GB 5413.12-2010 English PDF (GB5413.12-2010)

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GB 5413.12-2010: National food safety standard -- Determination of vitamin B2 in foods for infants and young children, milk and milk products
GB 5413.12-2010
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard
Determination of Vitamin B2 in Foods for Infants and
Young Children, Milk and Milk Products
ISSUED ON. MARCH 26 2010
IMPLEMENTED ON. JUNE 1, 2010
Issued by. Ministry of Health of the People’s Republic of China
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Normative References ... 4 
3 Principle ... 4 
4 Reagents and Materials ... 4 
5 Apparatus and Instruments ... 5 
6 Analytical Steps ... 6 
7 Expression of Results ... 7 
8 Precision ... 7 
9 Others ... 7 
Appendix A (Informative) Liquid Chromatogram of Vitamin B2 Standard
Solution ... 8 
Foreword
This Standard replaces GB/T 5413.12-1997 Milk Powder and Formula Foods for
Infant and Young Children - Determination of Vitamin B2 Content.
Compared with GB/T 5413.12-1997, this Standard mainly has the following changes.
--- Modify the name of Standard into Determination of Vitamin B2 in Foods for
Infants and Young Children, Milk and Milk Products.
--- Delete the method- fluorescence spectrophotometry.
--- Modify the structure of original standard.
--- In the mixed enzyme solution, ADD acid phosphatase, so as to resolve the
sample determination that riboflavin phosphate is added as the enhancer of
Vitamin B2.
--- In the sample treatment method, ADD the notice of “Avoid direct irradiation of
strong light”.
--- External standard quantification adopts multiple points standard curve method.
--- In the result calculation, CLARIFY that the content of Vitamin B2 in sample is
measured based on riboflavin.
--- ADD Appendix A – Liquid Chromatograms of Standard Solution.
This Standard’s Appendix A is informative.
The historical editions replaced by this Standard are as follows.
--- GB 5413-1985 and GB/T 5413.12-1997.
National Food Safety Standard
Determination of Vitamin B2 in Foods for Infants and
Young Children, Milk and Milk Products
1 Scope
This Standard specifies the method for determination of Vitamin B2 in foods for infants
and young children, milk and milk products.
This Standard is applicable to the determination of Vitamin B2 in foods for infants and
young children, milk and milk products.
2 Normative References
The following documents are essential to the application of this document. For the
dated documents, only the versions with the dates indicated are applicable to this
document; for the undated documents, only the latest version (including all the
amendments) are applicable to this standard.
3 Principle
In hydrochloric acid, sample is thermostat-hydrolysis and enzymolysis; then it is
separated through C18 reversed phase chromatographic column. Use fluorescence
detector (Ex. 462 nm; Em. 522 nm) to detect, and quantify with external standard
method.
4 Reagents and Materials
Unless otherwise specified, purity of all reagents used in this method is analytically
pure, and water is the first-graded water specified in GB/T 6682.
4.1 Hydrochloric acid.
4.2 Sodium acetate trihydrate.
4.3 Glacial acetic acid.
5.2 Autoclave.
5.3 pH meter. Precision is 0.01.
5.4 Tissue grinder.
5.5 0.45 µm micropore aqueous phase filter membrane.
5.6 Balance. sensitivity of 1 mg and 0.1 mg.
6 Analytical Steps
6.1 Treatment of sample
Weigh 5~10 g of sample (accurate to 0.01 g) (if necessary, the sample can be ground
in the grinder; the sample contains more than 5µg of Vitamin B2) in a 100 mL conical
flask; then add 60 mL of 0.1 mol/L hydrochloric acid solution (4.7). Shake to mix well.
Seal it with cotton stopper and Kraft paper; then transfer it into an autoclave; keep it at
121°C for 30 minutes. Take it out after it is cooled to below 40°C. Shake slightly for a
few times. Use 2.0 mol/L sodium acetate solution (4.10) to adjust the pH value to
about 4.0; add 2.0 ml of mixed enzyme solution (4.11). Shake it to mix well. Keep it in
an incubator at 37°C for overnight. Transfer the enzymolysis solution to a 100 mL
volumetric flask; use water to fix the volume to mark. Filter it through quantitative filter
paper; take the filtrate then filter through 0.45µm membrane (5.5); store the filtrate for
later use.
Note. During the operation, irradiation of strong light shall be avoided.
6.2 Determination
6.2.1 Reference chromatography conditions
Chromatographic column. C18 reversed phase chromatographic column (particle
diameter 5µm, 250mm×4.6 mm) or other columns with the same performance.
Mobile phase. 0.05 mol/L sodium acetate solution (4.9) methanol (4.4) = 65+35.
Flow rate. 1.0 mL/min.
Detection wavelength. excitation wavelength. 462 nm; emission wavelength. 522 nm.
Injection volume. 20µL.
6.2.2 Drawing of standard curve
Sequentially conduct the chromatography determination to the series of standard
working solutions of Vitamin B2 (4.12.3) (see Figure A.1 of Appendix A for the standard