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Acacia

Draft monographs and

general texts for comment
IMPORTANT NOTICE
This section contains proposals for new and revised monographs and general texts, intended for inclusion in the
European Pharmacopoeia and submitted for public comment. You can comment through the appropriate national
authority at the address listed on the inside-back cover of this issue. Readers from countries that are not members of the
European Pharmacopoeia Commission should send their comments directly to the European Directorate for the Quality
of Medicines. In order to facilitate the work of the Secretariats of the National Authorities and the European Directorate
for the Quality of Medicines who collect the comments, please mention in any correspondence the PA/PH reference
number indicated at the beginning of each text. Comments that propose modifications of limits should be supported by
analytical data obtained on a significant number of batches. Proposed changes of methodology should be supported by
experimental results of a comparative trial of the method published in Pharmeuropa for comment and the proposed
alternative. Only comments sent before 30 September 2006 will be considered before the final version is prepared.
It is stressed that these proposals have not been adopted by the European Pharmacopoeia Commission and must not
be regarded as official standards.
In the case of proposals for revision, text to be deleted is crossed out and replacements or additions are underlined.
In certain cases, draft monographs require, for appropriate checking, the use of a reference material that is not yet
commercially available; in exceptional circumstances, we will try to make the necessary substance available; please
enquire to the European Directorate for the Quality of Medicines.

Reference: PA/PH/Exp. 13B/T (06) 16 ANP

1-3 cm, frequently with a cracked surface, easily broken

into irregular, whitish or slightly yellowish angular
NOTE ON THE MONOGRAPH
fragments with conchoidal fracture and a glassy and
The European Pharmacopoeia Commission has decided
transparent appearance. In the centre of an unbroken
that more attention will be paid to functionality-related
tear there is sometimes a small cavity.
characteristics (FRCs) in monographs on excipients.
B. Reduce to a powder (355). The powder is white or
While these caracteristics are of importance for the
yellowish-white. Examine under a microscope using
intended use of many excipients, they are essentially a
glycerol R (50 per cent V/V). The powder presents
matter for agreement between the excipient supplier and
angular, irregular, colourless, transparent fragments.
the manufacturer of a medicinal product. For this reason,
Only traces of starch or vegetable tissues are visible. No
FRCs are to be dealt with in a specific non-mandatory
stratified membrane is apparent.
section of the monograph, and the particular use or uses
C. Examine the chromatograms obtained in the test for
for which a given FRC is relevant are to be specified.
glucose and fructose. The chromatogram obtained with
FRCs are very diverse in nature, and their treatment in
the test solution shows 3 zones due to galactose, arabinose
monographs will have to be adapted to each type.
and rhamnose. No other important zones are visible,
XXXX:0307
particularly in the upper part of the chromatogram.
D. Dissolve 1 g of the powdered drug (355) in 2 ml of water R
ACACIA
by stirring frequently for 2 h. Add 2 ml of ethanol (96 per
cent) R. After shaking, a white, gelatinous mucilage is
formed, which becomes fluid on adding 10 ml of water R.
Acaciae gummi
TESTS
Solution S. Dissolve 3.0 g of the powdered drug (355) in
25 ml of water R by stirring for 30 min. Allow to stand for
30 min and dilute to 30 ml with water R.
Insoluble matter. To 5.0 g of the powdered drug (355) add
100 ml of water R and 14 ml of dilute hydrochloric acid R,
CHARACTERS
boil
gently for 15 min, shaking frequently, and filter while
Acacia is almost completely but very slowly soluble, after
hot
through
a tared sintered-glass filter. Wash with hot
about 2 h, in twice its mass of water leaving only a very
water
R
and
dry
at 100-105 C. The residue weighs not more
small residue of vegetable particles ; the liquid obtained is
than
25
mg
(0.5
per
cent).
colourless or yellowish, dense, viscous, adhesive, translucent
Glucose and fructose. Examine by thin-layer chromatography
and weakly acid to blue litmus paper. Acacia is practically
(2.2.27), using a TLC silica gel plate R.
insoluble in ethanol (96 per cent).
It has the macroscopic and microscopic characters described Test solution. To 0.100 g of the powdered drug (355) in a
thick-walled centrifuge tube add 2 ml of a 100 g/l solution
under identification tests A and B.
of trifluoroacetic acid R, shake vigorously to dissolve
IDENTIFICATION
the forming gel, stopper the tube and heat the mixture at
120 C for 1 h. Centrifuge the hydrolysate, transfer the
A. Acacia occurs as yellowish-white, yellow or pale amber,
sometimes with a pinkish tint, friable, opaque, spheroidal, clear supernatant carefully into a 50 ml flask, add 10 ml
of water R and evaporate the solution to dryness under
oval or reniform pieces (tears) with a diameter of about

DEFINITION
Acacia is the air-hardened, gummy exudate flowing naturally
from or obtained by incision of the trunk and branches of
Acacia senegal L. Willdenow, other species of Acacia of
African origin and Acacia seyal Del.

PHARMEUROPA Vol. 18, No. 3, July 2006

381

Acacia, spray-dried

reduced pressure. To the resulting clear film add 0.1 ml of

water R and 0.9 ml of methanol R. Centrifuge to separate the
amorphous precipitate. Dilute the supernatant, if necessary,
to 1 ml with methanol R.
Reference solution. Dissolve 10 mg of arabinose R, 10 mg
of galactose R, 10 mg of glucose R, 10 mg of rhamnose R
and 10 mg of xylose R in 1 ml of water R and dilute to 10 ml
with methanol R.
Apply to the plate as bands 10 l of each solution. Develop
over a path of 10 cm using a mixture of 10 volumes of
a 16 g/l solution of sodium dihydrogen phosphate R,
40 volumes of butanol R and 50 volumes of acetone R.
Dry the plate in a current of warm air for a few minutes
and develop again over a path of 15 cm using the same
mobile phase. Dry the plate at 110 C for 10 min, spray
with anisaldehyde solution R and heat again at 110 C for
10 min. The chromatogram obtained with the reference
solution shows 5 clearly separated coloured zones due to
galactose (greyish-green to green), glucose (grey), arabinose
(yellowish-green), xylose (greenish-grey to yellowish-grey)
and rhamnose (yellowish-green), in order of increasing Rf
value. The chromatogram obtained with the test solution
shows no grey zone and no greyish-green zone between
the zones corresponding to galactose and arabinose in the
chromatogram obtained with the reference solution.
Starch, dextrin and agar. To 10 ml of solution S, previously
boiled and cooled, add 0.1 ml of 0.05 M iodine. No blue or
reddish-brown colour develops.
Sterculia gum
A. Place 0.2 g of the powdered drug (355) in a 10 ml
ground-glass-stoppered cylinder graduated in 0.1 ml. Add
10 ml of ethanol (60 per cent V/V) R and shake. Any gel
formed occupies not more than 1.5 ml.
B. To 1.0 g of the powdered drug (355) add 100 ml of water R
and shake. Add 0.1 ml of methyl red solution R. Not
more than 5.0 ml of 0.01 M sodium hydroxide is required
to change the colour of the indicator.
Tannins. To 10 ml of solution S add 0.1 ml of ferric chloride
solution R1. A gelatinous precipitate is formed, but neither
the precipitate nor the liquid shows a dark blue colour.
Tragacanth. Examine the chromatograms obtained in
the test for glucose and fructose. The chromatogram
obtained with the test solution shows no greenish-grey to
yellowish-grey zone corresponding to the zone of xylose in
the chromatogram obtained with the reference solution.
Loss on drying (2.2.32). Not more than 15.0 per cent,
determined on 1.000 g of the powdered drug (355) by drying
in an oven at 100-105 C.
Total ash (2.4.16). Not more than 4.0 per cent.
Microbial contamination. Total viable aerobic count (2.6.12)
not more than 104 micro-organisms per gram, determined
by plate count. It complies with the test for Escherichia
coli (2.6.13).
FUNCTIONALITY-RELATED CHARACTERISTICS
This section provides information on characteristics that
are recognised as being relevant control parameters for
one or more functions of the substance when used as
an excipient (see general chapter 5.15)(1). This section
is a non-mandatory part of the monograph and it is not
necessary to verify the characteristics to demonstrate
compliance. Control of these characteristics can however
contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process.

Where control methods are cited, they are recognised as

being suitable for the purpose, but other methods can also
be used. Wherever results for a particular characteristic
are reported, the control method must be indicated.
The following characteristic may be relevant for acacia
used as a viscosity-increasing agent and/or suspending
agent in aqueous preparations.
Apparent viscosity. Determine the dynamic viscosity using a
capillary viscometer (2.2.9) or a rotating viscometer (2.2.10)
on a 100 g/l solution of acacia (dried substance).

