EUROPEAN PHARMACOPOEIA 10.

0 Sodium hyaluronate

Glycerol and ethanol (96 per cent)-soluble substances :

maximum 1.0 per cent.
Shake 1.000 g with 25 mL of ethanol (96 per cent) R for
10 min. Filter. Evaporate the filtrate on a water-bath and dry
the residue at 70 °C for 1 h. The residue weighs not more than
10 mg.
Chlorides (2.4.4) : maximum 200 ppm.
Dilute 2.5 mL of solution S to 15 mL with water R.
Phosphates (2.4.11) : maximum 0.1 per cent.
O. ent-(17Z)-3β,11β,16α-trihydroxy-4β,8,14-trimethyl- Dilute 1 mL of solution S to 10 mL with water R. Dilute 1 mL
18-nor-5β,10α-cholesta-17(20),24-dien-21-oic acid of this solution to 100 mL with water R.
(deacetylfusidic acid). Sulfates (2.4.13): maximum 500 ppm.
Dilute 3 mL of solution S to 15 mL with water R.
Calcium : maximum 100 ppm, if intended for use in the
01/2017:1995 manufacture of parenteral preparations.
Inductively coupled plasma-atomic emission spectrometry
(2.2.57).
Test solution. Dissolve 0.50 g in a 1 per cent V/V solution of
nitric acid R and dilute to 50.0 mL with the same solution.
SODIUM GLYCEROPHOSPHATE, Reference solutions. Prepare the reference solutions using
HYDRATED calcium standard solution (100 ppm Ca) R, diluting with a
1 per cent V/V solution of nitric acid R.
Wavelength : 396.8 nm.
Natrii glycerophosphas hydricus Iron (2.4.9) : maximum 20 ppm.
Dilute 5 mL of solution S to 10 mL with water R.
Water (2.5.12) : 25.0 per cent to 35.0 per cent, determined on
0.100 g.
ASSAY
Dissolve 0.250 g in 30 mL of water R. Titrate with 0.05 M
C3H7Na2O6P,xH2O Mr 216.0 (anhydrous substance) sulfuric acid, determining the end-point potentiometrically
DEFINITION (2.2.20), (n1).
Mixture of variable proportions of hydrated disodium Calculate the percentage content of sodium glycerophosphate
(2RS)-2,3-dihydroxypropyl phosphate and hydrated disodium (anhydrous substance) using the following expression :
2-hydroxy-1-(hydroxymethyl)ethyl phosphate. The mixture n
may contain various amounts of other glycerophosphate esters. (
216.0 n1 - 2
4)
Content : 98.0 per cent to 105.0 per cent (anhydrous substance). m(100 - a)

CHARACTERS a = percentage content of water ;

Appearance : white or almost white, crystalline powder or n1 = volume of 0.05 M sulfuric acid used in the assay,
crystals. in millilitres ;
Solubility : freely soluble in water, practically insoluble in n2 = volume of 0.1 M hydrochloric acid used in the test
acetone and in ethanol (96 per cent). for alkalinity, in millilitres ;
m = mass of the substance to be examined, in grams.
IDENTIFICATION
A. Solution S (see Tests) gives reaction (a) of sodium (2.3.1).
B. To 0.1 g add 5 mL of dilute nitric acid R. Heat to boiling 01/2017:1472
and boil for 1 min. Cool. The solution gives reaction (b) corrected 10.0
of phosphates (2.3.1).
C. In a test-tube fitted with a glass tube, mix 0.1 g with 5 g of
potassium hydrogen sulfate R. Heat strongly and direct the
white vapour into 5 mL of decolorised fuchsin solution R.
A violet-red colour develops which becomes violet upon SODIUM HYALURONATE
heating for 30 min on a water-bath.
Natrii hyaluronas
TESTS
Solution S. Dissolve 10.0 g in carbon dioxide-free water R
prepared from distilled water R and dilute to 100 mL with the
same solvent.
Appearance of solution. Solution S is not more opalescent
than reference suspension II (2.2.1) and not more intensely
coloured than reference solution Y6 (2.2.2, Method II).
Alkalinity. To 10 mL of solution S add 0.2 mL of
phenolphthalein solution R. Not more than 1.0 mL of 0.1 M
hydrochloric acid is required to change the colour of the (C14H20NNaO11)n
indicator (n2). [9067-32-7]

www.webofpharma.com
General Notices (1) apply to all monographs and other texts 3821
Sodium hyaluronate EUROPEAN PHARMACOPOEIA 10.0

