EUROPEAN PHARMACOPOEIA 10.0 2.5.4.

Iodine value

2.5. ASSAYS Presumed value IOH Quantity of sample

(g)
Volume of
acetylating
reagent (mL)
01/2008:20501 200 - 250 0.75 5.0
corrected 8.6 250 - 300 0.60 or 1.20 5.0 or 10.0

300 - 350 1.0 10.0

350 - 700 0.75 15.0

2.5.1. ACID VALUE 700 - 950 0.5 15.0

The acid value IA is the number that expresses, in milligrams Heat the flask in a water-bath for 1 h keeping the level of the
the quantity of potassium hydroxide required to neutralise the water about 2.5 cm above the level of the liquid in the flask.
free acids present in 1 g of the substance. Withdraw the flask and allow to cool. Add 5 mL of water R
Dissolve 10.00 g of the substance to be examined, or the through the upper end of the condenser. If a cloudiness
quantity prescribed, (m g), in 50 mL of a mixture of equal appears add sufficient pyridine R to clear it, noting the volume
volumes of ethanol (96 per cent) R and light petroleum R3, added. Shake the flask and replace in the water-bath for
previously neutralised with 0.1 M potassium hydroxide 10 min. Withdraw the flask and allow to cool. Rinse the
or 0.1 M sodium hydroxide, unless otherwise specified, condenser and the walls of the flask with 5 mL of alcohol R,
using 0.5 mL of phenolphthalein solution R1 as indicator. If previously neutralised to phenolphthalein solution R1. Titrate
necessary, heat to about 90 °C to dissolve the substance to be with 0.5 M alcoholic potassium hydroxide using 0.2 mL of
examined. When the substance to be examined has dissolved, phenolphthalein solution R1 as indicator (n1 mL of 0.5 M
titrate with 0.1 M potassium hydroxide or 0.1 M sodium alcoholic potassium hydroxide). Carry out a blank test under
hydroxide until the pink colour persists for at least 15 s (n mL the same conditions (n2 mL of 0.5 M alcoholic potassium
of titrant). When heating has been applied to aid dissolution, hydroxide).
maintain the temperature at about 90 °C during the titration.
28.05(n2 - n1 )
5.611n IOH = + IA
IA = m
m
METHOD B
01/2008:20502 Introduce the prescribed quantity of the substance to be
examined (m g) into a perfectly dry 5 mL conical flask fitted
with a ground-glass or suitable plastic stopper and add 2.0 mL
of propionic anhydride reagent R. Close the flask and shake
gently to dissolve the substance. Allow to stand for 2 h unless
2.5.2. ESTER VALUE otherwise prescribed. Remove the stopper and transfer the
flask and its contents into a wide-mouthed 500 mL conical
The ester value IE is the number that expresses in milligrams flask containing 25.0 mL of a 9 g/L solution of aniline R in
the quantity of potassium hydroxide required to saponify the cyclohexane R and 30 mL of glacial acetic acid R. Swirl the
esters present in 1 g of the substance. It is calculated from the contents of the flask, allow to stand for 5 min, add 0.05 mL
saponification value IS and the acid value IA : of crystal violet solution R and titrate with 0.1 M perchloric
IE = IS - IA acid until an emerald-green colour is obtained (n1 mL of
0.1 M perchloric acid). Carry out a blank test under the same
conditions (n2 mL of 0.1 M perchloric acid).
01/2008:20503
5.610(n1 - n2 )
IOH =
m
To take account of any water present, determine this (y per
cent) by the semi-micro determination of water (2.5.12).
2.5.3. HYDROXYL VALUE
The hydroxyl value is then given by the equation :
The hydroxyl value IOH is the number that expresses in
milligrams the quantity of potassium hydroxide required IOH = (hydroxyl value as determined) - 31.1y
to neutralise the acid combined by acylation in 1 g of the
substance.
METHOD A
Introduce the quantity of the substance to be examined
shown in Table 2.5.3.-1 (m g) into a 150 mL acetylation 01/2008:20504
flask fitted with an air condenser, unless another quantity
is prescribed in the monograph. Add the quantity of acetic
anhydride solution R1 stated in Table 2.5.3.-1 and attach the
air condenser.
Table 2.5.3.-1 2.5.4. IODINE VALUE
Presumed value IOH Quantity of sample Volume of
(g) acetylating The iodine value II is the number that expresses in grams the
reagent (mL) quantity of halogen, calculated as iodine, that can be fixed in
10 - 100 2.0 5.0 the prescribed conditions by 100 g of the substance.
100 - 150 1.5 5.0 When the monograph does not specify the method to be used,
150 - 200 1.0 5.0
method A is applied. Any change from method A to method B
is validated.

