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GB 1903.52-2021: National Food Safety Standard - Food Nutrient Fortifier - Heme Chloride
GB 1903.52-2021
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Food Nutrient
Fortifier - Heme Chloride
ISSUED ON: SEPTEMBER 07, 2021
IMPLEMENTED ON: MARCH 07, 2022
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 ... 3 
Appendix A Inspection methods ... 5 
Appendix B Standard infrared spectrum of heme chloride ... 11 
Appendix C High performance liquid chromatogram of heme chloride standard
... 12 
National Food Safety Standard - Food Nutrient
Fortifier - Heme Chloride
1 Scope
This Standard applies to the food nutrient fortifier heme chloride that is made
by using glacial acetic acid method, enzymatic hydrolysis and acetic acid
combination method, with the blood and blood cell powder of qualified
quarantine animals as the main raw materials.
2 Molecular formula, structural formula and relative
molecular mass
2.1 Molecular formula
C34H32ClFeN4O4
2.2 Structural formula
2.3 Relative molecular mass
651.95 (according to the international relative atomic mass in 2018)
3 Technical requirements
3.1 Sensory requirements
Sensory requirements shall be in accordance with Table 1.
Table 1 – Sensory requirements
Item Requirement Inspection method
Color Blue or black
Appendix A 
Inspection methods
A.1 General provisions
Unless otherwise stated, only use confirmed analytical reagents and grade-3
water that is specified in GB/T 6682 during the analysis. The standard titration
solutions, standard solutions for impurity determination, preparations and
products, which are used in the test method, shall be prepared in accordance
with the provisions of GB/T 601, GB/T 602, and GB/T 603, when other
requirements are not specified. The used solution, if not indicated which solvent
is used, refers to aqueous solution.
A.2 Identification test
A.2.1 Reagents and materials
A.2.1.1 Nitric acid: analytically pure.
A.2.1.2 Sodium sulfide: analytically pure.
A.2.1.3 Sulfuric acid solution: Take 5 mL of concentrated sulfuric acid; slowly
add into about 80 mL of water; then, add water to make the volume up to 100
mL.
A.2.1.4 Hydrochloric acid solution: Take 50 mL of concentrated hydrochloric
acid; add water to dilute to make the volume 100 mL.
A.2.1.5 Ammonium thiocyanate solution: Dissolve 8 g of ammonium
thiocyanate in an appropriate amount of water; use water to dilute to 100 mL.
A.2.1.6 Pyridine sodium hydroxide solution: Dissolve 1.2 g of sodium hydroxide
in 200 mL of water; then, add 100 mL of pyridine and mix.
A.2.1.7 Ammonia solution: Take 400 mL of ammonia water; use water to dilute
to 1 000 mL.
A.2.2 Instruments and apparatuses
A.2.2.1 Water bath tank.
A.2.2.2 Analytical balance: Sensitivity is 0.1 g and 0.001 g.
A.2.3 Identification method
A.2.3.1 Color rendering test
A.2.3.1.1 Weigh 0.01 g (accurate to 0.001 g) of heme chloride sample; add 1
mL of sulfuric acid solution (A.2.1.3) and 1 mL of nitric acid (A.2.1.1) to dissolve;
evaporate to dryness in a water bath. Add 10 mL of hydrochloric acid solution
(A.2.1.4) to the residue to dissolve; add a few drops of ammonium thiocyanate
solution (A.2.1.5); shake well; the solution shall appear dark red.
A.2.3.1.2 Weigh 0.005 g (accurate to 0.001 g) of heme chloride sample; add 10
mL of pyridine sodium hydroxide solution (A.2.1.6) to dissolve; then, add 0.1 g
of sodium sulfide; shake well. The solution shall appear dark red.
A.2.3.1.3 Weigh 0.01 g (accurate to 0.001 g) of heme chloride sample; add 5
mL of nitric acid; shake well; heat. The solution shall appear yellow. After cooling,
use ammonia solution (A.2.1.7) to adjust the pH to alkaline; the solution shall
appear orange-yellow.
A.2.3.2 Infrared spectroscopy
Use potassium bromide pellet technique to test in accordance with the
provisions of GB/T 6040; the infrared spectrum of the sample shall be
consistent with the control spectrum. See Appendix B for the control spectrum.
A.3 Determination of heme chloride (C34H32ClFeN4O4) content
A.3.1 Ultraviolet spectrophotometry
A.3.1.1 Method summary
After the heme chloride is dissolved in 0.1 mol/L sodium hydroxide solution, use
the ultraviolet spectrophotometry for determination. There is a maximum
absorption peak at 386 nm ± 1 nm. Measure the absorbance value A; calculate
the sample content according to the concentration of the standard working
solution.
A.3.1.2 Reagents and materials
A.3.1.2.1 0.1 mol/L sodium hydroxide solution: Weigh 4.0 g of sodium hydroxide;
add an appropriate amount of water to dissolve; use water to dilute to 1 L.
A.3.1.2.2 Heme chloride standard substance (Hemin): C34H32ClFeN4O4, CAS:
16009-13-5, purity ≥97%.
A.3.1.2.3 Heme chloride standard stock solution: accurately weigh 20 mg
(accurate to 0.1 mg) of heme chloride standard substance; add 0.1 mol/L
sodium hydroxide solution (A.3.1.2.1) to a constant volume of 100 mL; prepare
a standard stock solution of a concentration of 200.0 mg/L. Store the solution
m – the mass of the sample represented by the final sample solution, in grams
(g).
The test results are based on the arithmetic mean of the parallel determination
results, and the absolute difference between two independent determination
results obtained under repeatability conditions is not more than 5% of the
arithmetic mean.
A.3.2 High-performance liquid chromatography
A.3.2.1 Method summary
After the heme chloride sample is dissolved and extracted with 0.1 mol/L
sodium hydroxide solution, use high performance liquid chromatography to
measure. The retention time is qualitative, and the peak area is quantified by
external standard method.
A.3.2.2 Reagents and materials
A.3.2.2.1 Methanol: chromatographic pure.
A.3.2.2.2 Acetic acid: chromatographic pure.
A.3.2.2.3 Acetic acid solution: draw 6 mL of acetic acid (A.3.2.2.2); use water
to dilute to 1 L.
A.3.2.2.4 Heme chloride standard substance (Hemin): See A.3.1.2.2.
A.3.2.2.5 Heme chloride standard stock solution: See A.3.1.2.3.
A.3.2.2.6 Heme chloride standard working solution: Use 0.1 mol/L sodium
hydroxide solution to dilute the heme chloride standard stock solution to
prepare the corresponding standard working solution before use.
A.3.2.2.7 Filter membrane: 0.45 μm, water phase.
A.3.2.3 Instruments and apparatuses
A.3.2.3.1 High performance liquid chromatograph: equipped with ultraviolet
detector or diode array detector.
A.3.2.3.2 Analytical balance: sensitivity of 0.1 g and 0.000 1 g.
A.3.2.4 Analysis steps
Weigh 0.02 g (accurate to 0.001 g) of sample in a 100 mL volumetric flask; use
0.1 mol/L sodium hydroxide solution to dissolve it and dilute it to the mark; shake
well; then, use it for HPLC determination.