DEFINITION Test solution (d). Weigh 10.0 g (m4p) of test solution (a) and
Sodium salt of hyaluronic acid, a glycosaminoglycan dilute with 30.0 g (m4s) of buffer solution at 25 °C. Mix this
consisting of D-glucuronic acid and N-acetyl-D-glucosamine solution by shaking for 20 min. Filter the solution through a
disaccharide units. It is extracted from cocks’ combs or sintered-glass filter (100) (2.1.2) and discard the first 10 mL.
obtained by fermentation from Streptococci, Lancefield Groups Determine the flow-times (2.2.9) for the buffer solution (t0)
A and C. and for the 4 test solutions (t1, t2, t3 and t4), at 25.00 ± 0.03 °C.
Content : 95.0 per cent to 105.0 per cent (dried substance). Use an appropriate suspended level viscometer (specifications :
viscometer constant about 0.005 mm2/s2, kinematic viscosity
Intrinsic viscosity : 90 per cent to 120 per cent of the value
of 1-5 mm2/s, internal diameter of tube R 0.53 mm, volume of
stated on the label.
bulb C 5.6 mL, internal diameter of tube N 2.8-3.2 mm) with a
PRODUCTION funnel-shaped lower capillary end. Use the same viscometer
Where applicable, the animals from which sodium hyaluronate for all measurements ; measure all outflow times in triplicate.
is derived must fulfil the requirements for the health of The test is not valid unless the results do not differ by more
animals suitable for human consumption. than 0.35 per cent from the mean and if the flow time t1 is not
less than 1.6 and not more than 1.8 times t0. If this is not the
When produced by fermentation of gram-positive bacteria, case, adjust the value of m0p and repeat the procedure.
the process must be shown to reduce or eliminate pyrogenic
or inflammatory components of the cell wall. Calculation of the relative viscosities
Since the densities of the sodium hyaluronate solutions and
CHARACTERS of the solvent are almost equal, the relative viscosities ηri
Appearance : white or almost white, very hygroscopic powder (being ηr1, ηr2, ηr3 and ηr4) can be calculated from the ratio
or fibrous aggregate. of the flow times for the respective solutions ti (being t1,
Solubility : sparingly soluble or soluble in water, practically t 2, t3and t4) to the flow time of the solvent t0, but taking

insoluble in acetone and in anhydrous ethanol. into account the kinetic energy correction factor for the
capillary (B = 30 800 s3), using the following expression :
IDENTIFICATION
B
A. Infrared absorption spectrophotometry (2.2.24). ti - 2
ti
Comparison : Ph. Eur. reference spectrum of sodium B
hyaluronate. t0 - 2
t0
B. It gives reaction (a) of sodium (2.3.1). Calculation of the concentrations
TESTS Calculate the concentration c1 (expressed in kg/m3) of sodium
Solution S. Weigh a quantity of the substance to be examined hyaluronate in test solution (a) using the following expression:
equivalent to 0.10 g of the dried substance and add 30.0 mL of
a 9 g/L solution of sodium chloride R. Mix gently on a shaker m0p ´ x ´ (100 - h) ´ m1p ´ ρ25
until dissolved (about 12 h). ( ) (
100 ´ 100 ´ m0p + m0s ´ m1p + m1s )
Appearance of solution. Solution S is clear (2.2.1) and its
absorbance (2.2.25) at 600 nm is not greater than 0.01. x = percentage content of sodium hyaluronate as
pH (2.2.3) : 5.0 to 8.5. determined under Assay ;
Dissolve the substance to be examined in carbon dioxide-free h = percentage loss on drying ;
water R to obtain a solution containing a quantity equivalent ρ25 = 1005 kg/m3 (density of the test solution at 25 °C).
to 5 mg of the dried substance per millilitre.
Calculate the concentration c2 (expressed in kg/m3) of sodium
Intrinsic viscosity. Sodium hyaluronate is very hygroscopic hyaluronate in test solution (b) using the following expression :
and must be protected from moisture during weighing.
Buffer solution (0.15 M sodium chloride in 0.01 M phosphate m 2p
c1 ´
buffer solution pH 7.0). Dissolve 0.78 g of sodium dihydrogen m 2s + m 2p
phosphate R and 4.50 g of sodium chloride R in water R and
Calculate the concentration c3 (expressed in kg/m3) of sodium
dilute to 500.0 mL with the same solvent (solution A). Dissolve
hyaluronate in test solution (c) using the following expression :
1.79 g of disodium hydrogen phosphate dodecahydrate R and
4.50 g of sodium chloride R in water R and dilute to 500.0 mL m3p
with the same solvent (solution B). Mix solutions A and B c1 ´
m3s + m3p
until a pH of 7.0 is reached. Filter through a sintered-glass
filter (4) (2.1.2). Calculate the concentration c4 (expressed in kg/m3) of sodium
Test solution (a). Weigh 0.200 g (m0p) (NOTE : this value is only hyaluronate in test solution (d) using the following expression :
indicative and should be adjusted after an initial measurement m4p
of the viscosity of test solution (a)) of the substance to be c1 ´
m4s + m4p
examined and dilute with 50.0 g (m0s) of buffer solution at
4 °C. Mix the solution by shaking at 4 °C during 24 h. Weigh Calculation of the intrinsic viscosity
5.00 g (m1p) of the solution and dilute with 100.0 g (m1s) of Calculate the intrinsic viscosity [η] by linear least-squares
buffer solution at 25 °C. Mix this solution by shaking for regression analysis using the Martin equation :
20 min. Filter the solution through a sintered-glass filter (100)
(2.1.2), and discard the first 10 mL. æ η - 1 ö÷
log10çç r ÷ = log [η] + k[η]c
Test solution (b). Weigh 30.0 g (m2p) of test solution (a) and ççè c ÷÷÷ø 10
dilute with 10.0 g (m2s) of buffer solution at 25 °C. Mix this
solution by shaking for 20 min. Filter the solution through a The decimal antilogarithm of the intercept is the intrinsic
sintered-glass filter (100) (2.1.2) and discard the first 10 mL. viscosity expressed in m3/kg.
Test solution (c). Weigh 20.0 g (m3p) of test solution (a) and Sulfated glycosaminoglycans : maximum 1 per cent, if the
dilute with 20.0 g (m3s) of buffer solution at 25 °C. Mix this product is extracted from cocks’ combs.
solution by shaking for 20 min. Filter the solution through a Appropriate safety precautions are to be taken when handling
sintered-glass filter (100) (2.1.2) and discard the first 10 mL. perchloric acid at elevated temperature.