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General Notices (1) apply to all monographs and other texts 167
2.5.5. Peroxide value EUROPEAN PHARMACOPOEIA 10.0

METHOD A volume of iodine chloride solution R stated in Table 2.5.4.-2.

Unless otherwise prescribed, use the following quantities Close the flask and keep it in the dark for 30 min, unless
(Table 2.5.4.-1) for the determination. otherwise prescribed, shaking frequently. Add 10 mL of a
100 g/L solution of potassium iodide R and 100 mL of water R.
Table 2.5.4.-1 Titrate with 0.1 M sodium thiosulfate, shaking vigorously until
Presumed value II Quantity of sample (g) the yellow colour is almost discharged. Add 5 mL of starch
1.0
solution R and continue the titration adding the 0.1 M sodium
less than 20
thiosulfate dropwise until the colour is discharged (n1 mL of
20 - 60 0.5 - 0.25 0.1 M sodium thiosulfate). Carry out a blank test under the
60 - 100 0.25 - 0.15
same conditions (n2 mL of 0.1 M sodium thiosulfate).
1.269 (n2 - n1 )
more than 100 0.15 - 0.10 II =
m
Introduce the prescribed quantity of the substance to be
examined (m g) into a 250 mL flask fitted with a ground-glass 01/2016:20505
stopper and previously dried or rinsed with glacial acetic
acid R, and dissolve it in 15 mL of chloroform R unless
otherwise prescribed. Add very slowly 25.0 mL of iodine
bromide solution R. Close the flask and keep it in the dark for
30 min unless otherwise prescribed, shaking frequently. Add 2.5.5. PEROXIDE VALUE
10 mL of a 100 g/L solution of potassium iodide R and 100 mL
of water R. Titrate with 0.1 M sodium thiosulfate, shaking The peroxide value IP is the number that expresses in
vigorously until the yellow colour is almost discharged. Add milliequivalents of active oxygen the quantity of peroxide
5 mL of starch solution R and continue the titration adding contained in 1000 g of the substance, as determined by the
the 0.1 M sodium thiosulfate dropwise until the colour is methods described below.
discharged (n1 mL of 0.1 M sodium thiosulfate). Carry out a When the monograph does not specify the method to be used,
blank test under the same conditions (n2 mL of 0.1 M sodium method A is applied. Any change from method A to method B
thiosulfate). is validated.
1.269 (n2 - n1 ) METHOD A
II =
m Place 5.00 g of the substance to be examined (m g) in a 250 mL
conical flask fitted with a ground-glass stopper. Add 30 mL
METHOD B of a mixture of 2 volumes of chloroform R and 3 volumes of
Unless otherwise prescribed, use the following quantities glacial acetic acid R. Shake to dissolve the substance and add
