GB 1903.44-2020 PDF English
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GB 1903.44-2020: National food safety standard - Food Nutritional Fortification Substance - Hydroxocobalamin
---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GB1903.44-2020
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Food
Nutritional Fortification Substance - Hydroxocobalamin
ISSUED ON: SEPTEMBER 11, 2020
IMPLEMENTED ON: MARCH 11, 2021
Issued by: National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
1 Scope ... 3
2 Molecular Formula, Structural Formula and Relative Molecular Mass ... 3
3 Technical Requirements ... 4
Appendix A Test Methods ... 5
Appendix B Reference Infrared Spectrum and Chromatogram of
Hydroxocobalamin ... 15
National Food Safety Standard – Food
Nutritional Fortification Substance – Hydroxocobalamin
1 Scope
This Standard is applicable to the food nutritional fortification substance
hydroxocobalamin, which is produced by the fermentation method of Ensifer
adhaerens through purifying and drying.
2 Molecular Formula, Structural Formula and Relative
Molecular Mass
2.1 Molecular formula
C62H89CoN13O15P
2.2 Structural formula
2.3 Relative molecular mass
1346.38 (according to 2018 international relative atomic mass)
Appendix A
Test Methods
A.1 General rules
Unless otherwise specified, only reagents confirmed to be analytically pure and Class-
1 water specified in GB/T 6682 are used in the analysis. The standard titration solutions,
standard solutions for impurity determination, preparations and products that are used
in the test methods shall be all prepared in accordance with the provisions of GB/T 601
and GB/T 602, when other requirements are not specified. The used solution refers to
an aqueous solution when it is not specified which solvent is used for preparation.
A.2 Identification test
A.2.1 Reagents and solutions
A.2.1.1 Potassium bromide.
A.2.1.2 Potassium pyrosulfate.
A.2.1.3 Hydrochloric acid.
A.2.1.4 Glacial acetic acid.
A.2.1.5 Sodium acetate.
A.2.1.6 Phenolphthalein indicator solution: 0.1g of phenolphthalein is dissolved in 95%
ethanol and make a constant volume to 100mL.
A.2.1.7 Sodium hydroxide solution: 2mol/L. Take 40g of sodium hydroxide; dissolve in
water and make constant volume to 500mL.
A.2.1.8 Acetic acid solution: 1mol/L. Pipette 58.8mL of glacial acetic acid in a 1L
volumetric flask and make constant volume by water.
A.2.1.9 1-nitroso-2-naphthol-3,6-disulfonate sodium solution: Dissolve 1g of 1-nitroso-
2-naphthol-3,6-disulfonate sodium in an appropriate amount of water; transfer to a
100mL volumetric flask and make constant volume with water.
A.2.2 Infrared spectra analysis
The infrared spectra analysis is performed according to 5.2.2 of GB/T 6040-2002. The
infrared absorption spectrum of the specimen shall be consistent with the standard
spectrum of hydroxocobalamin hydrochloride. The standard infrared spectrum of
hydroxocobalamin hydrochloride is shown in Figure B.1 of Appendix B.
A.3.2.8 Standard solution: According to the purity conversion, accurately take an
appropriate amount of hydroxocobalamin hydrochloride standard product to make it
contain hydroxocobalamin hydrochloride 0.02g, accurate to 0.0001g. And use the
diluent (A.3.2.7) to dilute it into a 200mL volumetric flask; then make constant volume
and use it as a standard solution. Filter it by a 0.45μm microporous membrane before
use. Parallel specimens are required.
A.3.2.9 Specimen solution: Accurately take 0.02g (calculated according to the sum of
the moisture and residual solvent) of the specimen, accurate to 0.0001g; and use the
diluent (A.3.2.7) to dissolve and make constant volume to 200mL as the specimen
solution; filter with 0.45μm microporous membrane before use. Parallel specimens are
required.
A.3.3 Apparatus
A.3.3.1 Electronic balance with a sensitivity of 0.1 mg.
A.3.3.2 High performance liquid chromatograph.
A.3.4 Reference chromatographic conditions
A.3.4.1 Detector: UV detector or diode array detector.
A.3.4.2 Detection wavelength: 351nm.
A.3.4.3 Chromatographic column: Take two columns of alkoxy silica gel (column length
10cm, inner diameter 4.6mm, large pores 2μm, mesopores 13nm) with extremely high
purity as fillers connected in series or equivalent columns.
A.3.4.4 Column temperature: 30°C.
A.3.4.5 Sampling volume: 20μL.
A.3.4.6 Mobile phase: A is methanol, B is buffer solution (A.3.2.6), and C is water. The
gradient elution conditions are shown in Table A.1.
- peak area of standard solution;
CU – concentration of hydroxocobalamin in the specimen solution, in mg/mL;
- molar mass of hydroxocobalamin hydrochloride, 1382.8, in g/mol.