www.webofpharma.com
3822 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 10.0 Sodium hyaluronate

Test solution. Introduce a quantity of the substance to be Bacterial endotoxins (2.6.14) : less than 0.5 IU/mg, if intended
examined equivalent to 50.0 mg of the dried substance into a for use in the manufacture of parenteral preparations without
test-tube 150 mm long and 16 mm in internal diameter and a further appropriate procedure for the removal of bacterial
dissolve in 1.0 mL of perchloric acid R. endotoxins ; less than 0.05 IU/mg, if intended for use in the
Reference solution. Dissolve 0.149 g of anhydrous sodium manufacture of intra-ocular preparations or intra-articular
sulfate R in water R and dilute to 100.0 mL with the same preparations without a further appropriate procedure for the
solvent. Dilute 10.0 mL of this solution to 100.0 mL with removal of bacterial endotoxins.
water R. Evaporate 1.0 mL in a test-tube 150 mm long and ASSAY
16 mm in internal diameter in a heating block at 90-95 °C,
and dissolve the residue in 1.0 mL of perchloric acid R. Determine the glucuronic acid content by reaction with
carbazole as described below.
Plug each test-tube with a piece of glass wool. Place the
test-tubes in a heating block or a silicone oil bath maintained Reagent A. Dissolve 0.95 g of disodium tetraborate R in
at 180 °C and heat until clear, colourless solutions are 100.0 mL of sulfuric acid R.
obtained (about 12 h). Remove the test-tubes and cool to Reagent B. Dissolve 0.125 g of carbazole R in 100.0 mL of
room temperature. Add to each test-tube 3.0 mL of a 33.3 g/L anhydrous ethanol R.
solution of barium chloride R, cap and shake vigorously. Allow Test solution. Prepare this solution in triplicate. Dissolve
the test-tubes to stand for 30 min. Shake each test-tube once 0.170 g of the substance to be examined in water R and dilute
again, and determine the absorbance (2.2.25) at 660 nm, using to 100.0 g with the same solvent. Dilute 10.0 g of this solution
water R as a blank. to 200.0 g with water R.
The absorbance obtained with the test solution is not greater Reference stock solution. Dissolve 0.100 g of D-glucuronic
than the absorbance obtained with the reference solution. acid R, previously dried to constant mass in vacuum over
Nucleic acids. The absorbance (2.2.25) of solution S at diphosphorus pentoxide R (2.2.32), in water R and dilute to
260 nm is maximum 0.5. 100.0 g with the same solvent.
Protein : maximum 0.3 per cent ; maximum 0.1 per cent, if Reference solutions. Prepare 5 dilutions of the reference
intended for use in the manufacture of parenteral preparations. stock solution containing between 6.5 μg/g and 65 μg/g of
D-glucuronic acid R.
Test solution (a). Dissolve the substance to be examined in
Place 25 test-tubes, numbered 1 to 25, in iced water. Add
water R to obtain a solution containing a quantity equivalent
1.0 mL of the 5 reference solutions in triplicate to the
to about 10 mg of the dried substance per millilitre.
test-tubes 1 to 15 (reference tubes), 1.0 mL of the 3 test
Test solution (b). Mix equal volumes of test solution (a) and solutions in triplicate to the test-tubes 16 to 24 (sample tubes),
water R. and 1.0 mL of water R to test-tube 25 (blank). Add to each
Reference solutions. Prepare a 0.5 mg/mL stock solution of test-tube 5.0 mL of freshly prepared reagent A, previously
bovine albumin R in water R. Prepare 5 dilutions of the stock cooled in iced water. Tightly close the test-tubes with plastic
solution containing between 5 μg/mL and 50 μg/mL of bovine caps, shake the contents, and place on a water bath for exactly
