Sodium Hyaluronate (from Microbial
Fermentation)
Sodium Hyaluronate (from Microbial Fermentation)
Published in: FCC 11 FCC 11 1S FCC 11 2S FCC 11 3S
First Published: FCC 9
Hyaluronic Acid Sodium
Hyaluronic Acid Sodium Salt
Haluronic Acid Na-salt
Sodium Hyaluronate (Streptococcus)
(C14H20NNaO11)n
Formula wt 8.02 × 104 to 4.01 × 106
CAS: [9067-32-7]
UNII: YSE9PPT4TH [sodium hyaluronate (from microbial fermentation)]
DESCRIPTION
Sodium Hyaluronate is the sodium salt of hyaluronic acid. Hyaluronic acid is a natural complex
carbohydrate of the glycosaminoglycan family and is a long-chain polymer containing D-glucuronic acid
and N-acetyl-D-glucosamine units. The molecular weight ranges from 8.02 × 104 to 4.01 × 106.
Sodium Hyaluronate occurs as a white or almost white, very hygroscopic powder or fibrous aggregate.
It is obtained by fermentation of glucose from nonhemolytic gram-positive bacteria Streptococcus equi
subsp. zooepidemicus. It is sparingly soluble in water; insoluble in acetone and in anhydrous ethanol.
Hyaluronate sodium is highly hygroscopic, and the user is cautioned to use extreme care to minimize
moisture absorption.
FUNCTION: Source of hyaluronate sodium
PACKAGING AND STORAGE: Store in tight, light-resistant containers, and avoid exposure to sunlight and
moisture. Store at 2°–8°, unless otherwise specified by the manufacturer.
IDENTIFICATION
• INFRARED SPECTRA, Spectrophotometric Identification Tests, Appendix IIIC
Reference standard: USP Sodium Hyaluronate for IR Identification RS
Sample and standard preparation: K
Acceptance criteria: The spectrum of the sample exhibits maxima at the same wavelengths as
those in the spectrum of the Reference standard.
• SODIUM, Appendix IIIA
Sample solution: 100 mg/mL
Acceptance criteria: Passes test
ASSAY
Change to read:
• SODIUM HYALURONATE CONTENT
[NOTE—This procedure determines sodium hyaluronate by calculating glucuronic acid content using
calibration curve and applying it in the formula described below. ]
[CAUTION— Sodium hyaluronate is highly hygroscopic, and the user is cautioned to use extreme
care to minimize moisture absorption. ]
Reference standard: USP D-Glucuronic Acid RS
Reagent A: Dissolve 0.95 g of disodium tetraborate in 100.00 mL of sulfuric acid.
Reagent B: Dissolve 0.125 g of carbazole in 100.00 mL of anhydrous ethanol.
Standard stock solution: Dissolve 0.100 g of Reference standard in water. Dilute with the same
solvent to 100.0 g.
Standard solution: Prepare five dilutions of the Standard stock solution containing 6.5–65 µg/g
of D-glucuronic acid.
Sample solution: [NOTE— Prepare the Sample solution in triplicate on the dried basis. ]
Dissolve 0.170 g of the substance to be examined in water, and dilute with the same solvent to
100.00 g. Dilute 10.00 g of this solution with water to 200.0 g.
Analysis: Place suitable test tubes, numbered 1–7, in iced water. Add a 1.0-mL aliquot of the
five dilutions of the Standard solution each, separately in test tubes 1–5; 1.0 mL of Sample
solution in test tube number 6; and 1.0 mL of water (blank) in test tube number 7. In each test
tube, gently add 5.0 mL of freshly prepared and previously cooled Reagent A. Secure the test
tubes with caps, mix the content, and place the test tubes in a boiling water bath for exactly 15
min. Cool the test tubes in ice water for exactly 15 min. Then add 0.20 mL of Reagent B in each
of the seven test tubes. Recap the test tubes, and mix well. Place the test tubes again in a boiling
water bath for exactly 15 min. Cool the test tubes at room temperature, and measure the
absorbance of the solutions at 530 nm against the blank.
Calculation: Establish a calibration curve with absorbancies versus concentrations of the
Standard solution to determine the concentration of D-glucuronic acid in the Sample solution.
Calculate the content of sodium hyaluronate, CS (mg/mL):
▲C = CG × Z × (M1/M2)▲ (ERR 1-Jan-2018)
S
CG = concentration of D-glucuronic acid in the Sample solution (mg/mL)
Z = purity value of USP D-Glucuronic Acid RS ▲
▲ (ERR 1-Jan-2018)
M1 = relative molecular mass of the disaccharide fragment, 401.3
M2 = relative molecular mass of glucuronic acid, 194.1
Acceptance criteria: NLT 95% sodium hyaluronate calculated on the dried basis
IMPURITIES
INORGANIC IMPURITIES
• ARSENIC, Arsenic Limit Test, Appendix IIIB
Acceptance criteria: NMT 2 mg/kg
• LEAD, Lead Limit Test, Flame Atomic Absorption Spectrophotometric Method, Appendix
IIIB
Acceptance criteria: NMT 1 mg/kg
• CHLORIDE, Chloride and Sulfate Limit Tests, Chloride Limit Test, Appendix IIIB
Sample: 1g
Acceptance criteria: NMT 0.5%
SPECIFIC TESTS
Change to read:
• INTRINSIC VISCOSITY
[CAUTION— Care must be taken while weighing hyaluronate sodium to minimize moisture
absorption. ]
Solution A: Dissolve 9.00 g of sodium chloride and 1.56 g of anhydrous sodium dihydrogen
phosphate in 1000 mL of water.
