2.5.14. Calcium in adsorbed vaccines EUROPEAN PHARMACOPOEIA 11.

strong sodium hydroxide solution R (6.5 mL to 7 mL). If a Reference solutions. Dissolve 0.100 g of bovine albumin R in
precipitate forms dissolve it by adding, dropwise, sufficient 100 mL of 0.1 M sodium hydroxide (stock solution containing
dilute sulfuric acid R. Transfer the solution to a 250 mL 1 g of protein per litre). Dilute 1 mL of the stock solution
conical flask, rinsing the combustion flask with 25 mL of to 20 mL with 0.1 M sodium hydroxide (working dilution
water R. Add 25.0 mL of 0.02 M sodium edetate, 10 mL of 1 : 50 mg of protein per litre). Dilute 1 mL of the stock solution
acetate buffer solution pH 4.4 R and a few glass beads and boil to 4 mL with 0.1 M sodium hydroxide (working dilution
gently for 3 min. Add 0.1 mL of pyridylazonaphthol solution R 2 : 250 mg of protein per litre). Place in 6 glass tubes 0.10 mL,
and titrate the hot solution with 0.02 M copper sulfate until the0.20 mL and 0.40 mL of working dilution 1 and 0.15 mL,
colour changes to purplish-brown. Carry out a blank titration 0.20 mL and 0.25 mL of working dilution 2. Make up the
omitting the vaccine. volume in each tube to 0.40 mL using 0.1 M sodium hydroxide.
1 mL of 0.02 M sodium edetate is equivalent to 0.5396 mg of Prepare a blank using 0.40 mL of 0.1 M sodium hydroxide.
Al. Add 2 mL of cupri-tartaric solution R3 to each tube, shake
and allow to stand for 10 min. Add to each tube 0.2 mL
of a mixture of equal volumes of phosphomolybdotungstic
01/2008:20514
reagent R and water R, prepared immediately before use.
Stopper the tubes, mix by inverting and allow to stand in
the dark for 30 min. The blue colour is stable for 60 min. If
necessary, centrifuge to obtain clear solutions.
Measure the absorbance (2.2.25) of each solution at 760 nm
2.5.14. CALCIUM IN ADSORBED using the blank as the compensation liquid. Draw a calibration
VACCINES curve from the absorbances of the 6 reference solutions and
the corresponding protein contents and read from the curve
All solutions used for this test must be prepared using water R. the content of protein in the test solution.
Determine the calcium by atomic emission spectrometry
(2.2.22, Method I). Homogenise the preparation to be 01/2008:20517
examined. To 1.0 mL add 0.2 mL of dilute hydrochloric acid R
and dilute to 3.0 mL with water R. Measure the absorbance
at 620 nm.