Reference: PA/PH/Exp. 13B/T (06) 17 ANP

NOTE ON THE MONOGRAPH
The European Pharmacopoeia Commission has decided
that more attention will be paid to functionality-related
characteristics (FRCs) in monographs on excipients. While
these characteristics are of importance for the intended
use of many excipients, they are essentially a matter
for agreement between the excipient supplier and the
manufacturer of a medicinal product. For this reason,
FRCs are to be dealt with in a specific non-mandatory
section of the monograph, and the particular use or uses
for which a given FRC is relevant are to be specified.
FRCs are very diverse in nature, and their treatment in
monographs will have to be adapted to each type.
XXXX:0308

ACACIA, SPRAY-DRIED
Acaciae gummi dispersione desiccatum
DEFINITION
Spray-dried acacia is obtained from a solution of acacia.
CHARACTERS
It dissolves completely and rapidly, after about 20 min, in
twice its mass of water. The liquid obtained is colourless or
yellowish, dense, viscous, adhesive, translucent, and weakly
acid to blue litmus paper. Spray-dried acacia is practically
insoluble in ethanol (96 per cent).
IDENTIFICATION
A. Examined under a microscope, in ethanol (96 per cent) R,
the powder is seen to consist predominantly of spheroidal
particles about 4-40 m in diameter, with a central cavity
containing one or several air-bubbles ; a few minute flat
fragments are present. Only traces of starch granules are
visible. No vegetable tissue is seen.
B. Examine the chromatograms obtained in the test for
glucose and fructose. The chromatogram obtained with
the test solution shows 3 zones due to galactose, arabinose
and rhamnose. No other important zones are visible,
particularly in the upper part of the chromatogram.
C. Dissolve 1 g of the drug to be examined in 2 ml of
water R by stirring frequently for 20 min. Add 2 ml of
ethanol (96 per cent) R. After shaking a white gelatinous
mucilage is formed, which becomes fluid on adding 10 ml
of water R.
TESTS
Solution S. Dissolve 3.0 g of the drug to be examined in
25 ml of water R by stirring for 10 min. Allow to stand for
20 min and dilute to 30 ml with water R.

(1) See draft in this issue.

382

PHARMEUROPA Vol. 18, No. 3, July 2006

Acemetacin

Glucose and fructose. Examine by thin-layer chromatography FUNCTIONALITY-RELATED CHARACTERISTICS

(2.2.27), using a TLC silica gel plate R.
This section provides information on characteristics that
are recognised as being relevant control parameters for
Test solution. To 0.100 g in a thick-walled centrifuge tube
one or more functions of the substance when used as
add 2 ml of a 100 g/l solution of trifluoroacetic acid R,
an excipient (see general chapter 5.15)(2). This section
shake vigorously to dissolve the forming gel, stopper the
is a non-mandatory part of the monograph and it is not
tube and heat the mixture at 120 C for 1 h. Centrifuge
necessary to verify the characteristics to demonstrate
the hydrolysate, transfer the clear supernatant carefully
compliance. Control of these characteristics can however
into a 50 ml flask, add 10 ml of water R and evaporate the
contribute to the quality of a medicinal product by
solution to dryness under reduced pressure. To the resulting improving the consistency of the manufacturing process.
clear film add 0.1 ml of water R and 0.9 ml of methanol R.
Where control methods are cited, they are recognised as
Centrifuge to separate the amorphous precipitate. Dilute the being suitable for the purpose, but other methods can also
supernatant, if necessary, to 1 ml with methanol R.
be used. Wherever results for a particular characteristic
are reported, the control method must be indicated.
Reference solution. Dissolve 10 mg of arabinose R, 10 mg
The following characteristic may be relevant for spray-dried
of galactose R, 10 mg of glucose R, 10 mg of rhamnose R
and 10 mg of xylose R in 1 ml of water R and dilute to 10 ml acacia used as a viscosity-increasing agent and/or
suspending agent or film former in aqueous preparations.
with methanol R.
Apparent viscosity. Determine the dynamic viscosity using a
Apply to the plate as bands 10 l of each solution. Develop capillary viscometer (2.2.9) or a rotating viscometer (2.2.10)
over a path of 10 cm using a mixture of 10 volumes of
on a 100 g/l solution of spray-dried acacia (dried substance).
a 16 g/l solution of sodium dihydrogen phosphate R,
40 volumes of butanol R and 50 volumes of acetone R.
Dry the plate in a current of warm air for a few minutes
and develop again over a path of 15 cm using the same
Reference: PA/PH/Exp. 10A/T (03) 56 ANP 1R
mobile phase. Dry the plate at 110 C for 10 min, spray
with anisaldehyde solution R and heat again at 110 C for
NOTE ON THE MONOGRAPH
10 min. The chromatogram obtained with the reference
A 1st draft monograph was published in Pharmeuropa 17.2,
solution shows 5 clearly separated coloured zones due to
and,
following the comments received, has now been
galactose (greyish-green to green), glucose (grey), arabinose
further modified.
(yellowish-green), xylose (greenish-grey to yellowish-grey)
and rhamnose (yellowish-green), in order of increasing Rf
Characters :
value. The chromatogram obtained with the test solution
the solubility is given for only 3 solvents ;
shows no grey zone and no greyish-green zone between
the substance shows polymorphism and the melting
the zones corresponding to galactose and arabinose in the
point has been deleted from the identification section : it
chromatogram obtained with the reference solution.
is now given for information only.
Starch, dextrin and agar. To 10 ml of solution S, previously Identification : the substance is not used in pharmacies, so
boiled and cooled, add 0.1 ml of 0.05 M iodine. No blue or
the 2nd series has been deleted.
reddish-brown colour develops.
Related substances : the method previously described lacked
Sterculia gum
reproducibility, so another LC method is now described.
Assay : the titration now proposed is simpler than the
A. Place 0.2 g in a 10 ml ground-glass-stoppered cylinder
previous one and the equivalence point is determined more
graduated in 0.1 ml. Add 10 ml of ethanol (60 per
easily.
cent V/V) R and shake. Any gel formed occupies not
XXXX:1686
more than 1.5 ml.

ACEMETACIN

B. To 1.0 g add 100 ml of water R and shake. Add 0.1 ml

of methyl red solution R. Not more than 5.0 ml of
0.01 M sodium hydroxide is required to change the
colour of the indicator.

Acemetacinum

Tannins. To 10 ml of solution S add 0.1 ml of ferric chloride

solution R1. A gelatinous precipitate is formed, but neither
the precipitate nor the liquid shows a dark blue colour.
Tragacanth. Examine the chromatograms obtained in
the test for glucose and fructose. The chromatogram
obtained with the test solution shows no greenish-grey to
yellowish-grey zone corresponding to the zone of xylose in
the chromatogram obtained with the reference solution.
Loss on drying (2.2.32). Not more than 10.0 per cent,
determined on 1.000 g by drying in an oven at 100-105 C.

C21H18ClNO6

Mr 415.8

DEFINITION
[[[1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3Total ash (2.4.16). Not more than 4.0 per cent.
yl]acetyl]oxy]acetic acid.
Microbial contamination. Total viable aerobic count (2.6.12) Content : 99.0 per cent to 101.0 per cent (dried substance).
not more than 104 micro-organisms per gram, determined
CHARACTERS
by plate count. It complies with the test for Escherichia
Appearance : yellow or greenish-yellow, crystalline powder.
coli (2.6.13).
(2) See draft in this issue.

PHARMEUROPA Vol. 18, No. 3, July 2006

383

Acemetacin

Solubility : practically insoluble in water, very soluble or

freely soluble in dimethylformamide, soluble in acetone,
sparingly soluble in methanol and in methylene chloride,
slightly soluble in anhydrous ethanol.
It shows polymorphism.
mp : about 152 C.

