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GB 5009.85-2016

Chinese Standard: 'GB 5009.85-2016'
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Standard ID GB 5009.85-2016 (GB5009.85-2016)
Description (Translated English) Determination of riboflavin in foods
Sector / Industry National Standard
Classification of Chinese Standard X09
Date of Issue 2016-12-23
Date of Implementation 2017-06-23
Older Standard (superseded by this standard) GB 5413.12-2010; GB/T 5009.85-2003; GB/T 7629-2008; GB/T 9695.28-2008
Regulation (derived from) National Health and Family Planning Commission Notice No.17 of 2016

GB 5009.85-2016
National food safety standard -
Determination of vitamin B2 in foods
食品中维生素 B2的测定
Issued by. National Health and Family Planning Commission of the PRC;
China Food and Drug Administration.
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Principles ... 4 
3 Reagents and materials ... 4 
4 Instrument and equipment ... 6 
5 Analytical procedures ... 6 
6 Analysis results expression ... 7 
7 Precision... 8 
8 Others ... 8 
9 Principles ... 8 
10 Reagents and materials... 9 
11 Instrument and equipment ... 10 
12 Analytical procedures ... 11 
13 Analysis results expression ... 13 
14 Precision ... 13 
15 Others ... 13 
Appendix A Concentration calibration method of vitamin B2 standard solution
... 14 
Appendix B Liquid chromatogram of vitamin B2 ... 16 
This standard replaces GB/T 5009.85-2003 "Determination of riboflavin in
foods", GB/T 9695.28-2008 "Meat and meat products-Determination of vitamin
B2 content", GB/T 7629-2008" Determination of vitamin B2 in cereals "and GB
5413.12-2010" National food safety standard-Determination of vitamin B2 in
foods for infants and young children, milk and milk products".
As compared with GB/T 5009.85-2003, the main changes of this standard are
as follows.
-- The standard name is changed to "National food safety standard-
Determination of vitamin B2 in foods";
-- Add high performance liquid chromatography;
-- Delete microbiological method.
National food safety standard -
Determination of vitamin B2 in foods
1 Scope
This standard specifies the determination of vitamin B2 in foods.
The first method of this standard is high performance liquid chromatography
and the second method is fluorescence spectrophotometry, which apply to the
determination of vitamin B2 in various type of foods.
Method I. High performance liquid chromatography
2 Principles
The sample is hydrolyzed in dilute hydrochloric acid at constant temperature,
adjusted to pH 6.0-6.5; use papain and taka-amylase to carry out enzymolysis.
After filtered through constant-volume, the filter liquor is separated through
reversed phase column and detected by HPLC fluorescence detector. Use
external standard method to quantify.
3 Reagents and materials
Unless otherwise indicated, the reagents used in this method are analytical
pure, the water is the grade-1 water specified in GB/T 6682.
3.1 Reagents
3.1.1 Hydrochloric acid (HCl).
3.1.2 Glacial acetic acid (CH3COOH).
3.1.3 Sodium hydroxide (NaOH).
3.1.4 Sodium acetate trihydrate (CH3COONa • 3H2O).
3.1.5 Methanol (CH3OH). Chromatographically pure.
3.1.6 Papain. active unit ≥ 10 U/mg.
3.1.7 Taka-amylase. active units ≥ 100 U/mg, or similar performance.
10.2.5 Mixed enzyme solution. Accurately WEIGH 2.345 g of papain and 1.175
g of taka-amylase; ADD water to dissolve it; USE water to dilute it to 50 mL.
Prepare it before use.
10.2.6 Eluent. acetone-glacial acetic acid-water (5+2+9, volume ratio).
10.2.7 Potassium permanganate solution (30 g/L). Accurately WEIGH 3 g of
potassium permanganate; USE water to dissolve it and dilute it to 100 mL.
10.2.8 Hydrogen peroxide solution (3%). PIPETTE 10 mL of 30% hydrogen
peroxide, USE water to dilute it to 100 mL.
