QB/T 4576-2023 English PDF (QBT4576-2023)

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QB/T 4576-2023: Sodium hyaluronate
QB/T 4576-2023
QB
LIGHT INDUSTRY STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 67.180
CCS X 69
Replacing QB/T 4576-2013
Sodium hyaluronate
透明质酸钠
ISSUED ON. DECEMBER 20, 2023
IMPLEMENTED ON. JULY 01, 2024
Issued by. Ministry of Industry and Information Technology of the PRC
Table of Contents
Foreword... 3
1 Scope... 5
2 Normative references... 5
3 Terms and definitions... 6
4 Molecular formula, relative molecular mass, structural formula... 6
5 Requirements... 7
6 Test method... 8
7 Inspection rules... 10
8 Marking, packaging, transportation, storage... 11
Appendix A (Normative) Sodium hyaluronate content - Spectrophotometer method 13
Appendix B (Normative) High-performance liquid chromatography method for sodium
hyaluronate content... 16
Appendix C (Informative) High-performance liquid chromatogram of sodium
hyaluronate... 19
Sodium hyaluronate
1 Scope
This document specifies the identification, sensory, physical and chemical, safety and
other requirements of sodium hyaluronate; describes the corresponding test methods;
specifies the inspection rules, marking, packaging, transportation, storage content;
gives the information of molecular formula, relative molecular mass, structural formula.
This document applies to the production, inspection, and sale of sodium hyaluronate
produced by fermentation of Streptococcus equi subspecies zooepidemicus with
glucose, yeast powder, peptone, etc. as raw materials.
2 Normative references
The contents of the following documents constitute essential clauses of the text through
normative references in the text. Among them, for dated references, only the version
corresponding to that date applies to this document; for undated references, the latest
version (including all amendments) applies to this document.
GB/T 191 Packaging - Pictorial marking for handling of goods
GB/T 601 Chemical reagent - Preparations of reference titration solutions
GB/T 602 Chemical reagent - Preparations of standard solutions for impurity
GB/T 603-2023 Chemical reagent - Preparations of reagent solutions for use in test
methods
GB 4789.2 National food safety standard - Microbiological examination of food.
Aerobic plate count
GB 4789.3 National food safety standard - Food microbiological examination -
Enumeration of coliforms
GB 4789.15 National food safety standard - Food microbiological examination -
Enumeration of moulds and yeasts
GB 5009.3 National food safety standard - Determination of moisture in food
GB 5009.4 National food safety standard - Determination of ash in foods
GB 5009.11 National food safety standards - Determination of total arsenic and
Determine according to Appendix A or Appendix B, using Appendix A as the arbitration
method.
6.5 Moisture
Make measurement according to the method 1 "Direct drying method" of GB 5009.3,
where the drying time is 4 h.
6.6 Ash
Make measurement according to the method 1 "total ash" of GB 5009.4.
6.7 pH
6.7.1 Water
Distilled water without carbon dioxide. It is prepared according to 5.1.1.7 of GB/T 603-
2023.
6.7.2 Instruments and equipment
pH meter. Accuracy of 0.01.
Magnetic stirrer.
Thermostatic water bath. Accuracy of ±0.5 °C.
6.7.3 Determination steps
Weigh 0.1 g of sodium hyaluronate sample (accurate to 0.01 g). Add 100 mL of distilled
water without carbon dioxide. Dissolve by magnetic stirring or heating in a 40 °C water
bath. Measure the pH of the solution with a pH meter. The result is rounded to one
decimal place.
6.8 Lead (measured as Pb)
It is determined according to the method of GB 5009.12.
6.9 Arsenic (measured as As)
It is determined according to the method of GB 5009.11.
6.10 Mold and yeast
It is determined according to the method of GB 4789.15.If the sample cannot be
dissolved, add an appropriate amount of sterile hyaluronidase to help the sample
dissolve.
6.11 Total colony count
7.4 Exit-factory inspection
The exit-factory inspection items are sensory requirements, sodium hyaluronate content
(on a dry basis), pH, moisture, total colony count, mold and yeast, and coliform group.
7.5 Type inspection
The type inspection items are all the items specified in the requirements of this
document. Generally, type inspection is carried out every six months. Type inspection
shall also be carried out in any of the following situations.
a) Where there are major changes in raw and auxiliary materials;
b) Where key processes or equipment are changed;
c) When the new trial products is produced or when the production is restored after
production suspension of 3 months;
d) Where there is a big difference between the exit-factory inspection and the last
type inspection results;
e) Where the national market supervision agency needs to conduct random
inspections according to relevant requirements.