TESTS
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.100 g of the substance to be
examined in acetonitrile R and dilute to 20.0 ml with the
same solvent.
Reference solution (a). Dilute 5.0 ml of the test solution to
50.0 ml with acetonitrile R. Dilute 1.0 ml of this solution to
IDENTIFICATION
100.0 ml with acetonitrile R.
First identification : A, C.
Reference solution (b). Dissolve 5.0 mg of 4-chlorobenzoic
acid R (impurity A) and 10.0 mg of indometacin CRS
Second identification : A, B, D, E.
(impurity B) in 40 ml of acetonitrile R, add 5.0 ml of the test
A. Melting point (2.2.14) : 151 C to 154 C.
solution and dilute to 50.0 ml with acetonitrile R. Dilute
B. Ultraviolet and visible absorption spectrophotometry
1.0 ml of this solution to 50.0 ml with acetonitrile R.
(2.2.25).
Reference solution (c). Dissolve 10 mg of acemetacin for
Test solution. Dissolve 50.0 mg in methanol R and dilute system suitability CRS (containing impurities C, D, E and F)
in acetonitrile R and dilute to 2 ml with the same solvent.
to 50.0 ml with the same solvent. Dilute 1.0 ml of this
solution to 50.0 ml with methanol R.
Column :
Spectral range : 280-360 nm.
size : l = 0.25 m, = 4.0 mm,
Absorption maximum : at 318 nm.
stationary phase : base-deactivated octadecylsilyl silica
gel for chromatography R (5 m)(3),
Specific absorbance at the absorption maximum : 140
temperature : 30 C.
to 160.
Mobile phase :
C. Infrared absorption spectrophotometry (2.2.24).
mobile phase A : dissolve 2.72 g of potassium dihydrogen
Comparison : acemetacin CRS.
phosphate R in water R and dilute to 1000 ml with the
If the spectra obtained in the solid state show differences,
same solvent ; adjust to pH 2.5 with dilute phosphoric
dissolve the substance to be examined and the reference
acid R ;
substance separately in acetone R, evaporate to dryness
mobile phase B : acetonitrile R ;
and record new spectra using the residues.
Time
Mobile phase A
Mobile phase B
D. Thin-layer chromatography (2.2.27).
(min)
(per cent V/V)
(per cent V/V)
Test solution. Dissolve 25 mg of the substance to be
0 - 15
55
45
examined in 5 ml of methanol R.
15 - 18
55 20
45 80
Reference solution (a). Dissolve 25 mg of acemetacin CRS
in 5 ml of methanol R.
20
80
18 - 21
Reference solution (b). Dissolve 25 mg of the substance
21 - 22
20 55
80 45
to be examined and 25 mg of indometacin R in 5 ml of
Flow rate : 1.0 ml/min.
methanol R.
Detection : spectrophotometer at 235 nm.
Plate : TLC silica gel F254 plate R.
Injection : 20 l.
Mobile phase : glacial acetic acid R, hexane R, methyl
isobutyl ketone R (3:7:10 V/V/V).
Relative retention with reference to acemetacin
(retention time = about 20 min) : impurity A = about 0.3 ;
Application : 5 l.
impurity B = about 0.9 ; impurity F = about 1.1 ;
Development : over a path of 10 cm. Introduce the plate impurity C = about 1.2 ; impurity D = about 1.3 ;
into the tank in a way that the solvent vapours have direct impurity E = about 1.4.
contact with the layer.
System suitability : reference solution (c) :
Drying : in a current of warm air.
the chromatogram obtained is similar to the
Detection : examine in ultraviolet light at 254 nm.
chromatogram supplied with acemetacin for system
suitability CRS.
System suitability : reference solution (b) :
Limits :
the chromatogram shows 2 clearly separated spots.
Results : the principal spot in the chromatogram obtained correction factors : for the calculation of contents,
multiply the peak areas of the following impurities by
with the test solution is similar in position and size to
the corresponding correction factor : impurity C = 1.25 ;
the principal spot in the chromatogram obtained with
impurity D = 1.4 ; impurity F = 1.3 ;
the reference solution (a).
impurity A : not more than the area of the corresponding
E. Dissolve 0.1 g in 10 ml of ethanol (96 per cent) R, heating
peak in the chromatogram obtained with reference
slightly if necessary. To 0.5 ml of this solution add
solution (b) (0.1 per cent) ;
0.5 ml of dimethylaminobenzaldehyde solution R2. A

impurity
B : not more than the area of the corresponding
precipitate is formed that dissolves on shaking. Heat on a
peak in the chromatogram obtained with reference
water-bath. A bluish-green colour is produced. Continue
solution (b) (0.2 per cent) ;
to heat for 5 min and cool in iced water for 2 min. A
precipitate is formed and the colour changes to light
impurities C, D, F : for each impurity, not more than the
greyish-green. Add 3 ml of ethanol (96 per cent) R. The
area of the principal peak in the chromatogram obtained
solution is clear and violet-pink.
with reference solution (a) (0.1 per cent) ;
(3) Hypersil BDS C18 is suitable.

384

PHARMEUROPA Vol. 18, No. 3, July 2006

Acemetacin

impurity E : not more than 3 times the area of the

principal peak in the chromatogram obtained with
reference solution (a) (0.3 per cent) ;
any other impurity : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent) ;
total: not more than 4 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.4 per cent) ;
disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Liquid chromatography (2.2.29).
Test solution. Dissolve 0.100 g of the substance to be
examined in acetonitrile for chromatography R and dilute
to 20.0 ml with the same solvent.
Reference solution (a). Dilute 5.0 ml of the test solution
to 50.0 ml with acetonitrile for chromatography R. Dilute
1.0 ml of this solution to 100.0 ml with acetonitrile for
chromatography R.
Reference solution (b). Dissolve 5.0 mg of acemetacin
impurity A CRS and 10.0 mg of acemetacin impurity B CRS
in acetonitrile for chromatography R, and dilute to 50.0 ml
with the same solvent.
Reference solution (c). Dilute 1.0 ml of reference solution (b)
to 20.0 ml with acetonitrile for chromatography R.
Reference solution (d). To 1 ml of reference solution (b),
add 10 ml of the test solution and dilute to 20 ml with
acetonitrile for chromatography R.
Reference solution (e). Dissolve the contents of a vial of
acemetacin impurity mixture CRS (containing impurities C,
D, E and F) in 2 ml of the test solution.
Column :
size : l = 0.25 m, = 4 mm ;
stationary phase : spherical end-capped octadecylsilyl
silica gel for chromatography R (5 m)(4) ;
temperature : 40 C.
Mobile phase :
mobile phase A : dissolve 1.0 g of potassium dihydrogen
phosphate R in 900 ml of water R, adjust to pH 6.5
with 1 M sodium hydroxide, and dilute to 1000 ml with
water R ;
mobile phase B : acetonitrile for chromatography R ;

Time
(min)
0-1

Mobile phase A
(per cent V/V)
95

Mobile phase B
(per cent V/V)
5

1-5

95 65

5 35

5 - 12

65

35

12 - 24

65 20

35 80

24 - 30

20

80

30 - 31

20 95

80 5

31 - 40

95

Flow rate : 1 ml/min.

Detection : spectrophotometer at 235 nm.
Injection : 20 l of the test solution and reference
solutions (a), (c), (d) and (e).
Identification of impurities : use the chromatogram
supplied with acemetacin impurity mixture CRS and the
chromatogram obtained with reference solution (e) to
identify the peaks due to impurities C, D, E and F.
Relative retention with reference to acemetacin
(retention time = about 11 min) : impurity A = about 0.7 ;
impurity B = about 0.9 ; impurity F = about 1.4 ;
impurity C = about 1.5 ; impurity D = about 1.8 ;
impurity E = about 2.7.
System suitability : reference solution (d) :
resolution : minimum 1.5 between the peaks due to
impurity B and acemetacin.
Limits :
correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity C = 1.3 ;
impurity D = 1.4 ; impurity F = 1.3 ;
impurity A : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (c) (0.1 per cent) ;
impurity B : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (c) (0.2 per cent) ;
impurities C, D, F : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent) ;
impurity E : not more than 3 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.3 per cent) ;

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

Figure 1686.-1. Chromatogram for the test for related substances of acemetacin
(4) LiChrospher RP18e is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

385

Alginic acid

unspecified impurities : for each impurity, not more

than the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.10 per cent) ;
total: not more than 4 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.4 per cent) ;
disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test F. Prepare the reference solution
F. [[[[[7-(4-chlorobenzoyl)-5-methoxy-1H-indol-3using 2 ml of lead standard solution (10 ppm Pb) R.
yl]acetyl]oxy]acetyl]oxy]acetic acid.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 C.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Dissolve 0.300 g in 50 ml of dimethylformamide R
and add 0.5 ml of a 12.6 g/l solution of oxalic
acid R in dimethylformamide R. Titrate with 0.1 M
tetrabutylammonium hydroxide in 2-propanol, determining
the end-point potentiometrically (2.2.20). Read the volume
added between the first 2 points of inflexion.
1 ml of 0.1 M tetrabutylammonium hydroxide in 2-propanol
is equivalent to 41.58 mg of C21H18ClNO6.
Dissolve 0.350 g in 20 ml of acetone R and add 10 ml of
water R. Titrate with 0.1 M sodium hydroxide, determining
the end-point potentiometrically (2.2.20).
1 ml of 0.1 M sodium hydroxide is equivalent to 41.58 mg of
C21H18ClNO6.
STORAGE
Protected from light.
IMPURITIES
Specified impurities : A, B, C, D, E, F.

A. 4-chlorobenzoic acid,
B. indometacin,

Reference: PA/PH/Exp. 11/T (06) 16 ANP

NOTE ON THE MONOGRAPH
The European Pharmacopoeia Commission has decided
that more attention will be paid to functionality-related
characteristics (FRCs) in monographs on excipients.
While these characteristics are of importance for the
intended use of many excipients, they are essentially a
matter for agreement between the excipient supplier and
the manufacturer of a medicinal product. For this reason,
FRCs are to be dealt with in a specific non-mandatory
section of the monograph, and the particular use or uses
for which a given FRC is relevant are to be specified.
FRCs are very diverse in nature, and their treatment in
monographs will have to be adapted to each type.
XXXX:0591

ALGINIC ACID
Acidum alginicum
DEFINITION
Mixture of polyuronic acids [(C6H8O6)n] composed of residues
of D-mannuronic and L-guluronic acids, obtained mainly from
algae belonging to the Phaeophyceae. A small proportion of
the carboxyl groups may be neutralised.
Content : 19.0 per cent to 25.0 per cent of carboxyl groups
(CO2H) (dried substance).
CHARACTERS
Appearance : white or pale yellowish-brown, crystalline or
amorphous powder.
Solubility : very slightly soluble or practically insoluble
in ethanol (96 per cent), practically insoluble in organic
solvents. It swells in water but does not dissolve ; it dissolves
in solutions of alkali hydroxides.