(Table 2.5.4.-2) for the determination. 0.5 mL of saturated potassium iodide solution R. Shake for
Table 2.5.4.-2 exactly 1 min then add 30 mL of water R. Titrate with 0.01 M
sodium thiosulfate, adding the titrant slowly with continuous
Presumed Mass (g) Mass (g) Iodine
value II (corresponding (corresponding chloride
vigorous shaking, until the yellow colour is almost discharged.
to an excess of to an excess of solution (mL) Add 5 mL of starch solution R and continue the titration,
150 per cent 100 per cent ICl) shaking vigorously, until the colour is discharged (n1 mL of
ICl) 0.01 M sodium thiosulfate). Carry out a blank test under the
<3 10 10 25 same conditions (n2 mL of 0.01 M sodium thiosulfate). The
3 8.4613 10.5760 25 volume of 0.01 M sodium thiosulfate used in the blank titration
must not exceed 0.1 mL.
5 5.0770 6.3460 25
10(n1 - n2 )
10 2.5384 3.1730 20 Ip =
m
20 0.8461 1.5865 20 METHOD B
40 0.6346 0.7935 20 Carry out the operations avoiding exposure to actinic light.
60 0.4321 0.5288 20 Place 50 mL of a mixture of 2 volumes of trimethylpentane R
and 3 volumes of glacial acetic acid R in a conical flask and
80 0.3173 0.3966 20 replace the stopper. Swirl the flask until the substance to be
100 0.2538 0.3173 20 examined (m g ; see Table 2.5.5.-1) has dissolved. Using a
suitable volumetric pipette, add 0.5 mL of saturated potassium
120 0.2115 0.2644 20 iodide solution R and replace the stopper. Allow the solution
140 0.1813 0.2266 20 to stand for 60 ± 1 s, thoroughly shaking the solution
continuously, then add 30 mL of water R.
160 0.1587 0.1983 20
Table 2.5.5.-1
180 0.1410 0.1762 20
Expected peroxide Mass of substance
200 0.1269 0.1586 20 value Ip to be examined (g)
0 to 12 5.00 to 2.00
The mass of the sample is such that there will be an excess of
iodine chloride solution R of 50 per cent to 60 per cent of the 12 to 20 2.00 to 1.20
amount added, i.e. 100 per cent to 150 per cent of the amount
20 to 30 1.20 to 0.80
absorbed.
Introduce the prescribed quantity of the substance to be 30 to 50 0.800 to 0.500
examined (m g) into a 250 mL flask fitted with a ground-glass 50 to 90 0.500 to 0.300
stopper and previously rinsed with glacial acetic acid R or
dried, and dissolve it in 15 mL of a mixture of equal volumes Titrate the solution with 0.01 M sodium thiosulfate (V1 mL),
of cyclohexane R and glacial acetic acid R, unless otherwise adding it gradually and with constant, vigorous shaking, until
prescribed. If necessary, melt the substance before dissolution the yellow iodine colour has almost disappeared. Add about
(melting point greater than 50 °C). Add very slowly the 0.5 mL of starch solution R1 and continue the titration, with