7.6 Judgment rules
If the inspection results show that one item of the product does not meet the
requirements of this document, samples shall be taken from twice the amount of
packaging for re-inspection; the re-inspection results shall prevail. If there is still one
unqualified item, the batch of products shall be judged as unqualified products. If the
inspection results show that two or more items of the product do not meet the
requirements of this document, the batch of products shall be judged as unqualified
products.
8 Marking, packaging, transportation, storage
8.1 Marking
The outer packaging marking shall comply with the provisions of GB/T 191.Pre-
packaged product labels shall comply with the provisions of GB 7718.For signs with
special requirements, they shall be marked as required by the purchaser.
8.2 Packaging
The packaging materials that meet the packaging requirements of the corresponding
industry products shall be used and can only be used after passing the inspection.
Strictly seal to prevent the product from absorbing moisture and leaking. For packaging
Appendix A
(Normative)
Sodium hyaluronate content - Spectrophotometer method
A.1 Principle
Hyaluronic acid contains N-acetylglucosamine and glucuronic acid in equal molar
ratios. Using borax as a catalyst to hydrolyze sodium hyaluronate with sulfuric acid can
separate glucuronic acid. Glucuronic acid reacts with carbazole to form an organic
complex, which shows a unique purple color; its absorbance is proportional to the mass
concentration of glucuronic acid. The content of sodium hyaluronate can be determined
by the content of glucuronic acid.
A.2 Reagents and solutions
A.2.1 Standard. D-glucuronic acid, CAS No.6556-12-3, purity not less than 98%.
A.2.2 Concentrated sulfuric acid. High purity.
A.2.3 Carbazole.
A.2.4 Borax.
A.2.5 Anhydrous ethanol.
A.2.6 Carbazole ethanol solution (1.25 g/L). Weigh 0.125 g of carbazole (accurate to
0.001 g); dissolve it in 100 mL of anhydrous ethanol; place it in a brown bottle. Store
in an explosion-proof refrigerator at 4 °C ~ 8 °C, which has a shelf life of 2 months; or
store in the dark at room temperature, which has a shelf life of 15 days.
A.2.7 D-glucuronic acid standard solution (0.2 mg/mL). Accurately weigh 20 mg of D-
glucuronic acid standard (accurate to 0.0001 g according to actual purity); place it in a
100 mL volumetric flask; add water to dissolve and dilute to the mark; shake well for
later use.
A.2.8 Borax-sulfuric acid solution (0.025 mol/L). Weigh 4.77 g of borax (accurate to
0.001 g); dissolve it in 500 mL concentrated sulfuric acid; store it in a sealed glass
narrow-necked bottle for later use.
A.3 Instruments and equipment
A.3.1 Vortex mixer.
A.3.2 Spectrophotometer. Capable of measuring absorbance at 530 nm.
A.4 Drawing of standard curve
Measure 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL of glucuronic acid standard solution
(A.2.7) respectively; place in a 10 mL volumetric flask; add water to dilute to the scale,
to obtain standard solutions with mass concentrations of 10 μg/mL, 20 μg/mL, 30
μg/mL, 40 μg/mL, 50 μg/mL. Take 6 stoppered graduated test tubes; add 1.0 mL of
standard solutions of different mass concentrations respectively; then add 5.0 mL of
borax sulfuric acid solution respectively; cool in an ice bath for 15 min. Shake gently
first; then mix thoroughly with a vortex mixer. Heat the test tube in boiling water for
10 min; remove and cool to room temperature in an ice water bath or running water.
Add 0.2 mL of carbazole solution (A.2.6) to the cooled test tube; mix well; heat in a
boiling water bath for another 15 min; cool to room temperature. Use a 10 mm cuvette
to measure the absorbance at a wavelength of 530 nm; draw a standard curve, using the
absorbance as the ordinate and the mass concentration as the abscissa.
A.5 Analysis steps
A.5.1 Weigh about 0.1 g (m, accurate to 0.0001 g) of sodium hyaluronate sample in a
stoppered conical flask; add water to 100 g (w1, accurate to 0.01 g); stir magnetically
until fully dissolved. Weigh about 4.0 g (w2, accurate to 0.01 g) of the above solution
in a 50 mL (V) volumetric flask; dilute to the mark with water; shake well.
A.5.2 Take 1 mL of sample solution; add 5 mL of borax-sulfuric acid solution; cool it
in an ice bath for 15 min. Shake gently first; then mix thoroughly with a vortex mixer.
Heat the test tube in boiling water for 10 min; then cool to room temperature in an ice
water bath or running water. Add 0.2 mL of carbazole test solution; mix well; heat in a
boiling water bath for another 15 min; then cool to room temperature. Measure the
absorbance at a wavelength of 530 nm, using a spectrophotometer with a 1 cm cuvette.
A.6 Calculation
The sodium hyaluronate content (on a dry basis) is calculated according to formula
(A.1).
Wherein.