C. R1 = Cl, R2 = H, R3 = CH2-CO2H : [[[1-(3,4-dichlorobenzoyl)5-methoxy-2-methyl-1H-indol-3-yl]acetyl]oxy]acetic acid,

IDENTIFICATION
A. To 0.2 g add 20 ml of water R and 0.5 ml of sodium
D. R1 = H, R2 = C(CH3)3, R3 = CH2-CO2H :
carbonate solution R. Shake and filter. To 5 ml of the
[[[1-(4-chlorobenzoyl)-6-(1,1-dimethylethyl)-5filtrate add 1 ml of calcium chloride solution R. A
methoxy-2-methyl-1H-indol-3-yl]acetyl]oxy]acetic acid,
voluminous gelatinous mass is formed.
E. R1 = R2 = H, R3 = CH2-CO-O-C(CH3)3 : 1,1-dimethylethyl B. To 5 ml of the filtrate obtained in identification test A add
0.5 ml of a 123 g/l solution of magnesium sulphate R.
[[[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3No voluminous gelatinous mass is formed.
yl]acetyl]oxy]acetate,
386

PHARMEUROPA Vol. 18, No. 3, July 2006

Altizide

C. To 5 mg add 5 ml of water R, 1 ml of a freshly prepared

10 g/l solution of 1,3-dihydroxynaphthalene R in
ethanol (96 per cent) R, and 5 ml of hydrochloric acid R.
Boil gently for 3 min, cool, add 5 ml of water R, and shake
with 15 ml of di-isopropyl ether R. Carry out a blank
test. The upper layer obtained with the substance to be
examined exhibits a deeper bluish-red colour than that
obtained with the blank.
TESTS
Chlorides : maximum 1,0 per cent.
To 2.50 g add 50 ml of dilute nitric acid R, shake for 1 h and
dilute to 100.0 ml with dilute nitric acid R. Filter. To 50.0 ml
of the filtrate add 10.0 ml of 0.1 M silver nitrate and 5 ml of
toluene R. Titrate with 0.1 M ammonium thiocyanate, using
2 ml of ferric ammonium sulphate solution R2 as indicator
and shaking vigorously towards the end-point.
1 ml of 0.1 M silver nitrate is equivalent to 3.545 mg of Cl.
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test F. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 15.0 per cent, determined
on 0.1000 g by drying in an oven at 100-105 C for 4 h.
Sulphated ash (2.4.14) : maximum 8.0 per cent (dried
substance), determined on 0.100 g.
Microbial contamination. Total viable aerobic count (2.6.12)
not more than 102 micro-organisms per gram, determined by
plate-count. It complies with the tests for Escherichia coli
and Salmonella (2.6.13).

Apparent viscosity. Determine the dynamic viscosity using

a rotating viscometer (2.2.10).
Prepare a 20 g/l suspension of alginic acid and add 0.1 M
sodium hydroxide until a solution is obtained.

Reference: PA/PH/Exp. 10B/T (06) 34 ANP

XXXX:2185

ALTIZIDE
Altizidum

C11H14ClN3O4S3

Mr 383.9

DEFINITION
(3RS)-6-Chloro-3,4-dihydro-3-[(prop-2-enylthio)methyl]2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide.
Content : 98.0 per cent to 102.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white powder.
Solubility : practically insoluble in water, soluble in
methanol, practically insoluble in dichloromethane.

ASSAY
To 0.2500 g add 25 ml of water R, 25.0 ml of 0.1 M sodium
hydroxide and 0.2 ml of phenolphthalein solution R. Titrate IDENTIFICATION
with 0.1 M hydrochloric acid.
Infrared absorption spectrophotometry (2.2.24).
1 ml of 0.1 M sodium hydroxide is equivalent to 4.502 mg
Comparison : altizide CRS.
of carboxyl groups ( CO2H).
TESTS
FUNCTIONALITY-RELATED CHARACTERISTICS
Impurity B. Thin layer chromatography (2.2.27).
This section provides information on characteristics that
Test
solution. Dissolve 0.200 g of the substance to be
are recognised as being relevant control parameters for
examined
in acetone R and dilute to 2.0 ml with the same
one or more functions of the substance when used as
solvent.
an excipient (see general chapter 5.15)(5). This section
Reference solution (a). Dissolve 10.0 mg of altizide
is a non-mandatory part of the monograph and it is not
impurity B CRS in acetone R and dilute to 25.0 ml with the
necessary to verify the characteristics to demonstrate
same solvent.
compliance. Control of these characteristics can however
contribute to the quality of a medicinal product by
Reference solution (b). To 1.0 ml of reference solution (a)
improving the consistency of the manufacturing process.
add 1.0 ml of the test solution.
Where control methods are cited, they are recognised as
Reference solution (c). Dilute 5.0 ml of reference solution (a)
being suitable for the purpose, but other methods can also to 10.0 ml with acetone R.
be used. Wherever results for a particular characteristic
Plate : TLC silica gel F254 plate R.
are reported, the control method must be indicated.
Mobile phase : acetone R, methylene chloride R (25:75
The following characteristics may be relevant for alginic
V/V).
acid used as a disintegrant and/or binder.
Application : 10 l of the test solution and reference
Particle-size distribution (2.9.31) or (2.9.38).
solutions (b) and (c).
Settling volume. Place 75 ml of water R in a 100 ml
Development : over 2/3 of the plate.
graduated cylinder and add 1.5 g of the substance to be
Drying : in air.
examined in 0.5 g portions, shaking vigorously after each
Detection : spray with a mixture of equal volumes of a 10 g/l
addition. Dilute to 100.0 ml with water R and shake again
until the substance is homogeneously distributed. Allow to solution of potassium permanganate R and a 50 g/l solution
of sodium carbonate R, prepared immediately before use.
stand for 4 h.
System
suitability : reference solution (b) :
The following characteristic may be relevant for alginic
acid used as a gelling agent or viscosity-increasing agent. the chromatogram shows 2 clearly separated spots.
(5) See draft in this issue.

PHARMEUROPA Vol. 18, No. 3, July 2006

387

Altizide

Limit : the spot due to impurity B is not more intense than

the principal spot in the chromatogram obtained with
reference solution (c) (0.2 per cent).

System suitability : reference solution (c) :

resolution : minimum 1.0 between the peaks due to
altizide and furosemide.
Limits :
Related substances. Liquid chromatography (2.2.29).
Prepare the solutions immediately before use, except
impurity A : not more than twice the area of the principal
reference solution (b).
peak in the chromatogram obtained with reference
solution (a) (0.2 per cent) ;
Test solution. Dissolve 50.0 mg of the substance to be
unspecified impurities : for each impurity, not more
examined in 2 ml of acetonitrile R and dilute to 25.0 ml with
than the area of the principal peak in the chromatogram
the mobile phase.
obtained with reference solution (a) (0.10 per cent) ;
total : not more than 3 times the area of the principal peak
Reference solution (a). Dilute 1.0 ml of the test solution
in the chromatogram obtained with reference solution (a)
to 100.0 ml with the mobile phase. Dilute 1.0 ml of this
(0.3 per cent) ;
solution to 10.0 ml with the mobile phase.
disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
Reference solution (b). Dissolve 50 mg of the substance to
(0.05 per cent).
be examined in 5 ml of acetonitrile R and dilute to 25 ml with
water R. Allow to stand for 30 min. The solution contains
Water (2.5.32) : maximum 0.5 per cent, determined on
altizide and impurity A obtained by in situ degradation.
50.0 mg.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
Reference solution (c). Dissolve 4 mg of furosemide CRS
on 1.0 g.
in 2 ml of acetonitrile R, add 2 ml of the test solution and
dilute to 100 ml with the mobile phase.
ASSAY
Column :
size : l = 0.15 m, = 3.9 mm ;
stationary phase : octadecylsilyl silica gel for
chromatography R (5 m)(6) ;
temperature : 30 C.
Mobile phase : mix 250 volumes of acetonitrile R and
750 volumes of water R previously adjusted to pH 2 with
perchloric acid R.

Liquid chromatography (2.2.29) as described in the test for

related substances, with the following modifications.
Test solution. Dissolve 25.0 mg of the substance to be
examined in 2 ml of acetonitrile R and dilute to 25.0 ml with
the mobile phase.
Reference solution. Dissolve 25.0 mg of altizide CRS in 2 ml
of acetonitrile R and dilute to 25.0 ml with the mobile phase.
Calculate the percentage content of C11H14ClN3O4S3.
IMPURITIES
Specified impurities : A, B.

Flow rate : 0.7 ml/min.

Detection : spectrophotometer at 270 nm.
Injection: 5 l.

A. 4-amino-6-chloro-1,3-benzenedisulphonamide,

Run time : twice the retention time of altizide.

Relative retention with reference to altizide (retention
time = about 25 min) : impurity A = about 0.15 ;
furosemide = about 1.05.