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168 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 10.0 2.5.8. Determination of primary aromatic amino-nitrogen

constant shaking especially near the end-point, to liberate all of 28.05(n2 - n1 )

IS =
the iodine from the solvent layer. Add the sodium thiosulfate m
solution dropwise until the blue colour just disappears.
Depending on the volume of 0.01 M sodium thiosulfate used,
it may be necessary to titrate with 0.1 M sodium thiosulfate.
NOTE : there is a 15 s to 30 s delay in neutralising the starch 01/2008:20507
indicator for peroxide values of 70 and greater, due to the
tendency of trimethylpentane to float on the surface of the
aqueous medium and the time necessary to adequately mix the
solvent and the aqueous titrant, thus liberating the last traces
of iodine. It is recommended to use 0.1 M sodium thiosulfate
for peroxide values greater than 150. A small amount (0.5 per
2.5.7. UNSAPONIFIABLE MATTER
cent to 1.0 per cent m/m) of high HLB emulsifier (for example The term “unsaponifiable matter” is applied to the substances
polysorbate 60) may be added to the mixture to retard the non-volatile at 100-105 °C obtained by extraction with an
phase separation and decrease the time lag in the liberation organic solvent from the substance to be examined after it has
of iodine. been saponified. The result is calculated as per cent m/m.
Carry out a blank determination (V0 mL). If the result of Use ungreased ground-glass glassware.
the blank determination exceeds 0.1 mL of titration reagent,
replace the reagents and repeat the determination. Introduce the prescribed quantity of the substance to be
examined (m g) into a 250 mL flask fitted with a reflux
condenser. Add 50 mL of 2 M alcoholic potassium hydroxide R
1000(V1 - V0)c
Ip = and heat on a water-bath for 1 h, swirling frequently. Cool
m to a temperature below 25 °C and transfer the contents of
c the flask to a separating funnel with the aid of 100 mL of
= concentration of the sodium thiosulfate solution,
water R. Shake the liquid carefully with 3 quantities, each of
in moles per litre.
100 mL, of peroxide-free ether R. Combine the ether layers
in another separating funnel containing 40 mL of water R,
shake gently for a few minutes, allow to separate and reject
the aqueous phase. Wash the ether phase with 2 quantities,
each of 40 mL, of water R then wash successively with 40 mL
of a 30 g/L solution of potassium hydroxide R and 40 mL of
01/2008:20506 water R ; repeat this procedure 3 times. Wash the ether phase
several times, each with 40 mL of water R, until the aqueous
phase is no longer alkaline to phenolphthalein. Transfer the
ether phase to a tared flask, washing the separating funnel
with peroxide-free ether R.

2.5.6. SAPONIFICATION VALUE Distil off the ether with suitable precautions and add 6 mL of
acetone R to the residue. Carefully remove the solvent in a
current of air. Dry to constant mass at 100-105 °C. Allow to
The saponification value IS is the number that expresses in cool in a desiccator and weigh (a g).
milligrams the quantity of potassium hydroxide required to
neutralise the free acids and to saponify the esters present in 100a
1 g of the substance. Unsaponifiable matter = per cent
m
Unless otherwise prescribed, use the quantities indicated in Dissolve the residue in 20 mL of alcohol R, previously
Table 2.5.6.-1 for the determination. neutralised to phenolphthalein solution R and titrate with
0.1 M ethanolic sodium hydroxide. If the volume of 0.1 M
Table 2.5.6.-1 ethanolic sodium hydroxide used is greater than 0.2 mL, the
Presumed value IS Quantity of sample (g) separation of the layers has been incomplete ; the residue
weighed cannot be considered as “unsaponifiable matter”. In
<3 20 case of doubt, the test must be repeated.
3 to 10 12 to 15

10 to 40 8 to 12

40 to 60 5 to 8
01/2008:20508
60 to 100 3 to 5

100 to 200 2.5 to 3

200 to 300 1 to 2

300 to 400 0.5 to 1

Introduce the prescribed quantity of the substance to be
examined (m g) into a 250 mL borosilicate glass flask fitted
with a reflux condenser. Add 25.0 mL of 0.5 M alcoholic
potassium hydroxide and a few glass beads. Attach the
condenser and heat under reflux for 30 min, unless otherwise
prescribed. Add 1 mL of phenolphthalein solution R1 and
titrate immediately (while still hot) with 0.5 M hydrochloric
acid (n1 mL of 0.5 M hydrochloric acid). Carry out a blank test
under the same conditions (n2 mL of 0.5 M hydrochloric acid).
2.5.8. DETERMINATION OF PRIMARY
AROMATIC AMINO-NITROGEN
Dissolve the prescribed quantity of the substance to be
examined in 50 mL of dilute hydrochloric acid R or in another
prescribed solvent and add 3 g of potassium bromide R. Cool
in ice-water and titrate by slowly adding 0.1 M sodium nitrite
with constant stirring.
Determine the end-point electrometrically or by the use of
the prescribed indicator.

