oC).
c. Working solution: dilute 10 ml intermediate solution to 100 ml with water. Prepare fresh for each assay. Solution contains 1 μg riboflavin per ml.
6.9 Extraction solution
Mix 300 mL methanol, 100 mL pyridine, 100 mL H2O, and 10 mL CH3COOH. (Proportionate amounts may be prepared.)
7. APPARATUS
7.1 Spectrofluorometer 7.2 Autoclave
7.3 pH-meter
7.4 Analytical balance 7.5 Erlenmeyer flask, 250 ml
7.6 Volumetric flask 10, 25, 50, 250, 500 and 1000 ml 7.7 Beaker 25 ml
7.8 Pipette
8. PROCEDURE
8.1 Sample extraction
Place measured amount of test portion in suitable size flask and proceed by one of following methods:
8.1.1 For dry or semidry products containing no appreciable amount of basic substances
(1) Weigh 1-5 g the sample into the Erlenmeyer flask
(2) Add 60 ml 0.1 N HCI (equal to >10 times dry weight test portion in g)
(3) Mix contents of flasks well so that the solids are dispersed in the liquid.
(4) Heat mixture in autoclave 30 min at 121-123oC and cool.
(5) Mix regularly to ensure that no lumping of solids occur.
8.1.2 For dry or semidry products containing appreciable amounts of basic substances
(1) Weigh 1-5 g the sample into the Erlenmeyer flask (2) Adjust mixture to pH 5.0-6.0 with dilute HCI.
(3) Add amount of deionized water such that total volume liquid is equal in ml to >10 times dry weight test portion in g.
Then add equivalent of 1.0 ml 10 N HCI/100ml liquid.
(4) Heat mixture in autoclave 30 min at 121-123oC and cool.
(5) Mix regularly to ensure that no lumping of solids occur.
8.1.3 For liquid products
(1) Weigh 1-5 g the sample into the Erlenmeyer flask
(2) Adjust pH to 5.0-6.0 with dilute HCI or, with vigorous agitation, NaOH solution.
(3) Add amount of deionized water such that total volume of liquid is equal in ml to >10 times dry weight test portion in g.
Then add equivalent of 1.0 ml 10 M HCI/100ml liquid.
(4) Heat mixture in autoclave 30 min at 121-123oC and cool.
(5) Mix regularly to ensure that no lumping of solids occurs.
8.1.4 For concentrates, premise, and multivitamin supplements.
(1) Place measured amount of test portion in flask
(2) Add volume extraction solution equal in ml to >10 times dry weight test portion in g. If test portion is not readily soluble, comminute so that it may be dispersed evenly in liquid.
Then agitate vigorously and wash down sides of flask with extraction solution.
(3) Reflux mixture 1 hour and cool.
(4) Mix regularly to ensure that no lumping of solids occurs.
8.2 Precipitation of interfering impurities
8.2.1 Adjust pH to 6.0-6.5 with 1 N sodium hydroxide.
Swirl the extract constantly during the addition of alkali to prevent local areas of high pH.
8.2.2 Add immediately 1 N hydrochloric acid until no further precipitation occurs (pH around 4.5).
8.2.3 Transfer quantitatively into a 100 ml amber volumetric flask and make up to the mark.
8.2.4 Filter solution.
8.2.5 To a 50 ml aliquot of the filtrate, add 1 N hydrochloric acid drop-wise until no precipitation of dissolved protein occurs.
8.2.6 Add 1 N sodium hydroxide with constant shaking to pH 6.8
8.2.7 Dilute the aliquot to 100 ml with water.
8.2.8 Filter solution again if necessary.
8.3 Determination
Tube No. S1 S2 S1+Std S2+Std
Sample assay (ml) 10 10 10 10
Std. Solution (1 ug/ml) - - 1 1
Deionized water (ml) 1 1 - -
Mix
Glacial acetic acid (ml) 1 1 1 1
Mix
4% KMnO4 (ml) 0.5 0.5 0.5 0.5
Mix and stand for 2 min.
3% H2O2 (ml) 0.5 0.5 0.5 0.5
Mix thoroughly (red permanganate color should disappear in 10 sec.) Shake vigorously until excess oxygen is expelled
Measure fluorescence Exc. 440 nm, Emiss. 565 nm.
[A] [A] [B] [B]
Sodium dithionite (mg) 20 20 20 20
Mix Measure fluorescence within 5 seconds [C] [C] [C] [C]
Note:
1. Check fluorometer between readings with the standard solution to ensure that it gives the reading that was initially obtained.
2. Quantity of Na2S2O4 appreciably > 20 mg may reduce foreign pigments and / or foreign fluorescing substances, thereby causing erroneous results.
9. CALCULATIONS
Riboflavin (mg/100 g) = A - C x 20 x 100 B - A W 1000 A = Fluorescence reading of test sample B = Fluorescence reading of standard C = Fluorescence reading of blank W = Sample weight (g)
10. ACCEPTANCE OF RESULTS
10.1 The sample results are acceptable if the control sample result is within the level of two standard deviations of the mean on QC chart.
10.2 Sample duplicate
where, d1 = duplicate 1, d2 = duplicate 2, and d1 > d2;
then, (d1-d2)x100 < 5%
d1
10.3 The standard addition or spiked recovery sample shall be between 90-110%.
11. APPENDIX
Since riboflavin is light sensitive and is most readily destroyed by light in the blue and UV regions, it is necessary to perform all operations in the absence of strong light, and to use amber glassware. Throughout all stages, pH of all solutions containing the vitamin must be below 7.0. Where directed to filter through paper, use paper known not to adsorb riboflavin [ash-free papers have been found satisfactory].
DETERMINATION OF THIAMIN AND RIBOFLAVIN