albumin R. 15 min. Cool in iced water, and add to each test tube 0.20 mL
Add 2.5 mL of freshly prepared cupri-tartaric solution R3 of reagent B. Recap the tubes, shake, and put them again on a
to test-tubes containing 2.5 mL of water R (blank), 2.5 mL water-bath for exactly 15 min. Cool to room temperature and
of the test solutions (a) or (b) or 2.5 mL of the reference measure the absorbance (2.2.25) of the solutions at 530 nm,
solutions. Mix after each addition. After about 10 min, add against the blank.
to each test-tube 0.50 mL of a mixture of equal volumes of From the calibration curve obtained with the mean
phosphomolybdotungstic reagent R and water R prepared absorbances read for each reference solution, determine the
immediately before use. Mix after each addition. After 30 min, mean concentrations of D-glucuronic acid in the test solutions.
measure the absorbance (2.2.25) of each solution at 750 nm Calculate the percentage content of sodium hyaluronate using
against the blank. From the calibration curve obtained with the following expression :
the 5 reference solutions determine the content of protein in
the test solutions. cg 100 401.3
´Z´ ´
Chlorides (2.4.4): maximum 0.5 per cent. cs 100 - h 194.1
Dissolve 67 mg in 100 mL of water R. cg = mean of concentrations of D-glucuronic acid in
Iron : maximum 80 ppm. the test solutions, in milligrams per gram ;
Atomic absorption spectrometry (2.2.23, Method II). cs = mean of concentrations of the substance to be
Test solution. Dissolve a quantity of the substance to be examined in the test solutions, in milligrams per
examined equivalent to 0.25 g of the dried substance in 1 mL gram ;
of nitric acid R by heating on a water-bath. Cool and dilute to Z = determined percentage content of C6H10O7 in
10.0 mL with water R. D-glucuronic acid R ;
Reference solutions. Prepare 2 reference solutions in the h = percentage loss on drying ;
same manner as the test solution, adding 1.0 mL and 2.0 mL
respectively of iron standard solution (10 ppm Fe) R to the 401.3 = relative molecular mass of the disaccharide
dissolved substance to be examined. fragment ;
Source : iron hollow-cathode lamp using a transmission band 194.1 = relative molecular mass of glucuronic acid.
of 0.2 nm.
Wavelength : 248.3 nm. STORAGE
Atomisation device : air-acetylene flame. In an airtight container, protected from light and humidity.
If the substance is sterile, store in a sterile, airtight,
Loss on drying (2.2.32) : maximum 20.0 per cent, determined tamper-evident container.
on 0.500 g by drying at 100-110 °C over diphosphorus
pentoxide R for 6 h. LABELLING
Microbial contamination The label states :
TAMC : acceptance criterion 102 CFU/g (2.6.12). Use 1 g of – the intrinsic viscosity ;
the substance to be examined. – the origin of the substance ;

www.webofpharma.com
General Notices (1) apply to all monographs and other texts 3823
Sodium hydrogen carbonate EUROPEAN PHARMACOPOEIA 10.0

– the intended use of the substance ; ASSAY

– where applicable, that the substance is suitable for Dissolve 0.750 g in 50 mL of carbon dioxide-free water R.
parenteral administration other than intra-articular Titrate with 1 M hydrochloric acid, determining the end-point
administration ; potentiometrically (2.2.20). Read the volume added at the
– where applicable, that the substance is suitable for 2nd point of inflection, or at the point of inflection if only
parenteral administration, including intra-articular 1 point is detected.
administration ; 1 mL of 1 M hydrochloric acid is equivalent to 84.0 mg
– where applicable that the material is suitable for intra-ocular of NaHCO3.
use.
01/2017:0677
01/2017:0195