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General Notices (1) apply to all monographs and other texts 169
2.5.9. Determination of nitrogen by sulfuric acid digestion EUROPEAN PHARMACOPOEIA 10.0

01/2008:20509 otherwise prescribed, carefully unstopper the flask. Wash the

ground parts and the walls of the flask, as well as the sample
carrier, with water R. Combine the combustion products and
the washings and proceed as prescribed in the monograph.

2.5.9. DETERMINATION OF
NITROGEN BY SULFURIC ACID 01/2008:20511
corrected 8.0
DIGESTION
SEMI-MICRO METHOD
Place a quantity of the substance to be examined (m g)
containing about 2 mg of nitrogen in a combustion flask, add
4 g of a powdered mixture of 100 g of dipotassium sulfate R, 2.5.11. COMPLEXOMETRIC
5 g of copper sulfate pentahydrate R and 2.5 g of selenium R, TITRATIONS
and three glass beads. Wash any adhering particles from the
neck into the flask with 5 mL of sulfuric acid R, allowing it ALUMINIUM
to run down the sides of the flask, and mix the contents by Introduce 20.0 mL of the prescribed solution into a 500 mL
rotation. Close the mouth of the flask loosely, for example by conical flask, add 25.0 mL of 0.1 M sodium edetate and
means of a glass bulb with a short stem, to avoid excessive 10 mL of a mixture of equal volumes of a 155 g/L solution of
loss of sulfuric acid. Heat gradually at first, then increase the ammonium acetate R and dilute acetic acid R. Boil for 2 min,
temperature until there is vigorous boiling with condensation then cool. Add 50 mL of ethanol R and 3 mL of a freshly
of sulfuric acid in the neck of the flask ; precautions should be prepared 0.25 g/L solution of dithizone R in ethanol R. Titrate
taken to prevent the upper part of the flask from becoming the excess of sodium edetate with 0.1 M zinc sulfate until the
overheated. Continue the heating for 30 min, unless otherwise colour changes from greenish-blue to reddish-violet.
prescribed. Cool, dissolve the solid material by cautiously 1 mL of 0.1 M sodium edetate is equivalent to 2.698 mg of Al.
adding to the mixture 25 mL of water R, cool again and place
in a steam-distillation apparatus. Add 30 mL of strong sodium BISMUTH
hydroxide solution R and distil immediately by passing steam Introduce the prescribed solution into a 500 mL conical flask.
through the mixture. Collect about 40 mL of distillate in Dilute to 250 mL with water R and then, unless otherwise
20.0 mL of 0.01 M hydrochloric acid and enough water R prescribed, add dropwise, with shaking, concentrated
to cover the tip of the condenser. Towards the end of the ammonia R until the mixture becomes cloudy. Add 0.5 mL
distillation, lower the receiver so that the tip of the condenser of nitric acid R. Heat to about 70 °C until the cloudiness
is above the surface of the acid. Take precautions to prevent disappears completely. Add about 50 mg of xylenol orange
any water on the outer surface of the condenser from reaching triturate R and titrate with 0.1 M sodium edetate until the
the contents of the receiver. Titrate the distillate with 0.01 M colour changes from pinkish-violet to yellow.