01/2008:20515 2.5.17. NUCLEIC ACIDS IN

corrected 11.0 POLYSACCHARIDE VACCINES
Test solution. Use a volumetric flask with a suitable volume for
preparation of a solution containing about 5 mg per millilitre
of dry polysaccharide. Transfer the contents of a container
quantitatively to the flask and dilute to volume with water R.
2.5.15. PHENOL IN IMMUNOSERA Dilute the test solution if necessary to obtain an absorbance
AND VACCINES value suitable for the instrument used. Measure the absorbance
(2.2.25) at 260 nm using water R as the compensation liquid.
Homogenise the preparation to be examined. Dilute an The absorbance of a 1 g/L solution of nucleic acid at 260 nm is
appropriate volume with water R so as to obtain a solution 20.
presumed to contain 15 μg of phenol per millilitre. Prepare
a series of reference solutions with phenol R containing 01/2008:20518
5 μg, 10 μg, 15 μg, 20 μg and 30 μg of phenol per millilitre
respectively. To 5 mL of the solution to be examined and
to 5 mL of each of the reference solutions respectively, add
5 mL of buffer solution pH 9.0 R, 5 mL of 4-aminoantipyrine
solution R and 5 mL of potassium ferricyanide solution R.
Allow to stand for 10 min and measure the intensity of colour 2.5.18. PHOSPHORUS IN
at 546 nm.
POLYSACCHARIDE VACCINES
Plot the calibration curve and calculate the phenol content of
the preparation to be examined. Test solution. Use a volumetric flask with a suitable volume for
preparation of a solution containing about 5 mg per millilitre
of dry polysaccharide. Transfer the contents of a container
01/2008:20516 quantitatively to the flask and dilute to volume with water R.
Dilute the solution so that the volume used in the test (1 mL)
contains about 6 μg of phosphorus. Transfer 1.0 mL of the
solution to a 10 mL ignition tube.
Reference solutions. Dissolve 0.2194 g of potassium dihydrogen
2.5.16. PROTEIN IN POLYSACCHARIDE phosphate R in 500 mL of water R to give a solution containing
the equivalent of 0.1 mg of phosphorus per millilitre. Dilute
VACCINES 5.0 mL of the solution to 100.0 mL with water R. Introduce
Test solution. Use a volumetric flask with a suitable volume for 0.5 mL, 1.0 mL and 2.0 mL of the dilute solution into 3
preparation of a solution containing about 5 mg per millilitre ignition tubes.
of dry polysaccharide. Transfer the contents of a container Prepare a blank solution using 2.0 mL of water R in an ignition
quantitatively to the flask and dilute to volume with water R. tube.
Place 1 mL of the solution in a glass tube and add 0.15 mL of a To all the tubes add 0.2 mL of sulfuric acid R and heat in an
400 g/L solution of trichloroacetic acid R. Shake, allow to stand oil bath at 120 °C for 1 h and then at 160 °C until white fumes
for 15 min, centrifuge for 10 min at 5000 r/min and discard appear (about 1 h). Add 0.1 mL of perchloric acid R and heat at
the supernatant. Add 0.4 mL of 0.1 M sodium hydroxide to the 160 °C until the solution is decolorised (about 90 min). Cool
centrifugation residue. and add to each tube 4 mL of water R and 4 mL of ammonium