Solution B: Dissolve 9.00 g of sodium chloride and 1.79 g of disodium hydrogen phosphate in
1000 mL of water.
Buffer: Mix Solution A with Solution B until a pH of 7.0 is achieved. [NOTE— Approximately 1: 1.9
volumes (Solution A and Solution B) ]
Pass through a sintered-glass filter of NMT 4-µm pore size.
Sample solution A: Weigh 0.2 g (m 0S) of material under investigation, and dilute with 50.0 g
(m0B) of Buffer. [NOTE— The weight of the sample may be adjusted after initial measurement of
viscosity of Sample solution A. ]
Mix by shaking at 4° for 24 h. Weigh 5.00 g (m1S) of this solution, and dilute with 100.0 g (m1B)
of Buffer. Mix by shaking at 25° for 20 min. Pass the solution through a sintered-glass filter of
NMT 100-µm pore size, and discard the first 10 mL.
[NOTE— For the Sample solution, a 0.45-µm PVDF filter may be used; discard the first few drops. ]
Sample solution B: Weigh 30.0 g (m2S) of Sample solution A, and dilute with 10.0 g (m2B) of
Buffer. Mix by shaking at 25° for 20 min. Pass the solution through a sintered-glass filter of NMT
100-µm pore size, and discard the first 10 mL.
Sample solution C: Weigh 20.0 g (m3S) of Sample solution A, and dilute with 20.0 g (m3B) of
Buffer. Mix by shaking at 25° for 20 min. Pass the solution through a sintered-glass filter of NMT
100-µm pore size, and discard the first 10 mL.
Sample solution D: Weigh 10.0 g (m4S) of Sample solution A, and dilute with 30.0 g (m4B) of
Buffer. Mix by shaking at 25° for 20 min. Pass the solution through a sintered-glass filter of NMT
100-µm pore size, and discard the first 10 mL.
Viscometer: Suspended level capillary type
Specifications
Viscometer constant: About 0.005 mm2/s2
Kinematic viscosity (k): 1–5 mm2/s
Internal diameter
Tube R: 0.53 mm
Tube N: 2.8–3.2 mm with a funnel-shaped lower capillary
Volume of bulb: 5.6 mL
Analysis: Determine the flow times for Buffer (t0), Sample solution A (t1), Sample solution B
(t2), Sample solution C (t3), and Sample solution D (t4), in triplicate at 25° ± 0.1°. The test is not
valid unless the flow time t1 is 1.6–1.8 times t0. If this is not the case, then adjust the value of
m0S, and repeat the procedure. Calculate the relative viscosity (ηr) of each solution:
▲η
r
= [ti − (B/ti2)]/[t0 − (B/t02)]▲ (ERR 1-Jan-2018)
ηr = ηr1, ηr2, ηr3, or ηr4
ti = t1, t2, t 3, or t4
B = kinetic energy correction factor for the capillary, 30,800 S3
Calculate the concentration (C1) of sodium hyaluronate in Sample solution A:
C 1 = (m0S) × (x/100) × [(100 − h)/100] × [1/(m0S + m0B )] × [m1S/(m1S + m1B )] × ρ25
m0S, m0B, m1S, and m1B as
indicated above
x = percentage of content of sodium hyaluronate as
determined in the Assay
h = percentage of loss on drying per Loss on Drying
ρ25 = density of solution at 25°, 1005 kg/m3
Calculate the concentration of sodium hyaluronate C2, C3, and C4 in Sample solution B, Sample
solution C, and Sample solution D, respectively:
C2 = C1 × [m2S/(m2B + m2S)]
C3 = C1 × [m3S/(m3B + m3S)]
C4 = C1 × [m4S/(m4B + m4S)]
m2S, m2B, m3S, m3B, m4S, and m4B as indicated above
Calculate the intrinsic viscosity (η) by linear least-squares regression analysis:
log[(ηr − 1)/c] = log [η] + k × [η] × c
m2S, m2B, m3S, m3B, m4S, and m4B as indicated above
c = concentrations (C1, C2, C3, or C4)
k = straight line fitting empirical constant1
The decimal antilogarithm of the intercept is the intrinsic viscosity expressed in m3/kg.
Acceptance criteria: 90%–120% of the labeled value
• LOSS ON DRYING, Appendix IIC: (105° for 6 h over diphosphorus pentoxide)
Sample: 0.5 g
Acceptance criteria: NMT 20%
• PH, pH Determination, Appendix IIB
Sample solution: 5 mg/mL prepared by dissolving a previously dried sample in carbon-dioxide
free water
Acceptance criteria: 6.0–8.0
1 The Rheology Handbook, by Thomas G. Mezger (2nd edition), page 220.
Please check for your question in the FAQs before contacting USP.
Topic/Question Contact Expert Committee
SODIUM HYALURONATE (FROM MICROBIAL Gina Clapper FI2015 Food Ingredients
FERMENTATION) Senior Scientific 2015
Liaison
+1 (301) 692-3626
Page Information
FCC 11 - page 1083
FCC 10 - page 1169
FCC 9 - page 1090