sodium hydroxide, using methyl red mixed solution R as
indicator (n1 mL of 0.01 M sodium hydroxide). 1 mL of 0.1 M sodium edetate is equivalent to 20.90 mg of Bi.
Repeat the test using about 50 mg of glucose R in place of the CALCIUM
substance to be examined (n2 mL of 0.01 M sodium hydroxide). Introduce the prescribed solution into a 500 mL conical
0.01401(n2 - n1 ) flask, and dilute to 300 mL with water R. Add 6.0 mL of
Content of nitrogen = per cent strong sodium hydroxide solution R and about 200 mg of
m calconecarboxylic acid triturate R. Titrate with 0.1 M sodium
edetate until the colour changes from violet to full blue.
01/2008:20510 1 mL of 0.1 M sodium edetate is equivalent to 4.008 mg of Ca.
MAGNESIUM
Introduce the prescribed solution into a 500 mL conical flask
and dilute to 300 mL with water R. Add 10 mL of ammonium
chloride buffer solution pH 10.0 R and about 50 mg of mordant
2.5.10. OXYGEN-FLASK METHOD black 11 triturate R. Heat to about 40 °C then titrate at this
temperature with 0.1 M sodium edetate until the colour
Unless otherwise prescribed the combustion flask is a conical changes from violet to full blue.
flask of at least 500 mL capacity of borosilicate glass with
a ground-glass stopper fitted with a suitable carrier for the 1 mL of 0.1 M sodium edetate is equivalent to 2.431 mg of Mg.
sample, for example in platinum or platinum-iridium. LEAD
Finely grind the substance to be examined, place the Introduce the prescribed solution into a 500 mL conical flask
prescribed quantity in the centre of a piece of filter paper and dilute to 200 mL with water R. Add about 50 mg of
measuring about 30 mm by 40 mm provided with a small strip xylenol orange triturate R and hexamethylenetetramine R until
about 10 mm wide and 30 mm long. If paper impregnated the solution becomes violet-pink. Titrate with 0.1 M sodium
with lithium carbonate is prescribed, moisten the centre of edetate until the violet-pink colour changes to yellow.
the paper with a saturated solution of lithium carbonate R
and dry in an oven before use. Envelop the substance to be 1 mL of 0.1 M sodium edetate is equivalent to 20.72 mg of Pb.
examined in the paper and place it in the sample carrier. ZINC
Introduce into the flask water R or the prescribed solution
designed to absorb the combustion products, displace the air Introduce the prescribed solution into a 500 mL conical
with oxygen by means of a tube having its end just above the flask and dilute to 200 mL with water R. Add about 50 mg
liquid, moisten the neck of the flask with water R and close of xylenol orange triturate R and hexamethylenetetramine R
with its stopper. Ignite the paper strip by suitable means with until the solution becomes violet-pink. Add 2 g of
the usual precautions. Keep the flask firmly closed during the hexamethylenetetramine R in excess. Titrate with 0.1 M
combustion. Shake the flask vigorously to completely dissolve sodium edetate until the violet-pink colour changes to yellow.
the combustion products. Cool and after about 5 min, unless 1 mL of 0.1 M sodium edetate is equivalent to 6.54 mg of Zn.