B. 3-[(2,2-dimethoxyethyl)thio]-prop-1-ene.

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

Figure 2185.-1. Chromatogram for the test for related substances of altizide : solution of altizide spiked with impurity A
(6) Symmetry Shield is suitable.

388

PHARMEUROPA Vol. 18, No. 3, July 2006

Aprotinin

Reference: PA/PH/Exp. 6/T (06) 15 ANP

Plate : TLC silica gel G plate R.

Mobile phase : water R, glacial acetic acid R (80:100 V/V)
NOTE ON THE MONOGRAPH
containing 100 g/l of sodium acetate R.
Protein impurities of higher molecular mass : this test is
Application
: 10 l.
replaced by :
Development
: over a path of 12 cm.
a capillary zone electrophoresis for the detection of
Drying
:
in
air.
des-Ala- and des-Ala-des-Gly-aprotinins ;
Detection : spray with a solution of 0.1 g of ninhydrin R
an LC for the detection of N-pyroglutamyl-aprotinin and
in a mixture of 6 ml of a 10 g/l solution of cupric
related compounds ;
chloride R, 21 ml of glacial acetic acid R and 70 ml of
a new size-exclusion chromatography for the detection
anhydrous ethanol R. Dry the plate at 60 C.
of oligomers.
Results : the principal spot in the chromatogram obtained
Assay : glass-silver-silver chloride electrodes are proposed
with the test solution is similar in position, colour and
as an alternative to glass and calomel electrodes.
size to the principal spot in the chromatogram obtained
Labelling : bacterial endotoxins are no longer mentioned
with the reference solution.
because this requirement is covered in the General Notices.
B.
Determine the ability of the substance to be examined to
XXXX:0580
inhibit trypsin activity using the method described below.
Test solution. Dilute 1 ml of solution S to 50 ml with
APROTININ
buffer solution pH 7.2 R.
Trypsin solution. Dissolve 10 mg of trypsin BRP in
Aprotininum
0.002 M hydrochloric acid and dilute to 100 ml with the
same acid.
Casein solution. Dissolve 0.2 g of casein R in buffer
solution pH 7.2 R and dilute to 100 ml with the same
buffer solution.
Precipitating solution : glacial acetic acid R, water R,
anhydrous ethanol R (1:49:50 V/V/V).
Mix 1 ml of the test solution with 1 ml of the trypsin
solution. Allow to stand for 10 min and add 1 ml of the
casein solution. Incubate at 35 C for 30 min. Cool in
iced water and add 0.5 ml of the precipitating solution.
Shake and allow to stand at room temperature for 15 min.
C284H432N84O79S7
Mr 6511
The solution is cloudy. Carry out a blank test under the
DEFINITION
same conditions using buffer solution pH 7.2 R instead of
Aprotinin is a polypeptide consisting of a chain of 58 amino
the test solution. The solution is not cloudy.
acids. It inhibits stoichiometrically the activity of several
TESTS
proteolytic enzymes such as chymotrypsin, kallikrein,
plasmin and trypsin. It contains not less than 3.0 Ph. Eur. U. Solution S. Prepare a solution of the substance to be
of aprotinin activity per milligram, calculated with reference examined containing 15 Ph. Eur. U./ml, calculated from the
to the dried substance.
activity stated on the label.
Appearance of solution. Solution S is clear (2.2.1).
PRODUCTION
The animals from which aprotinin is derived must fulfil the
Absorbance (2.2.25) : maximum 0.80 by measuring at the
requirements for the health of animals suitable for human
absorption maximum at 277 nm.
consumption to the satisfaction of the competent authority. Prepare a solution of the substance to be examined
The manufacturing process is validated to demonstrate the
containing 3.0 Ph. Eur. U./ml.
suitable inactivation or removal of any contamination by
Protein impurities of higher molecular mass. Size-exclusion
viruses or other infectious agents.
chromatography (2.2.30).
The method of manufacture is validated to demonstrate that
Use cross-linked dextran for chromatography R2. Use a
the product, if tested, would comply with the following tests.
180 g/l solution of anhydrous acetic acid R to swell the
Abnormal toxicity (2.6.9). Inject into each mouse a quantity gel and as the eluent. Prepare a column of gel 0.8 m to
of the substance to be examined containing 2 Ph. Eur. U.
1.0 m long and 25 mm in diameter, taking care to avoid the
dissolved in a sufficient quantity of water for injections R to introduction of air bubbles. Place at the top of the column
give a volume of 0.5 ml.
a quantity of the substance to be examined containing
Histamine (2.6.10) : maximum 0.2 g of histamine base per 300 Ph. Eur. U. dissolved in 1 ml of a 180 g/l solution of
anhydrous acetic acid R and allow to elute. Collect the
3 Ph. Eur. U.
eluate in fractions of 2 ml. Measure the absorbance (2.2.25)
CHARACTERS
of each fraction at the absorption maximum at 277 nm and
plot the values on a graph. The chromatogram obtained
Appearance : almost white hygroscopic powder.
does not present an absorption maximum before the elution
Solubility : soluble in water and in isotonic solutions,
of the aprotinin.
practically insoluble in organic solvents.
Des-Ala-aprotinin and des-Ala-des-Gly-aprotinin. Capillary
IDENTIFICATION
zone electrophoresis (2.2.47) : use the normalisation
A. Thin-layer chromatography (2.2.27).
procedure.
Test solution. Solution S (see Tests).
Test solution. Prepare a solution of the substance to be
Reference solution. Aprotinin solution BRP.
examined containing not less than 1 Ph. Eur. U./ml.
PHARMEUROPA Vol. 18, No. 3, July 2006

389

Aprotinin

Reference solution. Dilute aprotinin solution BRP to obtain

the same concentration as the test solution.
Capillary :
material : uncoated fused silica ;
size : effective length = 45-60 cm, = 75 m.
Temperature : 25 C.
CZE buffer. Dissolve 8.21 g of potassium dihydrogen
phosphate R in 400 ml of water R, adjust to pH 3.0 with
phosphoric acid R, dilute to 500.0 ml with water R and filter
through a membrane filter (0.45 m).
Detection : spectrophotometer at 214 nm.
Between-run rinsing : rinse the capillary for at least 1 min
with 0.1 M sodium hydroxide filtered through a membrane
filter (0.45 m) and for 2 min with the CZE buffer.
Injection : under pressure or vacuum (for example, 3 s at a
differential pressure of 3.5 kPa).
Migration : apply a field strength of 0.2 kV/cm, using the
CZE buffer as the electrolyte in both buffer reservoirs.
Run time : 30 min.
Identification of impurities : use the electropherogram
supplied with aprotinin solution BRP and the
electropherogram obtained with the reference solution to
identify the peaks due to impurities A and B.
Relative migration with reference to aprotinin (migration
time = about 22 min) : impurity A = about 0.98 ;
impurity B = about 0.99.
System suitability : reference solution after at least
6 injections :
migration time : aprotinin = 19.0 min to 25.0 min ;
resolution : minimum 0.8 between the peaks due to
impurities A and B ; minimum 0.5 between the peaks due
to impurity B and aprotinin ;
symmetry factor : maximum 1.3 for the peak due to
aprotinin ;
peak distribution : the electrophoregram obtained
is qualitatively and quantitatively similar to the
electropherogram supplied with aprotinin solution BRP ;
height of the principal peak : at least 1000 times the
height of the baseline noise. If necessary, adjust the
sample load to give peaks of sufficient height.
Limits :
impurity A : maximum 8.0 per cent ;
impurity B : maximum 7.5 per cent.
N-pyroglutamyl-aprotinin and related compounds. Liquid
chromatography (2.2.29) : use the normalisation procedure.
Test solution. Prepare a solution of the substance
to be examined in mobile phase A, containing about
5 Ph. Eur. U./ml.
Resolution solution. Dissolve the contents of a vial of
aprotinin for system suitability CRS 1 in mobile phase A to
obtain the same concentration as the test solution.
Column :
size : l = 0.075 m, = 7.5 mm ;
stationary phase : strong cation-exchange silica gel for
chromatography R (10 m)(7) ;
temperature : 40 C.
Mobile phase :
mobile phase A : dissolve 3.52 g of potassium dihydrogen
phosphate R and 7.26 g of disodium hydrogen phosphate
dihydrate R in 1000 ml of water ; filter and degas ;

mobile phase B : dissolve 3.52 g of potassium dihydrogen

phosphate R, 7.26 g of disodium hydrogen phosphate
dihydrate R and 66.07 g of ammonium sulphate R in
1000 ml of water ; filter and degas ;
Time
(min)
0 - 21

Mobile phase A
(per cent V/V)
92 64

Mobile phase B
(per cent V/V)
8 36

21 - 30

64 0

36 100

30 - 31

0 92

100 8

31 - 40

92

Flow rate : 1.0 ml/min.

