4 Requirement
4.1 Appearance
Test according to 5.1. The test sample should be white or off-white granules, powder, or fibrous solid.
4.2 Identification
Test according to 5.2. Infrared absorption spectrum should be consistent with the reference spectrum.
4.3 pH Value
Test according to 5.3. Solution at a concentration of 5 mg/mL should have a pH value between 5.0 and 8.5.
4.4 Solution Appearance
Test according to 5.4. The solution S should be clear. At the wavelength of 600 nm, the absorbance A600nm should not exceed 0.01.
4.5 Loss on Drying
Test according to 5.5. The loss on drying of the test sample should not exceed 15.0% (mass fraction).
4.6 Heavy Metal Content
Test according to 5.6. The heavy metal content of the test sample should not exceed 10 μg/g.
4.7 Iron Content
Test according to 5.7. The iron content of the test sample should not exceed 80 μg/g.
4.8 Hviscosity Number and Molecular Weight
4.8.1 The viscosity number should be between 90% and 120% of its labeled value.
4.8.2 The molecular weight is calculated based on the viscosity number.
4.9 Nucleic Acid
Test according to 5.9. At a wavelength of 260 nm, the absorbance A260nm should not exceed 0.5.
4.10 Chloride Content
Test according to 5.10. The chloride content of the test sample should not exceed 0.001%.
4.11 Microbial Limit
Weigh 5.0 g of the test sample and add it to 100 mL of phosphate buffer (pH 7.2) containing 45,000 units of hyaluronidase per milliliter in a sterile environment. Stir at 42°C for 30 minutes until dissolved, then prepare a 1:20 solution. Test according to the method specified in 1105 and 1106 of Part IV of the Chinese Pharmacopoeia (2020 edition).
4.12 Streptococcus pyogenes Hemolysis
Take 0.5 g of the test sample, dissolve it in 100 mL of sterile 0.9% sodium chloride solution in a conical flask, and mix well. Spread 0.5 mL of the solution on two blood agar plates. Incubate at 37°C for 48 hours.
4.13 Hemolytic Property
Take 0.5 g of the test sample, dissolve it in 100 mL of sterile 0.9% sodium chloride solution to make a test solution. Take 0.5 mL of the test solution and add 0.5 mL of 1% blood suspension to each of two test tubes; mix well. Use another set of test tubes containing 0.5 mL of sterile 0.9% sodium chloride solution and 0.5 mL of 1% blood suspension as blank control solutions. Use a set of test tubes with only 0.5 mL of sterilized pure water for the positive control solution. Incubate all samples, blank controls, and positive controls at 37°C in a constant temperature box for 2 hours. Observe results visually.
4.14 Hyaluronic Acid Sodium Content
4.14.1 Equipment
The equipment used is as follows:
- Analytical balance
- UV spectrophotometer or equivalent device
4.14.2 Solution Preparation
4.14.2.1 0.025 mol/L Boric acid sulfuric acid solution
Weigh 4.77 g of boric acid and dissolve it in concentrated sulfuric acid (AR grade) to a volume of 500 mL, then mix well.
4.14.3 Reference Solution Preparation
Accurately weigh about 0.1 g of glucose uronic acid reference substance dried at 105°C with phosphorus pentoxide as the drying agent until constant weight and prepare a solution containing 50 μg/mL by dissolving in water, then mix well.
4.14.4 Test Solution Preparation
Accurately weigh about 0.1 g of the test sample, place it in a 100 mL volumetric flask, dissolve with an appropriate amount of water until completely dissolved, and dilute to volume and mix well. Then accurately weigh about 4.0 g (the density of the solution is calculated as 1.000 g/mL, which is equivalent to 4.00 mL), place it in a 50 mL volumetric flask, dilute with water to volume, mix well, and use this as the test solution.
4.14.5 Measurement
Proceed according to the following steps:
a) Prepare a series of standard solutions for glucose uronic acid as shown in Table 2.
b) Place all tubes containing standard samples into an ice water bath. Slowly add 5.0 mL of 0.025 mol/L boric acid sulfuric acid solution to each tube, tightly close the cap, and mix well. Heat in boiling water for 10 minutes, then cool in ice water to room temperature. Precisely add 0.20 mL of carbazole reagent, shake well, heat again in boiling water for 15 minutes, and then cool back to room temperature using the blank control tube (No. 0) as a reference. Use a spectrophotometer to determine the absorbance at 530 nm for each standard tube and sample tube.
c) Draw an absorption-concentration curve with the standard tubes. From this curve, find the concentration of glucose uronic acid in the sample tubes.
4.14.6 Calculation
Calculate the hyaluronic acid sodium content according to Equation (5):
X = \(\frac{(401.3 \times C_i \times 100 \times 50 \times \rho \times 10^6)}{[194.1 \times C_5 \times C_6 \times 10^6 \times (100 - w)]} \times 100\%\)
Where:
X —— Hyaluronic acid sodium content, %;
401.3 —— Molecular weight of the hyaluronic acid disaccharide fragment;
Ci —— Concentration of glucose uronic acid obtained from the regression equation, μg/mL;
100 —— The first dilution volume of the test sample, mL;
50 —— Second dilution volume, mL;
ρ —— Density of the test solution at 25°C, 1.000 g/mL;
w5 —— Weight of the accurately weighed test sample, g;
w6 —— Weight of the sample after second dilution, g;
\(10^6\) —— Conversion factor from grams to micrograms;
194.1 —— Molecular weight of glucose uronic acid.
4.15 Bacterial Endotoxin
Prepare a 2 mg/mL solution of hyaluronic acid sodium using bacterial endotoxin test water, and test according to the method specified in 1143 of Part IV of the Chinese Pharmacopoeia (2020 edition).
4.16 Transdermal Absorption
Test as per GB/T 27818.
T/CSBM 0043-2023 history
2023T/CSBM 0043-2023 Sodium hyaluronate for nursing、protective and dental medical devices