SODIUM HYDROXIDE
SODIUM HYDROGEN CARBONATE
Natrii hydroxidum
Natrii hydrogenocarbonas
NaOH Mr 40.00
NaHCO3 Mr 84.0 [1310-73-2]
[144-55-8]
DEFINITION
DEFINITION Content : 97.0 per cent to 100.5 per cent.
Content : 99.0 per cent to 101.0 per cent.
CHARACTERS
CHARACTERS Appearance : white or almost white, crystalline masses,
Appearance : white or almost white, crystalline powder. supplied as pellets, sticks or slabs, deliquescent, readily
Solubility : soluble in water, practically insoluble in ethanol absorbing carbon dioxide.
(96 per cent). Solubility : very soluble in water, freely soluble in ethanol
When heated in the dry state or in solution, it gradually (96 per cent).
changes into sodium carbonate.
IDENTIFICATION
IDENTIFICATION A. pH (2.2.3): minimum 11.0.
A. To 5 mL of solution S (see Tests) add 0.1 mL of Dissolve 0.1 g in 10 mL of water R. Dilute 1 mL of the
phenolphthalein solution R. A pale pink colour is produced. solution to 100 mL with water R.
Heat ; gas is evolved and the solution becomes red. B. 2 mL of solution S (see Tests) gives reaction (a) of sodium
B. It gives the reaction of carbonates and bicarbonates (2.3.1). (2.3.1).
C. Solution S gives reaction (a) of sodium (2.3.1).
TESTS
TESTS Solution S. Carry out the procedure described below with
Solution S. Dissolve 5.0 g in 90 mL of carbon dioxide-free caution. Dissolve 5.0 g in 12 mL of water R. Add 17 mL of
water R and dilute to 100.0 mL with the same solvent. hydrochloric acid R1, adjust to pH 7 with a 103 g/L solution of
Appearance of solution. Solution S is clear (2.2.1) and hydrochloric acid R and dilute to 50 mL with water R.
colourless (2.2.2, Method II). Appearance of solution. The solution is clear (2.2.1) and
Carbonates. The pH (2.2.3) of freshly prepared solution S colourless (2.2.2, Method II).
is not greater than 8.6. Dissolve 1.0 g in 10 mL of water R.
Chlorides (2.4.4): maximum 150 ppm. Carbonates : maximum 2.0 per cent, calculated as Na2CO3 as
To 7 mL of solution S add 2 mL of nitric acid R and dilute to determined in the assay.
15 mL with water R. Chlorides (2.4.4) : maximum 200 ppm.
Sulfates (2.4.13) : maximum 150 ppm. Dissolve 0.25 g in 5 mL of water R, acidify the solution with
To a suspension of 1.0 g in 10 mL of distilled water R add about 4 mL of nitric acid R and dilute to 15 mL with water R.
hydrochloric acid R until neutral and about 1 mL in excess. The solution, without addition of dilute nitric acid R, complies
Dilute to 15 mL with distilled water R. with the test.
Ammonium (2.4.1) : maximum 20 ppm. Sulfates (2.4.13): maximum 200 ppm.
Dilute 10 mL of solution S to 15 mL with water R. Prepare the Dissolve 0.75 g in 6 mL of distilled water R, adjust to pH 7 with
standard using a mixture of 5 mL of water R and 10 mL of hydrochloric acid R and dilute to 15 mL with distilled water R.
ammonium standard solution (1 ppm NH4) R. Iron (2.4.9) : maximum 10 ppm, determined on solution S.
Arsenic (2.4.2, Method A) : maximum 2 ppm, determined on ASSAY
0.5 g.
Dissolve 2.000 g in about 80 mL of carbon dioxide-free water R.
Calcium (2.4.3) : maximum 100 ppm. Add 0.3 mL of phenolphthalein solution R and titrate with 1 M
To a suspension of 1.0 g in 10 mL of distilled water R add hydrochloric acid. Add 0.3 mL of methyl orange solution R and
hydrochloric acid R until neutral and dilute to 15 mL with continue the titration with 1 M hydrochloric acid.
distilled water R. 1 mL of 1 M hydrochloric acid used in the 2nd part of the
Iron (2.4.9) : maximum 20 ppm. titration is equivalent to 0.1060 g of Na2CO3.
Dissolve 0.5 g in 5 mL of dilute hydrochloric acid R and dilute 1 mL of 1 M hydrochloric acid used in the combined titrations
to 10 mL with water R. is equivalent to 40.00 mg of total alkali, calculated as NaOH.

www.webofpharma.com
3824 See the information section on general monographs (cover pages)