Detection : spectrophotometer at 210 nm.
Injection : 40 l.
Relative retention with reference to aprotinin (retention
time = 17.0 min to 20.0 min) : impurity C = about 0.9.
System suitability :
retention time : aprotinin = 17.0 min to 20.0 min ;
resolution : minimum 1.0 between the peaks due to
impurity C and aprotinin ;
symmetry factor : maximum 1.3 for the peak due to
aprotinin.
Limits :
impurity C : maximum 1.0 per cent ;
any other impurity : maximum 0.5 per cent ;
sum of impurities other than C : maximum 1.0 per cent.
Aprotinin oligomers. Size-exclusion chromatography
(2.2.30) : use the normalisation procedure.
Test solution. Prepare a solution of the substance to be
examined containing about 5 Ph. Eur. U./ml.
Resolution solution. Dissolve the contents of a vial of
aprotinin for system suitability CRS 2 to obtain the same
concentration as the test solution.
Column : 3 columns coupled in series :
size : l = 0.30 m, = 7.8 mm ;
stationary phase : hydrophilic silica gel for
chromatography R of a grade suitable for fractionation of
globular proteins in the relative molecular mass range of
20 000 to 10 000 000 (8 m)(8).
Mobile phase : acetonitrile R, glacial acetic acid R, water R
(2:2:6 V/V/V) ; filter and degas.
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 277 nm.
Injection : 100 l.
Run time : 40 min.
Relative retention with reference to aprotinin monomer
(retention time = 24.5 min to 25.5 min) : aprotinin
dimer = about 0.9.
System suitability : resolution solution :
retention time : aprotinin monomer = 24.5 min to
25.5 min ;
resolution : minimum 1.3 between the peaks due to
aprotinin dimer and monomer ;
symmetry factor : maximum 2.5 for the peak due to
aprotinin monomer.
Limit :
total : maximum 1.0 per cent.
Loss on drying (2.2.32) : maximum 6.0 per cent, determined
on 0.100 g by drying in vacuo.

(7) Tosoh TSKgel SP5-PW is suitable.

(8) Tosoh TSKgel G4000SWXL is suitable.

390

PHARMEUROPA Vol. 18, No. 3, July 2006

Aprotinin concentrated solution

Bacterial endotoxins (2.6.14) : less than 0.14 IU per

STORAGE
European Pharmacopoeia Unit of aprotinin, if intended for
In an airtight, tamper-proof container, protected from light.
use in the manufacture of parenteral dosage forms without a
further appropriate procedure for the removal of bacterial
LABELLING
endotoxins.
The label states :
ASSAY
the number of European Pharmacopoeia Units of
The activity of aprotinin is determined by measuring its
aprotinin activity per milligram.
inhibitory action on a solution of trypsin of known activity.
where applicable, that the substance is free from bacterial
The inhibiting activity of the aprotinin is calculated from
endotoxins.
the difference between the initial activity and the residual
activity of the trypsin.
IMPURITIES
The inhibiting activity of aprotinin is expressed in European
Pharmacopoeia Units. 1 Ph. Eur. U. inhibits 50 per cent of
the enzymatic activity of 2 microkatals of trypsin.
Use a reaction vessel with a capacity of about 30 ml, provided
with :
a device that will maintain a temperature of 25 0.1 C ;
a stirring device, such as a magnetic stirrer ;
a lid with 5 holes for accommodating the electrodes, the
tip of a burette, a tube for the admission of nitrogen and
the introduction of the reagents.
An automatic or manual titration apparatus may be used. In
A. des-57-glycine-des-58-alanine-aprotinin,
the latter case the burette is graduated in 0.05 ml and the
pH-meter is provided with a wide reading scale and glass and
calomel or glass-silver-silver chloride electrodes.
Test solution. Prepare a solution of the substance to be
examined in 0.0015 M borate buffer solution pH 8.0 R
expected to contain 1.67 Ph. Eur. U./ml (about 0.6 mg
(m mg) per millilitre).
Trypsin solution. Prepare a solution of trypsin BRP
containing about 0.8 microkatals per millilitre (about
1 mg/ml), using 0.001 M hydrochloric acid as the solvent.
Use a freshly prepared solution and keep in iced water.
Trypsin and aprotinin solution. To 4.0 ml of the trypsin
solution add 1.0 ml of the test solution. Dilute immediately
B. des-58-alanine-aprotinin,
to 40.0 ml with 0.0015 M borate buffer solution pH 8.0 R.
Allow to stand at room temperature for 10 min and then
keep in iced water. Use within 6 h of preparation.
Dilute trypsin solution. Dilute 0.5 ml of the trypsin solution
to 10.0 ml with 0.0015 M borate buffer solution pH 8.0 R.
Allow to stand at room temperature for 10 min and then
keep in iced water.
Maintain an atmosphere of nitrogen in the reaction flask and
stir continuously ; introduce 9.0 ml of 0.0015 M borate buffer
solution pH 8.0 R and 1.0 ml of a freshly prepared 6.9 g/l
solution of benzoylarginine ethyl ester hydrochloride R.
Adjust to pH 8.0 with 0.1 M sodium hydroxide. When the
temperature has reached equilibrium at 25 0.1 C, add
1.0 ml of the trypsin and aprotinin solution and start a
C. N-pyroglutamyl-aprotinin.
timer. Maintain at pH 8.0 by the addition of 0.1 M sodium
hydroxide and note the volume added every 30 s. Continue
the reaction for 6 min. Determine the number of millilitres of
0.1 M sodium hydroxide used per second (n1 ml). Carry out,
under the same conditions, a titration using 1.0 ml of the
dilute trypsin solution. Determine the number of millilitres
Reference: PA/PH/Exp. 6/T (06) 26 ANP
of 0.1 M sodium hydroxide used per second (n2 ml).
Calculate the aprotinin activity in European Pharmacopoeia NOTE ON THE MONOGRAPH
Units per milligram using the following expression :
Protein impurities of higher molecular mass : this test is
replaced by :
a capillary zone electrophoresis for the detection of
des-Ala- and des-Ala-des-Gly-aprotinins ;
a LC for the detection of N-pyroglutamyl-aprotinin and
The estimated activity is not less than 90 per cent and not
related compounds ;
more than 110 per cent of the activity stated on the label.
PHARMEUROPA Vol. 18, No. 3, July 2006

391

Aprotinin concentrated solution

Detection : spray with a solution of 0.1 g of ninhydrin R

in a mixture of 6 ml of a 10 g/l solution of cupric
chloride R, 21 ml of glacial acetic acid R and 70 ml of
anhydrous ethanol R. Dry the plate at 60 C.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with the reference solution.
B. Determine the ability of the preparation to be examined to
APROTININ CONCENTRATED
inhibit trypsin activity using the method described below.
SOLUTION
Test solution. Dilute 1 ml of solution S to 50 ml with
buffer solution pH 7.2 R.
Trypsin solution. Dissolve 10 mg of trypsin BRP in
Aprotinini solutio concentrata
0.002 M hydrochloric acid and dilute to 100 ml with the
same acid.
Casein solution. Dissolve 0.2 g of casein R in buffer
solution pH 7.2 R and dilute to 100 ml with the same
buffer solution.
Precipitating solution : glacial acetic acid R, water R,
anhydrous ethanol R (1:49:50 V/V/V).
Mix 1 ml of the test solution with 1 ml of the trypsin
solution. Allow to stand for 10 min and add 1 ml of the
casein solution. Incubate at 35 C for 30 min. Cool in
iced water and add 0.5 ml of the precipitating solution.
Shake and allow to stand at room temperature for 15 min.
C284H432N84O79S7
Mr 6511
The solution is cloudy. Carry out a blank test under the
same conditions using buffer solution pH 7.2 R instead of
DEFINITION
the test solution. The solution is not cloudy.
Aprotinin concentrated solution is a solution of aprotinin, a
TESTS
polypeptide consisting of a chain of 58 amino acids, which
inhibits stoichiometrically the activity of several proteolytic Solution S. Prepare a solution containing 15 Ph. Eur. U./ml,
enzymes such as chymotrypsin, kallikrein, plasmin and
if necessary by dilution, on the basis of the activity stated
trypsin. It contains not less than 15.0 Ph. Eur. U. of aprotinin on the label.
activity per millilitre.
Appearance of solution. Solution S is clear (2.2.1).
PRODUCTION
Absorbance (2.2.25) : maximum 0.80 by measuring at the
absorption maximum at 277 nm.
The animals from which aprotinin is derived must fulfil the
Prepare a solution containing 3.0 Ph. Eur. U./ml.
requirements for the health of animals suitable for human
consumption to the satisfaction of the competent authority. Protein impurities of higher molecular mass. Size-exclusion
The manufacturing process is validated to demonstrate the
chromatography (2.2.30).
suitable inactivation or removal of any contamination by
Freeze-dry the preparation to be examined using a pressure
viruses or other infectious agents.
of 2.7 Pa and a temperature of 30 C ; the operation,
The method of manufacture is validated to demonstrate that including freeze-drying and a period of drying at 15-25 C,
the product, if tested, would comply with the following tests. takes 6-12 h.
Abnormal toxicity (2.6.9). Inject into each mouse a quantity Use cross-linked dextran for chromatography R2. Use a
180 g/l solution of anhydrous acetic acid R to swell the
of the preparation to be examined containing 2 Ph. Eur. U.
diluted with a sufficient quantity of water for injections R to gel and as the eluent. Prepare a column of gel 0.8 m to
1.0 m long and 25 mm in diameter, taking care to avoid the
give a volume of 0.5 ml.
introduction of air bubbles. Place at the top of the column
Histamine (2.6.10) : maximum 0.2 g of histamine base per a quantity of the preparation to be examined containing
3 Ph. Eur. U.
300 Ph. Eur. U. dissolved in 1 ml of a 180 g/l solution of
anhydrous acetic acid R and allow to elute. Collect the
CHARACTERS
eluate in fractions of 2 ml. Measure the absorbance (2.2.25)
Appearance : clear, colourless liquid.
of each fraction at the absorption maximum at 277 nm and
plot the values on a graph. The chromatogram obtained
IDENTIFICATION
does not present an absorption maximum before the elution
A. Thin-layer chromatography (2.2.27).
of the aprotinin.
Test solution. Solution S (see Tests).
Des-Ala-aprotinin and des-Ala-des-Gly-aprotinin. Capillary
zone electrophoresis (2.2.47) : use the normalisation
Reference solution. Aprotinin solution BRP.
procedure.
Plate : TLC silica gel G plate R.
Test solution. Dilute the preparation to be examined to a
Mobile phase : water R, glacial acetic acid R (80:100 V/V) concentration of not less than 1 Ph Eur. U./ml.
containing 100 g/l of sodium acetate R.
Reference solution. Dilute aprotinin solution BRP to obtain
Application : 10 l.
the same concentration as the test solution.
Development : over a path of 12 cm.
Capillary :
Drying : in air.
material: uncoated fused silica ;
a new size-exclusion chromatography for the detection
of oligomers.
Assay : glass-silver-silver chloride electrodes are proposed
as an alternative to glass and calomel electrodes.
Labelling : bacterial endotoxins are no longer mentioned
because this requirement is covered in the General Notices.
XXXX:0579