X1 - Sodium hyaluronate content in the sample (on a dry basis), in grams per hundred
grams (g/100 g)
ρ1 - According to the absorbance of the sample, the corresponding mass
concentration of glucuronic acid as found from the standard curve, in micrograms
per milliliter (μg/mL);
Appendix B
(Normative)
High-performance liquid chromatography method for sodium hyaluronate
content
B.1 Principle
Hyaluronidase can act on the β-1,4-glycosidic bond of sodium hyaluronate to hydrolyze
sodium hyaluronate, to produce N-acetylglucosamine-glucuronic acid disaccharide.
The content of N-acetylglucosamine-glucuronic acid disaccharide product is
determined by high-performance liquid chromatography. The standard curve is drawn,
using the mass concentration of sodium hyaluronate as the abscissa and the peak area
of N-acetylglucosamine-glucuronic acid disaccharide generated by the hydrolysis of
sodium hyaluronate as the ordinate. The content of sodium hyaluronate in the sample
can be calculated by the peak area of N-acetylglucosamine-glucuronic acid
disaccharide generated by hydrolysis.
B.2 Reagents and consumables
B.2.1 Sodium hyaluronate reference substance. CAS No.9067-32-7, purity not less than
99%.
B.2.2 Hyaluronidase. Enzyme activity not less than 4000 IU/mL.
B.2.3 Sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O).
B.2.4 Sodium hydrogen phosphate dodecahydrate (NaH2PO4·12H2O).
B.2.5 Phosphoric acid (H3PO4).
B.2.6 Phosphate buffer solution (0.2 mol/L, pH 6.0). Weigh 27.4 g sodium dihydrogen
phosphate dihydrate (B.2.3) and 8.8 g sodium dihydrogen phosphate dehydrate (B.2.4)
in a 1000 mL beaker; dissolve in water and transfer to a 1000 mL volumetric flask;
adjust the pH to 6.0 with 1 mol/L phosphoric acid solution or 1 mol/L sodium hydroxide
solution; add water to make it reach to the mark; shake well.
B.2.7 Sodium hyaluronate reference solution (1.0 mg/mL). Weigh 50 mg of sodium
hyaluronate reference (accurate to 0.1 mg according to actual purity) in a volumetric
flask; add 40 mL of phosphate buffer solution to dissolve; ultrasonicate until fully
dissolve it; use phosphate buffer solution to make it reach to the mark; shake well.
B.2.8 Hyaluronidase solution (1000 IU/mL). Pipette an appropriate amount of
hyaluronidase (B.2.2) into a 10 mL volumetric flask; dissolve and make the volume
reach to the mark with phosphate buffer. Prepare it immediately before use.
B.2.9 Mobile phase (1% phosphoric acid solution). Pipette 10.0 mL of phosphoric acid
(B.2.5) into 800 mL of water; mix well; transfer to a 1000 mL volumetric flask; add
water to make the volume reach to the mark; mix well.
B.3 Instruments and equipment
B.3.1 High performance liquid chromatography. Equipped with a UV detector.
B.3.2 Chromatographic column. Sulfonated cross-linked styrene divinylbenzene
copolymer strong cation exchange chromatography column (300 mm × 8 mm), or other
equivalent chromatographic columns.
B.3.3 Reference chromatographic conditions. Flow rate 0.6 mL/min; injection volume
20 µL; column temperature 40 °C; detection wavelength 232 nm.
B.4 Analysis steps
B.4.1 Drawing of standard curve
Pipette 0.05 mL, 0.1 mL, 0.2 mL, 0.5 mL, 1.0 mL, 2.0 mL of sodium hyaluronate
reference solution into 15 mL stoppered graduated test tubes; add phosphate buffer
solution (B.2.6) to 9 mL; then add 1 mL of hyaluronidase solution (B.2.8); mix well;
enzymolyze it in a 37 °C ~ 42 °C water bath for 1 hour. Boil in water bath for 2 min to
terminate the reaction. After cooling to room temperature, filter with a 0.22 µm filter
membrane; analyze according to the chromatographic conditions described in B.3.3;
record the peak area. The mass concentrations of the reference solution are 0.005
mg/mL, 0.010 mg/mL, 0.020 mg/mL, 0.050 mg/mL, 0.100 mg/mL, 0.200 mg/mL,
respectively. Draw a standard curve, using the mass concentration of the sodium
hyaluronate reference substance series working solution as the abscissa and the peak
area as the ordinate.
B.4.2 Sample pretreatment
Weigh 0.1 g of sample (accurate to 0.001 g) into a 100 mL volumetric flask; add 80 mL
of phosphate buffer solution; ultrasonic it in a 42 °C water bath until fully dissolved;
use phosphate buffer solution to make the volume reach to the mark.
B.4.3 Enzymatic hydrolysis reaction
Pip...