45.1.09
—Dissolve 15 g HPO3 in 1 L
AOAC Official Method 981.15
Riboflavin in Foods
and Vitamin Preparations
Automated Method
First Action 1981
Final Action 1982
AACC–AOAC Method
[Applicable to products for which manual method, 970.65 (see
45.1.08), is applicable and which contain ³0.1 mg/g.]
A. Apparatus
(a) Automatic analyzer.—Technicon AutoAnalyzer II system
with flow scheme as in Figure 981.15 (Pulse Instrumentation (1992)
Ltd., 433 Birch Cresent, Saskatoon, SK, Canada S7N 2K2;
www.autoanalyzer.com), or equivalent. Prepare in accordance with
manufacturer’s directions.
(b) Fluorometer.—Technicon Fluoronephelometer with
LY-013-B008-01-C flow cell or Aminco Fluoro-Colorimeter with
J4-7413 flow cell and J4-7125 4 watt lamp, or equivalent.
(c) Filters.—Primary: with band pass of 50% T at 430 nm
(Technicon 518-7004); secondary: sharp cut 37% T at 513–527 nm
and 80% T at 557 nm (Technicon 518-7032).
(d) Glass electrode.—Small diameter for adjusting pH in
volumetric flask.
(e) Filter medium.—Glass fiber paper, Whatman GF/A, or
equivalent, shown not to adsorb riboflavin.
B. Reagents
( a ) We t t i n g a g e n t . — 3 0 % a q u e o u s B r i j 3 5 s o l u t i o n
(Aldrich-Sigma, Cat. No. B4184).
Figure 981.15. Flow diagram for automated determination of riboflavin.
(b) Metaphosphoric acid solution.
H2O and adjust pH to 1.9 with NaCH3COO solution. Add 30 g NaCl
and 1 mL wetting agent, and filter. Prepare fresh weekly.
(c) So dium ac e tate so lu tion.—1.25M. Dis solve 345 g
NaCH3COO×3H2O in and dilute to 2 L with H2O.
(d) Metaphosphoric acid buffer.—pH 4.3. Dissolve 15 g HPO3 in
1 L H2O and adjust to pH 4.3 with crystalline NaCH3COO. Prepare
fresh daily.
(e) Wash solution.—Adjust pH of 2 L 0.1M HCl to 4.3 with
1.25M NaCH3COO (ca 240 mL). Add pH 4.3 HPO3 buffer, (d), to
make 4 L and add 4 mL wetting agent. Prepare fresh daily.
(f) Dialysate receiving solution.—Mix equal volumes wash
solution, (e), and HPO3 solution, (b).
(g) So dium ac e tate so lu tion.—0.4%. Dis solve 4.0 g
NaCH3COO×3H2O and dilute to 1 L with H2O.
(h) Potassium permanganate solution.—1%. Dissolve 2.0 g
KMnO4 in 200 mL H2O by magnetic stirring 30 min, and filter
through glass fiber GF/A filter. Store in dark bottle and prepare fresh
weekly.
(i) Sodium bisulfite solution.—Dissolve 1.1 g NaHSO3 in 50 mL
pH 7 phosphate buffer (4.55 g KH2PO4 and 9.45 g Na2HPO4/L). In
use, adjust NaHSO3 solution concentration visually in continuous
flow system so that there is ca 10% excess over that required to
reduce KMnO4. Avoid large excess of NaHSO3. Once NaHSO3 level
is determined, it is not necessary to repeat adjustment unless pump
tubes are changed.
(j) Sodium hydrosulfite solution.—Dissolve 2.0 g Na2S2O4 in
100 mL 0.4% NaCH3COO solution. Keep solution in ice bath when
in use (stable ca 2 h).
( k ) R i b o f l a v i n s t a n d a rd s o l u t i o n s . — ( 1 ) St o c k
solution.—50 mg/mL. Dissolve 50.0 mg riboflavin (stored in dark in
desiccator) in ca 800 mL 0.1M HCl (overnight magnetic stirring
required) and dilute to 1 L with 0.1M HCl. Store under toluene in dark.
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Prepare fresh every 2 weeks. (2) Intermediate solution.—10 mg/mL.
Dilute 40 mL stock solution to 200 mL with 0.1M HCl. (3) Working
solution.—1 mg/mL. Dilute 10 mL intermediate solution to 100 mL
with 0.1M HCl. Prepare solutions (2) and (3) fresh daily. Prepare
standards to accompany test solutions by pipetting 1, 3, 5, 10, and
15 mL working standard solution into 100 mL amber volumetric
flasks and adding 49, 47, 45, 40, and 35 mL 0.1M HCl, respectively, to
ob tain con cen trates of 0.01, 0.03, 0.05, 0.10, and 0.15 mg
riboflavin/mL in determination. Autoclave and treat samples and
standards alike.
C. Preparation of Test Sample
Grind representative portion to pass No. 40 sieve.
D. Determination
Transfer accurately weighed test portion (1.5 g maximum)
containing ca 10 mg riboflavin to 100 mL amber volumetric flask,
and add 50.0 mL 0.1M HCl, washing down sides of flask and
dispersing test portion. Cover flasks with foil and autoclave 30 min
at 121°C. Exhaust autoclave slowly to prevent bumping. Let cool to
room temperature. (Solutions may be stored at this point.) For
products which spatter during hydrolysis, use Erlenmeyer and
transfer to volumetric flask after autoclaving.
Add predetermined aliquot of 1.25M NaCH3COO with swirling.
(Reproducible dispensing unit is convenient for this step. Adjust
unit so that when aliquot of 1.25M NaCH3COO solution is added to
50.0 mL 0.1M HCl, pH is 4.3 ± 0.1 [ca 6.0 mL].) Add ca 35 mL
pH 4.3 HPO3 buffer. Check pH with meter and adjust any test sample
hydrolysate differing from standards by ³0.1 pH unit. (If solution is
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also to be used for thiamin determination, add 5.0 mL Takadiastase
solution [5% in pH 4.3 CH3COOH] and incubate overnight at 37°C
before diluting to volume. Enzyme hydrolysis will not affect
riboflavin determination, but enzyme solution must also be added to
standards because enzyme contains small amount of riboflavin.)
Dilute solutions to volume with pH 4.3 HPO3 buffer solution, add
drop of wetting agent, and filter through glass fiber paper.
Pump high standard solution (0.15 mg/mL) through system and
set recorder pen at 100% with standard calibration adjustment on
fluorometer. Aspirate and pump set of standards and test portion
filtrates through system. Use one 0.10 mg/mL standard with every
series of 20 test solutions to correct for any drift. If test solution is
more concentrated than highest standard, dilute with wash solution
to bring peak height into range of standards. After all test solutions
have been run, replace NaCH3COO solution with Na2S2O4. Let run
to stable baseline, adjust to original baseline, and resample filtrates
to obtain corresponding blanks.
Standards provide linear standard curve passing through origin.
Subtract peak height of blank from peak height of test solution and
determine concentration, C, mg/mL, from standard curve.
Riboflavin/100 g, mg = C ´ 10/W
where W = g test portion.
References: J. Agric. Food Chem. 23, 815(1975).
JAOAC 62, 1041(1979).
CAS-83-88-5 (riboflavin)
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