Intrinsic Viscosity Measurements of Sodium Hyaluronate

according to EU Pharmacopoeia
Relevant for: EU Pharmacopoeia, Polymers, Zero Shear Viscosity, Intrinsic Viscosity

Sodium hyaluronate is an important raw material for various pharmaceutical and medical
applications. To ensure consistent quality and a safe application sodium hyaluronate undergoes
various tests described in the EU Pharmacopoeia. The intrinsic viscosity is one of the required
test parameters for sodium hyaluronate and can easily be measured using a Lovis 2000 M.

1.1 Tests required in EU Pharmacopoeia

The EU Pharmacopoeia is a collection of test methods

for the identification and quality control of several
pharmaceuticals/medicines. The qualitative and
quantitative composition and the tests to be carried
out on medicines, raw materials used in production,
and on the intermediates of synthesis are described.
All producers of medicines and/or substances for
pharmaceutical use must therefore apply these quality
standards in order to market their products.

Tip: Since the release of version 9.3 in July 2017, the

Rolling Ball Principle is included as part of the general
chapter 2.2.49 “Falling Ball and Automatic Rolling Ball
1 Quality Control of Sodium Hyaluronate
Viscometer Methods”.

Sodium hyaluronate is the sodium salt of hyaluronic

acid, which is naturally present in the human body. Apart from other parameters, sodium hyaluronate has
For medicinal applications, hyaluronic acid either has to be tested for its intrinsic viscosity, which has to be
to be extracted from rooster combs or is synthesized stated additionally when labeling the substance.
by bacteria under lab conditions. Sodium hyaluronate
can be classified into different grades and is widely 1.2 Lovis 2000 M/ME in pharmaceutical industry
used in several industries. Some applications are
listed in the table below. Lovis 2000 M/ME is a reliable partner in strongly
regulated fields such as the pharmaceutical industry,
Industry Application as it is compliant with 21 CFR Part 11 and a Pharma
Qualification Package can be offered.
Active pharmaceutical
Pharma Industry ingredient and pharmaceutical
excipient in various drugs In addition to dynamic and kinematic viscosity,
Injectable for joint disorders, Lovis 2000 M/ME offers an integrated polymer
Medical Industry filler in plastic surgery, gels for software for automatic calculation of polymer
wound healing
parameters such as intrinsic viscosity and molar
Cosmetics Industry
Moisturizer in creams, lotions mass.
and gels
Dietary supplement in
Food Industry combination with vitamins, food Tip: Lovis 2000 M/ME can easily be combined with
additive several other Anton Paar instruments, e.g. density
Table 1: Uses of sodium hyaluronate meters, refractometers, and pH meters, to gain more
parameters from one sample. Sample changers for
automatic filling and cleaning are an additional option.

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2 Samples and Sample Preparation 3 Measurements

Nine samples of sodium hyaluronate were This section describes the instrument setup and the
investigated for their intrinsic viscosity [m3/kg]. used method settings.
Sample preparation was performed as described in
the related monograph of the European 3.1 Instrumental setup
Pharmacopoeia and is described below.
For the measurement of the intrinsic viscosity [m3/kg]
2.1 Sample preparation of the hyaluronate solutions a Lovis 2000 M in flow-
through mode was used.
For the intrinsic viscosity measurements, a buffer
solution was prepared for the following dilutions of  Capillary: diameter 1.59 mm /
sodium hyaluronate. borosilicate glass
 Ball: diameter 1.5 mm / steel
2.1.1 Buffer solution  O-rings: Viton extreme