392

PHARMEUROPA Vol. 18, No. 3, July 2006

Aprotinin concentrated solution

size : effective length = 45-60 cm, = 75 m.

Temperature : 25 C.
CZE buffer. Dissolve 8.21 g of potassium dihydrogen
phosphate R in 400 ml of water R, adjust to pH 3.0 with
phosphoric acid R, dilute to 500.0 ml with water R and filter
through a membrane filter (0.45 m).
Detection : spectrophotometer at 214 nm.
Between-run rinsing : rinse the capillary for at least 1 min
with 0.1 M sodium hydroxide filtered through a 0.45 m
membrane filter (0.45 m) and for 2 min with the CZE buffer.
Injection : under pressure or vacuum (for example, 3 s at a
differential pressure of 3.5 kPa).
Migration : apply a field strength of 0.2 kV/cm, using the
CZE buffer as the electrolyte in both buffer reservoirs.
Run time : 30 min.
Identification of impurities : use the electropherogram
supplied with aprotinin solution BRP and the
electropherogram obtained with the reference solution to
identify the peaks due to impurities A and B.
Relative migration with reference to aprotinin (migration
time = about 22 min) : impurity A = about 0.98 ; impurity B =
about 0.99.
System suitability : reference solution after at least 6
injections :
migration time : aprotinin = 19.0 min to 25.0 min ;
resolution : minimum 0.8 between the peaks due to
impurities A and B ; minimum 0.5 between the peaks due
to impurity B and aprotinin ;
symmetry factor : maximum 1.3 for the peak due to
aprotinin ;
peak distribution : the electrophoregram obtained
is qualitatively and quantitatively similar to the
electropherogram supplied with aprotinin solution BRP ;
height of the principal peak : at least 1000 times the
height of the baseline noise. If necessary, adjust the
sample load to give peaks of a sufficient height.
Limits :
impurity A : maximum 8.0 per cent ;
impurity B : maximum 7.5 per cent.
N-pyroglutamyl-aprotinin and related compounds. Liquid
chromatography (2.2.29) : use the normalisation procedure.
Test solution. Dilute the preparation to be examined in
mobile phase A to a concentration of about 5 Ph. Eur. U./ml.
Resolution solution. Dissolve the contents of a vial of
aprotinin for system suitability CRS 1 in mobile phase A to
obtain the same concentration as the test solution.
Column :
size : l = 0.075 m, = 7.5 mm ;
stationary phase : strong cation-exchange siilica gel for
chromatography R (10 m)(9) ;
temperature : 40 C.
Mobile phase :
mobile phase A : dissolve 3.52 g of potassium dihydrogen
phosphate R and 7.26 g of disodium hydrogen phosphate
dihydrate R in 1000 ml of water ; filter and degas ;
mobile phase B : dissolve 3.52 g of potassium dihydrogen
phosphate R, 7.26 g of disodium hydrogen phosphate
dihydrate R and 66.07 g of ammonium sulphate R in
1000 ml of water ; filter and degas ;

Time
(min)
0 - 21

Mobile phase A
(per cent V/V)
92 64

Mobile phase B
(per cent V/V)
8 36

21 - 30

64 0

36 100

30 - 31

0 92

100 8

31 - 40

92

Flow rate : 1.0 ml/min.

Detection : spectrophotometer at 210 nm.
Injection : 40 l.
Relative retention with reference to aprotinin (retention
time = 17.0 min to 20.0 min) : impurity C = about 0.9.
System suitability :
retention time : aprotinin = 17.0 min to 20.0 min ;
resolution : minimum 1.0 between the peaks due to
impurity C and aprotinin ;
symmetry factor : maximum 1.3 for the peak due to
aprotinin.
Limits :
impurity C : maximum 1.0 per cent ;
any other impurity : maximum 0.5 per cent ;
sum of impurities other than C : maximum 1.0 per cent.
Aprotinin oligomers. Size-exclusion chromatography
(2.2.30) : use the normalisation procedure.
Test solution. Dilute the preparation to be examined to a
concentration of about 5 Ph. Eur. U./ml.
Resolution solution. Dissolve the contents of a vial of
aprotinin for system suitability CRS 2 to obtain the same
concentration as the test solution.
Column : 3 columns coupled in series :
size : l = 0.30 m, = 7.8 mm ;
stationary phase : hydrophilic silica gel for
chromatography R of a grade suitable for fractionation of
globular proteins in the relative molecular mass range of
20 000 to 10 000 000 (8 m)(10).
Mobile phase : water R, glacial acetic acid R, acetonitrile R
(2:2:6 V/V/V) ; filter and degas.
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 277 nm.
Injection : 100 l.
Run time : 40 min.
Relative retention with reference to aprotinin monomer
(retention time = 24.5 min to 25.5 min) : aprotinin
dimer = about 0.9.
System suitability : resolution solution :
retention time : aprotinin monomer = 24.5 min to
25.5 min ;
resolution : minimum 1.3 between the peaks due to
aprotinin dimer and monomer ;
symmetry factor : maximum 2.5 for the peak due to
aprotinin monomer.
Limit :
total : maximum 1.0 per cent.
Specific activity of the dry residue : minimum 3.0 Ph. Eur. U.
of aprotinin activity per milligram of dry residue.
Evaporate 25.0 ml to dryness in a water-bath, dry the residue
at 110 C for 15 h and weigh. From the mass of the residue
and the activity determined as described below, calculate the

(9) Tosoh TSKgel SP5-PW is suitable.

(10) Tosoh TSKgel G4000SWXL is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

393

Aprotinin concentrated solution

number of European Pharmacopoeia Units per milligram

of dry residue.

Calculate the aprotinin activity in European Pharmacopoeia

Units per millilitre using the following expression :

Bacterial endotoxins (2.6.14) : less than 0.14 IU per

European Pharmacopoeia Unit of aprotinin, if intended for
use in the manufacture of parenteral dosage forms without a
further appropriate procedure for the removal of bacterial
endotoxins.
D
ASSAY
The activity of aprotinin is determined by measuring its
inhibitory action on a solution of trypsin of known activity.
The inhibiting activity of the aprotinin is calculated from
the difference between the initial activity and the residual
activity of the trypsin.
The inhibiting activity of aprotinin is expressed in European
Pharmacopoeia Units. 1 Ph. Eur. U. inhibits 50 per cent of
the enzymatic activity of 2 microkatals of trypsin.
Use a reaction vessel with a capacity of about 30 ml, provided
with :
a device that will maintain a temperature of 25 0.1 C ;
a stirring device, such as a magnetic stirrer ;

dilution factor of the aprotinin concentrated

solution to be examined in order to obtain a
solution containing 1.67 Ph. Eur. U./ml.