A.3.8 Precision
The absolute difference between two independent determination results obtained
under repeatability conditions shall not exceed 2% of its arithmetic mean.
A.4 Determination of related substances (by anhydrous and solvent-free)
A.4.1 Reagents and solutions
A.4.1.1 Methanol: chromatographically pure.
A.4.1.2 Sodium dihydrogen phosphate (NaH2PO4·12H2O).
A.4.1.3 Phosphoric acid.
A.4.1.4 Buffer solution: Take 15.6g of sodium dihydrogen phosphate; pour it into a
1000mL volumetric flask through a funnel; add a small amount of water to dissolve;
and add water to the mark. Shake well, filter through 0.45μm microporous membrane;
adjust the pH to 3.0 with 1:100 phosphoric acid solution; and put it in an ultrasonic
water bath to degas for 10min.
A.4.1.5 Specimen dissolving solution: Accurately measure 820mL of water filtered
through a 0.45μm microporous filter membrane; take 100mL of buffer solution (A.4.1.4)
and 80mL of methanol to mix; shake well; and put it in an ultrasonic water bath to
degas for 10min.
A.4.1.6 System suitability solution: 0.75mg/mL. After conversion of purity, accurately
take an appropriate amount of hydroxocobalamin hydrochloride standard product
(A.3.2.4), and dissolve it by the specimen dissolving solution (A.4.1.5) to a 50mL
volumetric flask; make the concentration is 0.75mg/mL as the system suitability
solution.
A.4.1.7 Quantitative solution: 7.5μg/mL. Respectively accurately pipette 0.5mL of the
system suitability solution (A.4.1.6); and use the specimen dissolving solution (A.4.1.5)
to dissolve and make constant volume to a 50mL volumetric flask.
A.4.1.8 Limited solution: 0.75μg/mL. Accurately pipette 5mL of a quantitative solution
(A.4.1.7); and dissolve with the specimen solution (A.4.1.5) and make constant volume
to a 50 mL volumetric flask as the limited solution.
A.4.1.9 Specimen solution: 0.75mg/mL. Accurately take 0.0375g (after calculating
A.5.4 Reference chromatographic conditions
A.5.4.1 Chromatographic column: Capillary column (30m×0.53mm×3.0μm, the fixative
solution is 6% cyanopropyl phenyl-94% polydimethylsiloxane), or equivalent column.
A.5.4.2 Column temperature: 40°C, keep 13min.
A.5.4.3 Detector temperature: 250°C; inlet temperature: 140°C.
A.5.4.4 Carrier gas: N2; column flow: 4mL/min; air flow: 400mL/min; hydrogen flow:
60mL/min; makeup gas flow: 25mL/min; split ratio: 2:1.
A.5.4.5 Sampling mode: Headspace injection, with an injection volume of 1.0mL.
A.5.4.6 Conditions of headspace sampler: Headspace bottle equilibrium temperature
80°C, equilibrium time 60 min; temperature of quantitative loop 100°C; temperature of
transfer line 100°C.
A.5.5 System suitability test
Carry out the system suitability test before the specimen test; use the acetone standard
use solution (A.5.2.2) for 5 consecutive injections for repeatability test; and calculate
the relative standard deviation (RSD) of the peak area. The relative standard deviation
(RSD) of repeatability test is required to be no more than 10.0%. At the same time,
record the number of theoretical plates of the acetone peak in the first injection of
acetone standard use solution (A.5.2.2). The number of theoretical plates shall be no
less than 5000. After the conditions are met, the normal test of the specimen is carried
out; if it is not met, the system suitability test is continued after the cause is found out,
and the specimen test is carried out after passing the system suitability test. Take the
acetone standard use solution (A.5.2.2) for injection for each 5 batches of specimens
and before the end of the test; and calculate it together with the peak area of the first
5-needle system suitability and the acetone standard peak area confirmed each time
in the middle. The relative standard deviation (RSD) of the repeatability test is no more
than 10.0%; and the number of theoretical plates of the acetone peak in the acetone
standard use solution (A.5.2.2) shall be no less than 5000. If the above conditions are
not met, the test data in between is invalid. Re-test the system suitability, and re-test
after the system suitability is qualified. The system suitability is valid for 48h.
A.5.6 Analysis steps
After the system applicability 5-needle repeatability is qualified, one needle of water
sample is injected; and then 2 needles of reference solution (A.5.2.4) and 2 needles of
specimen solution (A.5.2.3) are injected in sequence. The prepared reference solution
(A.5.2.4) and specimen solution (A.5.2.3) are equilibrated at 80°C for 60min; and 1.0
mL of headspace gas is injected into the sample. Measure under the above
chromatographic conditions and record the chromatogram.
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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