45.1.

08
AOAC Official Method 970.65
Riboflavin (Vitamin B2)
in Foods and Vitamin Preparations
Fluorometric Method
First Action 1970
Final Action 1971

A. Apparatus

Photofluorometer.Use fluorometer suitable for accurately

measuring fluorescence of solutions containing riboflavin in concentrates of 0.050.2 g/mL. Input filter of narrow T range with
maximum ca 440 nm and output filter of narrow T range with
maximum ca 565 nm have been found satisfactory.
B. Reagents

(Do not shake standard solutions stored under toluene.)

(a) Riboflavin standard solutions.(1) Stock solution.100
g/mL. Dissolve 50 mg USP Riboflavin Reference Standard, previously dried and stored in dark in desiccator over P2O5, in 0.02N
CH3COOH to make 500 mL. (To facilitate solution, warm with ca
300 mL 0.02N CH3COOH on steam bath with constant stirring until
dissolved, cool, and add 0.02N CH3COOH to make 500 mL.) Store
under toluene at ca 10.
(2) Intermediate solution.10 g/mL. Dilute 100 mL stock
solution to 1 L with 0.02N CH3COOH. Store under toluene at ca
10.
(3) Working solution I.1 g/mL. Dilute 10 mL intermediate
solution to 100 mL with H2O. Prepare fresh for each assay. Use as
standard solution in 970.65D.
(4) Working solution II.0.1 g/mL. Dilute 10 mL intermediate
solution to 1 L with H2O. Prepare fresh for each assay. Use as
standard solution in 970.65E.
(b) Sodium hydrosulfite.High purity and stored to avoid undue
exposure to light and air. Check suitability as follows: To each of 2
tubes add 10 mL H2O and 1 mL standard riboflavin solution containing 20 g/mL, and proceed as in 970.65D with respect to addition
of CH3COOH, KMnO4 solution, and H2O2 solution. Then when 8
mg Na2S2O4 is added with mixing, riboflavin should be completely
reduced in 5 s.
(c) Extraction solution.Mix 300 mL methanol, 100 mL pyridine, 100 mL H2O, and 10 mL CH3COOH. (Proportionate amounts
may be prepared.)
C. Preparation of Sample Solution

(Throughout all stages, protect solutions from undue exposure to

light and keep at pH <7.0. Where directed to filter through paper,
use paper known not to adsorb riboflavin [ash-free papers have been
found satisfactory].)
Place measured amount of sample in suitable size flask and
proceed by one of following methods:
(a) For dry or semidry materials containing no appreciable
amount of basic substances.Add volume 0.1N HCl equal in mL
to 10 times dry weight sample in g; resulting solution must contain
0.1 mg riboflavin/mL. If material is not readily soluble, comminute
so that it may be evenly dispersed in liquid. Then agitate vigorously
and wash down sides of flask with 0.1N HCl.
Heat mixture in autoclave 30 min at 121123 and cool. If lumping
occurs, agitate until particles are evenly dispersed. Adjust, with
vigorous agitation, to pH 6.06.5 with NaOH solution; then imme-