Solution 1: 0.78 g of sodium dihydrogen phosphate

and 4.50 g of sodium chloride were diluted in 500 mL Tip: All polymer parameters derive from the “relative
of deionized water. viscosity”, which is a comparison of the runtime of the
solvent and the polymer dissolved in that solvent.
Solution 2: 1.79 g of disodium hydrogen phosphate Therefore, only the runtime is required and
and 4.50 g of sodium chloride were diluted in 500 mL Lovis 2000 M features as the perfect solution.
of deionized water.
Buffer solution: Solution 1 and 2 were mixed until a pH
value of 7.00 was reached. This buffer solution was 3.2 Method settings
filtered through a sintered glass filter prior to use.
The method Lovis Polymer (Multi Concentration) was
used for the intrinsic viscosity determination of all
2.1.2 Sodium hyaluronate dilutions
solutions. Method settings as well as special polymer
settings in the software are listed below.
Four dilutions of each sodium hyaluronate sample
were prepared from a stock solution as described
below.  Measurement mode: Polymer
 Temperature: 25 °C
 Temperature equilibration: 20 s
Stock solution: 0.2 g of sodium hyaluronate was
 Measurement cycles: 3
dissolved in 50.0 g of buffer solution by stirring the
 Manual angle: Automatic
solution at 4 °C for 24 hours.
 Measuring distance: Automatic
 Variation coefficient: 0.3 %
The dilutions A, B, C, and D were prepared by diluting
a subsequent amount of the stock solution (for dilution
Polymer settings
A) and of dilution A (for dilution B, C, and D) with
buffer solution (Table 2). All dilutions were stirred for  Calculation method: Runtime ratio
20 min and filtered through a sintered glass filter  Angle count
discarding the first 10 mL. (Zero Shear Scan): 4
 Use Zero Shear Scan: Yes
 Concentration unit: kg/m3
Dilution Stock solution Dilution A
Amount buffer  Calculation method: Martin
solution
Dilution A 5.0 g - 100.0 g
Tip: Lovis has an integrated Zero Shear Scan
Dilution B - 30.0 g 10.0 g
function. This means that a solution is automatically
Dilution C - 20.0 g 20.0 g measured at various angles and the result is
Dilution D - 10.0 g 30.0 g extrapolated to a hypothetical zero shear rate.
Table 2: Preparation of the sodium hyaluronate solutions A, B, C,
and D

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3.3 Results 4 Conclusion

In the following the results of the intrinsic viscosity

measurements using the Lovis 2000 M are presented.
Four dilutions of each sample (1 to 9) were measured,
from which the intrinsic viscosity [m3/kg] was
calculated according to Martin (Table 3)
The Lovis 2000 M is well suited for measurements in
the pharmaceutical industry. The measuring principle
is an accepted method in the EU Pharmacopoeia and
US Pharmacopeia and the instrument delivers precise
results from very small sample volumes. Combination
with sample changers for automatic filling and
cleaning can facilitate users' lab routine.

3
Sample Intrinsic viscosity [m /kg]
Sodium hyaluronate 1 1.484
Sodium hyaluronate 2 2.558
Sodium hyaluronate 3 0.830
Sodium hyaluronate 4 1.538
Sodium hyaluronate 5 1.536
Sodium hyaluronate 6 2.539
Sodium hyaluronate 7 1.518
Sodium hyaluronate 8 2.278
Sodium hyaluronate 9 2.138
3
Table 3: Intrinsic viscosity [m /kg] of nine sodium hyaluronate
samples

Table 4 shows the excellent repeatability of seven Figure 1: Lovis 2000 M in flow-through filling mode
subsequent measurements of the same sample.

3
Sample Intrinsic viscosity [m /kg]
Sodium hyaluronate 1 (a) 1.484
5 References
Sodium hyaluronate 1 (b) 1.529
Sodium hyaluronate 1 (c) 1.523
1. European Pharmacopoeia 9.0, Monograph "Sodium
Sodium hyaluronate 1 (d) 1.523 Hyaluronate"
Sodium hyaluronate 1 (e) 1.553
Sodium hyaluronate 1 (f) 1.516 Acknowledgement
Sodium hyaluronate 1 (g) 1.515 Special thanks to Mr. Jisa kamil, Ph.D., from Contipro
3 Pharma a.s., who performed the measurements
Mean value [m /kg] 1.52
3
Standard deviation [m /kg] 0.02
Contact Anton Paar GmbH
RSD [%] 1.35
Table 4: Repeatability in [%] of the subsequent measurement of
sodium hyaluronate sample 1 support-visco@anton-paar.com

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