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170 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 10.0
04/2018:20512 stirring. Titrate the excess of reagent using methanol R or the
prescribed solvent, containing an accurately known quantity
of water.
2.5.12. WATER: SEMI-MICRO
DETERMINATION
The semi-micro determination of water is based upon the
quantitative reaction of water with sulfur dioxide and iodine
in a suitable anhydrous medium in the presence of a base with determined using the reagent/solvent system chosen.
sufficient buffering capacity.
APPARATUS
The apparatus consists of a titration vessel with :
– 2 identical platinum electrodes ;
– tight inlets for introduction of solvent and titrant ;
– an inlet for introduction of air via a desiccant ;
– a sample inlet fitted with a stopper or, for liquids, a septum.
Inlet systems for introduction of dry nitrogen or for aspiration
of solvents may also be fitted.
The titration is carried out according to the instrument
supplier’s instructions. Care is taken throughout the
determination to avoid exposure of reagents and solvents to
atmospheric moisture. The end-point is determined using
2 identical indicator electrodes connected to an electrical
source that maintains between the electrodes either a constant Calculate the regression line. The x-axis represents the
current (2.2.65. Voltametric titration) or a constant voltage
(2.2.19. Amperometric titration). Where direct titration is
used (method A), addition of titrant causes either a decrease
in voltage where constant current is maintained or an increase Calculate the slope (b), the intercept with the y-axis (a) and
in current where constant voltage is maintained, until the
end-point is reached. Instruments with automatic end-point
detection are commonly used. Instrument qualification
is carried out according to established quality system
procedures, for example using a suitable certified reference
material (sodium aminosalicylate dihydrate for equipment
qualification CRS may be used).
STANDARDISATION
To the titration vessel, add methanol R, dried if necessary,
or the solvent recommended by the supplier of the titrant.
Where applicable for the apparatus used, eliminate residual
water from the measurement cell or carry out a pre-titration.
Introduce a suitable amount of water in an appropriate form
(water R or a certified reference material) and carry out the
titration, stirring for the necessary time. The water equivalent
is not less than 80 per cent of that indicated by the supplier.
Standardise the titrant before the first use and at suitable
intervals thereafter.
Unless otherwise prescribed, use Method A.
METHOD A
Introduce into the titration vessel methanol R, or the solvent
indicated in the monograph or recommended by the supplier
of the titrant. Where applicable for the apparatus used,
eliminate residual water from the measurement cell or carry
out a pre-titration. Introduce the substance to be examined
rapidly and carry out the titration, stirring for the necessary
extraction time.
METHOD B
Introduce into the titration vessel methanol R, or the solvent
indicated in the monograph or recommended by the supplier
of the titrant. Where applicable for the apparatus used,
eliminate residual water from the measurement cell or carry
out a pre-titration. Introduce the substance to be examined
rapidly and in a suitable state of division. Add an accurately
measured volume of the titrant, sufficient to give an excess
of about 1 mL or the prescribed volume. Allow to stand
protected from light for 1 min or the prescribed time, with
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General Notices (1) apply to all monographs and other texts
2.5.13. Aluminium in adsorbed vaccines
SUITABILITY
The accuracy of the determination with the chosen titrant
must be verified for each combination of substance, titrant and
solvent to be examined. The following procedure, given as an
example, is suitable for samples containing 2.5-25 mg of water.
The water content of the substance to be examined is
Thereafter, in the same titration vessel, sequential known
amounts of water, corresponding to about 50-100 per cent
of the amount found in the substance to be examined, are
added in an appropriate form (at least 5 additions) and the
water content is determined after each addition. Calculate the
percentage recovery (r) after each addition using the following
expression :
W
r = 100 2
W1
W1 = amount of water added, in milligrams ;
W2 = amount of water found, in milligrams.
Calculate the mean percentage recovery ( r ). The
reagent/solvent system is considered to be acceptable if r is
between 97.5 per cent and 102.5 per cent.
cumulative water added whereas the y-axis represents the sum
of the initial water content determined for the substance (M)
and the cumulative water determined after each addition.
the intercept of the extrapolated calibration line with the
x-axis (d).
Calculate the percentage errors (e1 and e2) using the following
expressions :
a-M
e1 = 100
M
d -M
e 2 = 100
M
a = the y-axis intercept, in milligrams of water ;
d = the x-axis intercept, in milligrams of water ;
M = water content of the substance, in milligrams of
water.
The reagent/solvent system is considered to be acceptable if :
– e1 and e 2 are not greater than 2.5 per cent ;
– b is between 0.975 and 1.025.
01/2008:20513
2.5.13. ALUMINIUM IN ADSORBED
VACCINES
Homogenise the preparation to be examined and transfer
a suitable quantity, presumed to contain 5 mg to 6 mg of
aluminium, to a 50 mL combustion flask. Add 1 mL of sulfuric
acid R, 0.1 mL of nitric acid R and some glass beads. Heat
the solution until thick, white fumes are evolved. If there is
charring at this stage add a few more drops of nitric acid R
and continue boiling until the colour disappears. Allow to
cool for a few minutes, carefully add 10 mL of water R and
boil until a clear solution is obtained. Allow to cool, add
0.05 mL of methyl orange solution R and neutralise with
strong sodium hydroxide solution R (6.5 mL to 7 mL). If a
171
2.5.14. Calcium in adsorbed vaccines EUROPEAN PHARMACOPOEIA 10.0