10.2.9 Sodium dithionite solution (200 g/L). Accurately WEIGH 20 g of sodium
dithionite, USE water to dissolve it and dilute it to 100 mL. This solution is
prepared before use and stored in an ice-water bath, valid for 4h.
10.3 Standard products
Vitamin B2 (C17H20N4O6, CAS number. 83-88-5). Purity≥98%.
10.4 Standard solution preparation
10.4.1 Vitamin B2 standard stock solution (100 μg/mL). PLACE the vitamin B2
standard in a vacuum desiccator or dryer with phosphorus pentoxide; after
drying for 24h, accurately WEIGH 10 mg (accurate to 0.1 mg) Vitamin B2
standard; ADD 2 mL of hydrochloric acid solution (1+1) to dissolve it in
ultrasound; immediately USE water to transfer it and dilute it to 100 mL. After
mixing it uniformly, TRANSFER it into a brown glass container; STORE it in a
4°C refrigerator with shelf life of 2 months. The standard stock solution requires
concentration correction before use. The method of correction sees Appendix
10.4.2 Vitamin B2 standard intermediate solution (10 μg/mL). Accurately
PIPETTE 10.00 mL of Vitamin B2 standard stock solution; USE water to dilute
it to 100 mL. STORE it in a 4°C refrigerator with shelf life of 1 months.
10.4.3 Vitamin B2 standard solution (1 μg/mL). Accurately PIPETTE 10 mL of
vitamin B2 standard intermediate solution; USE water to dilute it to 100 mL. This
solution is equivalent to 1.00 μg of vitamin B2 per ml. Store it in a 4°C refrigerator
with shelf life of 1 week.
11 Instrument and equipment
11.1 Fluorescence Spectrophotometer.
11.2 Balance. sensitivities are 1 mg and 0.01 mg.
According to riboflavin content in test sample TAKE out a certain volume of
sample extract (containing about 1 μg ~ 10 μg of vitamin B2) and vitamin B2
standard solution; PLACE it respectively into 20 mL graduated test tube with
cover; ADD water to 15 mL. ADD 0.5 mL of glacial acetic acid in each tube; MIX
it uniformly. ADD 0.5 mL of 30 g/L potassium permanganate solution; after
shaking it up, PLACE it for 2 min to get rid of impurities through oxidation. ADD
dropwise 3% hydrogen peroxide solution until the color of potassium
permanganate fades away. SHAKE out the tube vigorously to allow excess
oxygen to escape.
12.3 Adsorption and elution of vitamin B2
12.3.1 Vitamin B2 adsorption column
Use wet method to fill approximately 1 g of silica-magnesium adsorbent into the
column, accounting for 1/2 ~ 2/3 of the column length (about 5 cm) (use a small
group of absorbent cotton pads to absorb the lower end of the adsorption
column). Do not generate bubbles in the column; adjust flow rate to about 60
Note. USE equivalent commercial column.
12.3.2 Column and elution
After all the oxidized sample solution and standard solution pass through the
adsorption column, USE about 20 mL of hot water to rinse the impurities in
sample solution. USE 5 mL of eluant to elute vitamin B2 of the sample into 10mL
volumetric flask; then USE 3 mL ~ 4 mL of water to wash adsorption column;
the eluant is combined into the volumetric flask; USE water to dilute it to the
mark; WAIT for determination after mixing it uniformly.
12.4 Preparation of standard curve
Respectively, accurately pipette the standard solution of vitamin B2 0.3 mL, 0.6
mL, 0.9 mL, 1.25 mL, 2.5 mL, 5.0 mL, 10.0 mL, 20.0 mL (equivalent to 0.3 μg,
0.6 μg, 0.9 μg, 1.25 μg, 2.5 μg, 5.0 μg, 10.0 μg, 20.0 μg Vitamin B2) or pipette
the solution that the single point standard is similar to the sample content
according to the operation in 12.2 and 12.3.
12.5 Determination of sample solution
When excitation wavelength is 440 nm and emission wavelength is 525 nm,
measure the fluorescence value of sample tube and stan......
Related standard: GB 5009.91-2017    GB 5009.93-2017
Related PDF sample: GB 5009.84-2016    GB 5009.87-2016