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

EUROPEAN PHARMACOPOEIA 11.0 2.5.21. Methylpentoses in polysaccharide vaccines

molybdate reagent R. Heat in a water-bath at 37 °C for 90 min 01/2008:20520

and cool. Adjust the volume to 10.0 mL with water R. The
blue colour is stable for several hours.
Measure the absorbance (2.2.25) of each solution at 820 nm
using the blank solution as the compensation liquid. Draw
a calibration curve with the absorbances of the 3 reference 2.5.20. HEXOSAMINES IN
solutions as a function of the quantity of phosphorus in the POLYSACCHARIDE VACCINES
solutions and read from the curve the quantity of phosphorus
in the test solution. Test solution. Use a volumetric flask with a suitable volume for
preparation of a solution containing about 5 mg per millilitre
of dry polysaccharide. Transfer the contents of a container
quantitatively to the flask and dilute to volume with water R.
Dilute the solution so that the volumes used in the test contain
125 μg to 500 μg of glucosamine (hexosamine). Introduce
1.0 mL of the diluted solution into a graduated tube.
01/2008:20519 Reference solutions. Dissolve 60 mg of glucosamine
corrected 10.0 hydrochloride R in 100 mL of water R (stock solution
containing 0.500 g of glucosamine per litre). Introduce
0.25 mL, 0.50 mL, 0.75 mL, and 1.0 mL of the working dilution
into 4 graduated tubes.
Prepare a blank using 1 mL of water R.
Make up the volume in each tube to 1 mL with water R. Add
2.5.19. O-ACETYL IN 1 mL of a solution of hydrochloric acid R (292 g/L) to each
POLYSACCHARIDE VACCINES tube. Stopper the tubes and place in a water-bath for 1 h.
Cool to room temperature. Add to each tube 0.05 mL of
a 5 g/L solution of thymolphthalein R in alcohol R ; add a
Test solution. Use a volumetric flask with a suitable volume for solution of sodium hydroxide R (200 g/L) until a blue colour
preparation of a solution containing about 5 mg per millilitre is obtained and then 1 M hydrochloric acid until the solution
of dry polysaccharide. Transfer the contents of a container is colourless. Dilute the volume in each tube to 10 mL with
quantitatively to the flask and dilute to volume with water R. water R (neutralised hydrolysates).
Dilute the solution so that the volumes used in the test contain
In a second series of 10 mL graduated tubes, place 1 mL of each
30 μg to 600 μg of acetylcholine chloride (O-acetyl). Introduce
neutralised hydrolysate. Add 1 mL of acetylacetone reagent
0.3 mL, 0.5 mL and 1.0 mL in duplicate into 6 tubes (3 reaction
(a mixture, prepared immediately before use, of 1 volume
solutions and 3 correction solutions).
of acetylacetone R and 50 volumes of a 53 g/L solution of
Reference solutions. Dissolve 0.150 g of acetylcholine chloride R anhydrous sodium carbonate R) to each tube. Stopper the
in 10 mL of water R (stock solution containing 15 g of tubes and place in a water-bath at 90 °C for 45 min. Cool to
acetylcholine chloride per litre). Immediately before use, room temperature. Add to each tube 2.5 mL of alcohol R and
dilute 1 mL of the stock solution to 50 mL with water R 1.0 mL of dimethylaminobenzaldehyde solution (immediately
(working dilution 1 : 300 μg of acetylcholine chloride per before use dissolve 0.8 g of dimethylaminobenzaldehyde R in
millilitre). Immediately before use, dilute 1 mL of the stock 15 mL of alcohol R and add 15 mL of hydrochloric acid R)
solution to 25 mL with water R (working dilution 2 : 600 μg and dilute the volume in each tube to 10 mL with alcohol R.
of acetylcholine chloride per millilitre). Introduce 0.1 mL Stopper the tubes, mix by inverting and allow to stand in the
and 0.4 mL of working dilution 1 in duplicate (reaction and dark for 90 min. Measure the absorbance (2.2.25) of each
correction solutions) into 4 tubes and 0.6 mL and 1.0 mL solution at 530 nm using the blank as the compensation liquid.
of working dilution 2 in duplicate (reaction and correction Draw a calibration curve from the absorbances for the
solutions) into another 4 tubes. 4 reference solutions and the corresponding content of
hexosamine and read from the curve the quantity of
Prepare a blank using 1 mL of water R. hexosamine in the test solution.
Make up the volume in each tube to 1 mL with water R. Add
1.0 mL of a 4 M solution of hydrochloric acid prepared from 01/2008:20521
hydrochloric acid R to each of the correction tubes and to the
blank. Add 2.0 mL of alkaline hydroxylamine solution R to
each tube. Allow the reaction to proceed for exactly 2 min and
add 1.0 mL of the 4 M solution of hydrochloric acid to each
of the reaction tubes. To each tube, add 1.0 mL of a 100 g/L
solution of ferric chloride R in a 0.1 M solution of hydrochloric 2.5.21. METHYLPENTOSES IN
acid prepared from hydrochloric acid R, stopper the tubes and
shake vigorously to remove bubbles. POLYSACCHARIDE VACCINES
Test solution. Use a volumetric flask with a suitable volume
Measure the absorbance (2.2.25) of each solution at 540 nm
for preparation of a solution containing about 5 mg per
using the blank as the compensation liquid. For each reaction
millilitre of dry polysaccharide. Transfer the contents of a
solution, subtract the absorbance of the corresponding
container quantitatively to the flask and dilute to volume
correction solution. Draw a calibration curve from the
with water R. Dilute the solution so that the volumes used in
corrected absorbances for the 4 reference solutions and the
the test contain 2 μg to 20 μg of rhamnose (methylpentoses).
corresponding content of acetylcholine chloride and read
Introduce 0.25 mL, 0.50 mL and 1.0 mL of the diluted solution
from the curve the content of acetylcholine chloride in the
into 3 tubes.
test solution for each volume tested. Calculate the mean of
the 3 values. Reference solutions. Dissolve 0.100 g of rhamnose R in 100 mL
of water R (stock solution containing 1 g of methylpentose
1 mole of acetylcholine chloride (181.7 g) is equivalent to per litre). Immediately before use, dilute 1 mL of the stock
1 mole of O-acetyl (43.05 g). solution to 50 mL with water R (working dilution : 20 mg

General Notices (1) apply to all monographs and other texts 185