diately add dilute HCl until no further precipitation occurs (usually

ca pH 4.5, isoelectric point of many proteins).
Dilute mixture to measured volume containing >0.1 g riboflavin/mL and filter through paper. (In case of mixture difficult to filter,
centrifuging and/or filtering through fritted glass, using suitable
analytical filter-aid, may often be substituted for, or may precede,
filtering through paper. Ash-free filter paper pulp and Celite Analytical Filter-Aid have been found satisfactory.) Take aliquot of clear
filtrate and check for dissolved protein by adding dropwise, first
dilute HCl, and if no precipitate forms, then, with vigorous agitation,
NaOH solution, and proceed as follows:
(1) If no further precipitation occurs, add, with vigorous agitation, NaOH solution to pH 6.8, dilute solution to final measured
volume containing ca 0.1 mg riboflavin/mL, and if cloudiness
occurs, filter again.
(2) If further precipitation occurs, adjust solution again to point
of maximum precipitation, dilute to measured volume containing
>0.1 g riboflavin/mL, and then filter. Take aliquot of clear filtrate
and proceed as in (1).
If riboflavin content of sample is so low that these requirements
cannot be met, concentrate clear filtrate obtained at ca pH 4.5 to
suitable volume with heat under reduced pressure. Filter if necessary
and proceed as in (1).
(b) For dry or semidry materials containing appreciable amounts
of basic substances.Adjust mixture to pH 5.06.0 with dilute HCl.
Add amount of H2O such that total volume liquid is equal in mL to
10 times dry weight sample in g. (Resulting solution must contain
0.1 mg riboflavin/mL.) Then add equivalent of 1.0 mL 10N
HCl/100 mL liquid and proceed as in (a), beginning with second
sentence.
(c) For liquid materials.Adjust pH to 5.06.0 with dilute HCl
or, with vigorous agitation, NaOH solution, and proceed as in (b),
beginning with second sentence.
(d) For concentrates, premixes, and multivitamin supplements.
Place measured amount of sample in flask and add volume extraction
solution equal in mL to 10 times dry weight sample in g; resulting
solution must contain 0.1 mg riboflavin/mL. If sample 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.
Reflux mixture 1 h and cool. If lumping occurs, agitate mixture
until particles are dispersed evenly. Dilute mixture to measured
volume with extraction solution and let any undissolved particles
settle, or filter or centrifuge, if necessary. Take aliquot of clear
solution and dilute with H2O to measured volume containing ca 0.1
g riboflavin/mL and filter if solution is not clear. Proceed with
determination, 970.65E.
D. Determination

To each of 4 tubes (or reaction vessels) add 10 mL sample

solution. (If fluorometer is type that requires tubular cuvets, all
reactions may be carried out in matched set of these cuvets.) To
each of 2 tubes add 1 mL standard riboflavin working solution I,
(a)(3), and mix, and to each of 2 remaining tubes, add 1 mL H2O
and mix. To each tube add 1 mL CH3COOH and mix; add, with
mixing, 0.5 mL 4.0% KMnO4 solution (volume may be increased
for sample solutions that contain excess of oxidizable material, but
0.5 mL in excess of that required to complete oxidation of foreign
material should be added). Let stand 2 min; then to each tube add,
with mixing, 0.5 mL 3.0% H2O2 solution; permanganate color must

be destroyed within 10 s. Shake vigorously until excess O is expelled. If gas bubbles remain on sides of tubes after foaming stops,
remove by tipping tubes so that solution flows slowly from end to
end.
In fluorometer, measure fluorescence (X) of sample solution containing 1 mL added standard riboflavin working solution I. Next,
measure fluorescence (B) of sample solution containing 1 mL added
H2O. Add, with mixing, 20 mg powdered Na2S2O4 to 2 tubes,
measure minimum fluorescence (C) within 5 s. Calculate on basis
of aliquots taken as follows:
mg Riboflavin/mL final sample solution =
[(B C)/(X B)] 0.10 0.001
[Value of (B C)/(X B) must be 0.66 and 1.5.]
Note: Quantity of Na2S2O4 appreciably >20 mg may reduce foreign
pigments and/or foreign fluorescing substances, thereby causing
erroneous results.
E. Alternative Determination

(Applicable to high potency samples.)

Add 10 mL sample solution to 2 cuvets. Add 10 mL working
standard solution II, (a)(4), to each of another set of 2 cuvets. Add
1 mL CH3COOH to each tube and mix. Measure fluorescence of
sample and standard solutions in fluorometer. Add, with mixing, 20
mg powdered Na2S2O4 to 1 tube each of standard and sample and
measure minimum fluorescence within 5 s. Calculate on basis of
aliquots taken as follows:
mg Riboflavin/mL final sample solution =
[(I Q)/(I Q)] (0.1 0.001)
where I and I = fluorescence intensities of sample and standard,
respectively, and Q and Q = fluorescences of sample and standard,
respectively, after Na2S2O4 addition.
References: JAOAC 23, 346(1940); 24, 413(1941); 25,
459(1942); 26, 81(1943); 27, 540(1944); 30,
392(1947); 31, 701(1948); 32, 108, 461(1949); 33,
88, 632(1950); 37, 770(1954); 43, 42(1960); 53,
542(1970).
CAS-83-88-5 (riboflavin)