2 : 250 mg of protein per litre). Place in 6 glass tubes 0.10 mL,

precipitate forms dissolve it by adding, dropwise, sufficient
0.20 mL and 0.40 mL of working dilution 1 and 0.15 mL,
dilute sulfuric acid R. Transfer the solution to a 250 mL
conical flask, rinsing the combustion flask with 25 mL of0.20 mL and 0.25 mL of working dilution 2. Make up the
water R. Add 25.0 mL of 0.02 M sodium edetate, 10 mL ofvolume in each tube to 0.40 mL using 0.1 M sodium hydroxide.
acetate buffer solution pH 4.4 R and a few glass beads and boil
Prepare a blank using 0.40 mL of 0.1 M sodium hydroxide.
gently for 3 min. Add 0.1 mL of pyridylazonaphthol solution R
Add 2 mL of cupri-tartaric solution R3 to each tube, shake
and titrate the hot solution with 0.02 M copper sulfate until the
and allow to stand for 10 min. Add to each tube 0.2 mL
colour changes to purplish-brown. Carry out a blank titration
of a mixture of equal volumes of phosphomolybdotungstic
omitting the vaccine. reagent R and water R, prepared immediately before use.
1 mL of 0.02 M sodium edetate is equivalent to 0.5396 mg of Al.
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
01/2008:20514 necessary, centrifuge to obtain clear solutions.
Measure the absorbance (2.2.25) of each solution at 760 nm
using the blank as the compensation liquid. Draw a calibration
curve from the absorbances of the 6 reference solutions and
the corresponding protein contents and read from the curve
2.5.14. CALCIUM IN ADSORBED the content of protein in the test solution.
VACCINES 01/2008:20517
All solutions used for this test must be prepared using water R.
Determine the calcium by atomic emission spectrometry
(2.2.22, Method I). Homogenise the preparation to be
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 2.5.17. NUCLEIC ACIDS IN
at 620 nm. POLYSACCHARIDE VACCINES
01/2008:20515 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 test solution if necessary to obtain an absorbance
2.5.15. PHENOL IN IMMUNOSERA value suitable for the instrument used. Measure the absorbance
(2.2.25) at 260 nm using water R as the compensation liquid.
AND VACCINES The absorbance of a 1 g/L solution of nucleic acid at 260 nm is
Homogenise the preparation to be examined. Dilute an 20.
appropriate volume with water R so as to obtain a solution
presumed to contain 15 μg of phenol per millilitre. Prepare 01/2008:20518
a series of reference solutions with phenol R containing
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 aminopyrazolone solution R 2.5.18. PHOSPHORUS IN
and 5 mL of potassium ferricyanide solution R. Allow to stand
for 10 min and measure the intensity of colour at 546 nm. POLYSACCHARIDE VACCINES
Plot the calibration curve and calculate the phenol content of Test solution. Use a volumetric flask with a suitable volume for
the preparation to be examined. 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
Reference solutions. Dissolve 0.100 g of bovine albumin R in molybdate reagent R. Heat in a water-bath at 37 °C for 90 min
100 mL of 0.1 M sodium hydroxide (stock solution containing and cool. Adjust the volume to 10.0 mL with water R. The
1 g of protein per litre). Dilute 1 mL of the stock solution blue colour is stable for several hours.
to 20 mL with 0.1 M sodium hydroxide (working dilution Measure the absorbance (2.2.25) of each solution at 820 nm
1 : 50 mg of protein per litre). Dilute 1 mL of the stock solution using the blank solution as the compensation liquid. Draw
to 4 mL with 0.1 M sodium hydroxide (working dilution a calibration curve with the absorbances of the 3 reference

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