2.5.22. Uronic acids in polysaccharide vaccines EUROPEAN PHARMACOPOEIA 10.

of methylpentose and read from the curve the quantity of Reference solutions. Use the reference solutions prescribed in
methylpentose in the test solution for each volume tested. the monograph.
Calculate the mean of the 3 values. Prepare 2 series of 3 test-tubes, place in the tubes of each
series 0.5 mL, 1.0 mL and 1.5 mL respectively, of the reference
solution corresponding to the type of vaccine to be examined
01/2008:20522 and adjust the volume in each tube to 2.0 mL with water R.
Prepare blank solutions using 2.0 mL of water R in each of
2 test-tubes.
To all the tubes add 5.0 mL of resorcinol reagent R. Heat at
105 °C for 15 min, cool in cold water and transfer the tubes
2.5.22. URONIC ACIDS IN to a bath of iced water. To each tube add 5 mL of isoamyl
POLYSACCHARIDE VACCINES alcohol R and mix thoroughly. Place in the bath of iced
water for 15 min. Centrifuge the tubes and keep them in
Test solution. Use a volumetric flask with a suitable volume for the bath of iced water until the examination by absorption
preparation of a solution containing about 5 mg per millilitre spectrophotometry. Measure the absorbance (2.2.25) of each
of dry polysaccharide. Transfer the contents of a container supernatant solution at 580 nm and 450 nm using isoamyl
quantitatively to the flask and dilute to volume with water R. alcohol R as the compensation liquid. For each wavelength,
Dilute the solution so that the volumes used in the test contain calculate the absorbance as the mean of the values obtained
4 μg to 40 μg of glucuronic acid (uronic acids). Introduce with 2 identical solutions. Subtract the mean value for the
0.25 mL, 0.50 mL and 1.0 mL of the diluted solution into blank solution from the mean values obtained for the other
3 tubes. solutions.
Reference solutions. Dissolve 50 mg of sodium glucuronate R Draw a graph showing the difference between the absorbances
in 100 mL of water R (stock solution containing 0.4 g of at 580 nm and 450 nm of the reference solutions as a function
glucuronic acid per litre). Immediately before use, dilute 5 mL of the content of N-acetylneuraminic acid and read from the
of the stock solution to 50 mL with water R (working dilution : graph the quantity of N-acetylneuraminic acid (sialic acid) in
40 mg of glucuronic acid per litre). Introduce 0.10 mL, the test solution.
0.25 mL, 0.50 mL, 0.75 mL, and 1.0 mL of the working dilution
into 5 tubes.
Prepare a blank using 1 mL of water R. 01/2011:20524
Make up the volume in each tube to 1 mL with water R. Place
the tubes in iced water and add dropwise and with continuous
stirring to each tube 5.0 mL of borate solution R. Stopper the
tubes and place in a water-bath for 15 min. Cool to room
temperature. Add 0.20 mL of a 1.25 g/L solution of carbazole R
in ethanol R to each tube. Stopper the tubes and place in a
2.5.24. CARBON DIOXIDE IN GASES
water-bath for 15 min. Cool to room temperature. Measure Gases absorb light at one or more specific wavelengths. This
the absorbance (2.2.25) of each solution at 530 nm using the property is widely used to allow highly selective measurement
blank as the compensation liquid. of their concentrations.
Draw a calibration curve from the absorbances for the Description and principle of measurement. The
5 reference solutions and the corresponding content of concentration of carbon dioxide in other gases can be
glucuronic acid and read from the curve the quantity of determined using an infrared analyser.
glucuronic acid in the test solution for each volume tested. The infrared analyser generally consists of a light source
Calculate the mean of the 3 values. emitting broadband infrared radiation, an optical device,
a sample cell and a detector. The optical device may be
positioned either before or after the sample cell and it consists
01/2008:20523 of one or several optical filters, through which the broadband
radiation is passed. The optical device in this case is selected
for carbon dioxide. The measurement light beam passes
through the sample cell and may also pass through a reference
cell if the analyser integrates such a feature (some use an
2.5.23. SIALIC ACID IN electronic system instead of a reference cell).
POLYSACCHARIDE VACCINES When carbon dioxide is present in the sample cell, absorption
of energy in the measurement light beam will occur according
Test solution. Transfer quantitatively the contents of one or to the Beer-Lambert law and this produces a change in
several containers to a volumetric flask of a suitable volume the detector signal. This measurement signal is compared
that will give a solution with a known concentration of about to a reference signal to generate an output related to the
250 μg per millilitre of polysaccharide and dilute to volume concentration of carbon dioxide. The generated signal is
with water R. Using a syringe, transfer 4.0 mL of this solution linearised in order to obtain the carbon dioxide concentration.
to a 10 mL ultrafiltration cell suitable for the passage of To prevent the entry of particles into the sensors, which could
molecules of relative molecular mass less than 50 000. Rinse cause stray-light phenomena, the apparatus is fitted with a
the syringe twice with water R and transfer the rinsings to the suitable filter.
ultrafiltration cell. Carry out the ultrafiltration, with constant
Required technical specifications. When used for a limit
stirring, under nitrogen R at a pressure of about 150 kPa. Refill
test, the infrared analyser meets the following technical
the cell with water R each time the volume of liquid in it has
specifications :
decreased to 1 mL and continue until 200 mL has been filtered
and the remaining volume in the cell is about 2 mL. Using a – limit of detection : (generally defined as a signal-to-noise
syringe, transfer this residual liquid to a 10 mL volumetric ratio of 2) maximum 20 per cent of the maximum
flask. Wash the cell with 3 quantities, each of 2 mL, of water R, admissible concentration ;
transfer the washings to the flask and dilute to 10.0 mL with – repeatability : maximum relative standard deviation of
water R (test solution). In each of 2 test-tubes place 2.0 mL of 10 per cent of the maximum admissible concentration,
the test solution. determined on 6 measurements ;

174 See the information section on general monographs (cover pages)