The estimated activity is not less than 90 per cent and not
more than 110 per cent of the activity stated on the label.
STORAGE
In an airtight, tamper-proof container, protected from light.
LABELLING
The label states :
the number of European Pharmacopoeia Units of
aprotinin activity per millilitre.
where applicable, that the solution is free from bacterial
endotoxins.
IMPURITIES

a lid with 5 holes for accommodating the electrodes, the

tip of a burette, a tube for the admission of nitrogen and
the introduction of the reagents.
An automatic or manual titration apparatus may be used. In
the latter case the burette is graduated in 0.05 ml and the
pH-meter is provided with a wide reading scale and glass and
calomel or glass-silver-silver chloride electrodes.
Test solution. With 0.0015 M borate buffer solution
pH 8.0 R prepare an appropriate dilution (D) of the aprotinin
concentrated solution expected, on the basis of the stated
potency, to contain 1.67 Ph. Eur. U./ml.
Trypsin solution. Prepare a solution of trypsin BRP
containing about 0.8 microkatals per millilitre (about
1 mg/ml), using 0.001 M hydrochloric acid as the solvent.
Use a freshly prepared solution and keep in iced water.

A. des-57-glycine-des-58-alanine--aprotinin,

Trypsin and aprotinin solution. To 4.0 ml of the trypsin

solution add 1.0 ml of the test solution. Dilute immediately
to 40.0 ml with 0.0015 M borate buffer solution pH 8.0 R.
Allow to stand at room temperature for 10 min and then
keep in iced water. Use within 6 h of preparation.
Dilute trypsin solution. Dilute 0.5 ml of the trypsin solution
to 10.0 ml with 0.0015 M borate buffer solution pH 8.0 R.
Allow to stand at room temperature for 10 min and then
keep in iced water.
B. des-58-alanine--aprotinin,
Maintain an atmosphere of nitrogen in the reaction flask and
stir continuously ; introduce 9.0 ml of 0.0015 M borate buffer
solution pH 8.0 R and 1.0 ml of a freshly prepared 6.9 g/l
solution of benzoylarginine ethyl ester hydrochloride R.
Adjust to pH 8.0 with 0.1 M sodium hydroxide. When the
temperature has reached equilibrium at 25 0.1 C, add
1.0 ml of the trypsin and aprotinin solution and start a
timer. Maintain at pH 8.0 by the addition of 0.1 M sodium
hydroxide and note the volume added every 30 s. Continue
the reaction for 6 min. Determine the number of millilitres of
0.1 M sodium hydroxide used per second (n1 ml). Carry out,
under the same conditions, a titration using 1.0 ml of the
dilute trypsin solution. Determine the number of millilitres
of 0.1 M sodium hydroxide used per second (n2 ml).
C. N-pyroglutamyl-aprotinin.
394

PHARMEUROPA Vol. 18, No. 3, July 2006

Atropine

Reference: PA/PH/Exp. 11/T (06) 43 ANP

D. Place about 3 mg in a porcelain crucible and add 0.2 ml

of fuming nitric acid R. Evaporate to dryness on a
water-bath. Dissolve the residue in 0.5 ml of a 30 g/l
NOTE ON THE MONOGRAPH
solution of potassium hydroxide R in methanol R. A
Related substances : the column used in the test is no
violet
colour develops.
longer available at a suitable quality, so a new method that
shows better specificity and improved peak symmetry has E. Optical rotation (see Tests).
been developed for the monographs on Atropine (2056)
and Atropine sulphate (0068).
TESTS
XXXX:2056 Optical rotation (2.2.7) : 0.70 to + 0.05 (measured in
a 2 dm tube).
ATROPINE
Dissolve 1.25 g in ethanol (96 per cent) R and dilute to
25.0 ml with the same solvent.
Atropinum
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50 mg of the substance to be
examined in mobile phase A and dilute to 50 ml with mobile
phase A. Dilute 10 ml of the solution to 50 ml with mobile
phase A.
Reference solution (a). Dilute 1.0 ml of the test solution to
100.0 ml with mobile phase A. Dilute 1.0 ml of this solution
to 10.0 ml with mobile phase A.
C17H23NO3
Mr 289.4 Reference solution (b). Dissolve 5 mg of atropine for system
suitability CRS in mobile phase A and dilute to 25 ml with
mobile phase A.
DEFINITION
Column :
(1R,3r,5S)-8-Methyl-8-azabicyclo[3.2.1]oct-3-yl
(2RS)-3-hydroxy-2-phenylpropanoate.
size : l = 0.125 m, = 4 mm,
Content : 99.0 per cent to 101.0 per cent (dried substance).
stationary phase : octylsilyl silica gel for
chromatography R (4 m)(11).
CHARACTERS
Mobile phase :
Appearance : white or almost white, crystalline powder or
mobile phase A : to 606 ml of a 7.0 g/l solution of
colourless crystals.
potassium dihydrogen phosphate R adjusted to pH 3.3
Solubility : very slightly soluble in water, freely soluble in
with 0.05 M phosphoric acid, add 3.5 g of sodium
ethanol (96 per cent) and in methylene chloride.
dodecyl sulphate R and 320 ml of acetonitrile R,
mobile phase B : acetonitrile R,
IDENTIFICATION
First identification : A, B, E.
Second identification : A, C, D, E.
A. Melting point (2.2.14) : 115 C to 119 C.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : Ph. Eur. reference spectrum of atropine.
C. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 10 mg of the substance to be
examined in methanol R and dilute to 10 ml with the
same solvent.
Reference solution. Dissolve 10 mg of atropine
sulphate CRS in methanol R and dilute to 10 ml with
the same solvent.
Plate : TLC silica gel plate R.
Mobile phase : concentrated ammonia R, water R,
acetone R (3:7:90 V/V/V).
Application : 10 l.
Development : over half of the plate.
Drying : at 100-105 C for 15 min.
Detection : after cooling, spray with dilute potassium
iodobismuthate solution R.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with the reference solution.

Time
(min)
0 - 12

Mobile phase A
(per cent V/V)
100

Mobile phase B
(per cent V/V)
0

12 - 25

100 70

0 30

25 - 26

70 100

30 0

26 - 30

100

Flow rate : 1 ml/min.

Detection : spectrophotometer at 210 nm.
Injection : 10 l.
System suitability : reference solution (b) :
the chromatogram obtained is similar to the
chromatogram supplied with atropine for system
suitability CRS,
peak-to-valley ratio : minimum 20, where Hp = height
above the baseline of the peak due to impurity B and
Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to atropine.
Limits : locate the impurities by comparing with the
chromatogram obtained with reference solution (b) and
with the chromatogram supplied with atropine for system
suitability CRS :
impurity B : not more than 3 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.3 per cent),

(11) Merck Superspher 60 RP select B is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

395

Atropine

impurity A : not more than 4 times the area of the

principal peak in the chromatogram obtained with
reference solution (a) (0.4 per cent),
any other impurity : not more than twice the area of
the principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent),
total: not more than 15 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (1.5 per cent),
disregard limit : the area of the principal peak in the
chromatogram obtained with reference solution (a)
(0.1 per cent) ; disregard peaks due to the blank.
Test solution. Dissolve 60.0 mg of the substance to be
examined in mobile phase A and dilute to 50.0 ml with
mobile phase A. Dilute 10.0 ml of this solution to 50.0 ml
with mobile phase A.
Reference solution (a). Dilute 5.0 ml of the test solution to
100.0 ml with mobile phase A. Dilute 5.0 ml of this solution
to 50.0 ml with mobile phase A.
Reference solution (b). Dilute 5.0 ml of reference solution (a)
to 25.0 ml with mobile phase A.
Reference solution (c). Dissolve 5.0 mg of noratropine
sulphate CRS (impurity B) in the test solution and dilute to
20.0 ml with the test solution. Dilute 5.0 ml of this solution
to 25.0 ml with mobile phase A.
Reference solution (d). Dissolve 5.0 mg of atropine for
peak identification CRS (containing impurities A, B, C, D,
E, F, G and H) in mobile phase A and dilute to 20.0 ml with
mobile phase A.
Column :
size : l = 0.10 m, = 4.6 mm ;
stationary phase : octadecylsilyl silica gel for
chromatography R (3 m)(12) ;
temperature : 25 1 C.
Mobile phase :
mobile phase A : dissolve 3.5 g of sodium dodecyl
sulphate R in 606 ml of a 7.0 g/l solution of potassium
dihydrogen phosphate R previously adjusted to pH 3.3
with 0.05 M phosphoric acid, and mix with 320 ml of
acetonitrile R ;

mobile phase B : acetonitrile R ;

Time
(min)
0-2

Mobile phase A
(per cent V/V)
95

Mobile phase B
(per cent V/V)
5

2 - 20

95 70

5 30

20 - 20.1

70 95

30 5

20.1 - 25

95

Flow rate : 1 ml/min.

Detection : spectrophotometer at 210 nm.
Injection : 10 l.
Relative retention with reference to atropine (retention
time = about 11 min) : impurity C = about 0.2 ;
impurity E = about 0.67 ; impurity D = about 0.73 ;
impurity F = about 0.8 ; impurity B = about 0.89 ;
impurity H = about 0.93 ; impurity G = about 1.1 ;
impurity A = about 1.7.
System suitability : reference solution (c) :
resolution : minimum 2.5 between the peaks due to
impurity B and atropine ;
symmetry factor : minimum 2.5 for the peak due to
atropine.
Limits :
correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity A = 0.6 ;
impurity C = 0.6 ;
impurities A, C, D, E, F, G, H : for each impurity, not
more than twice the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.2 per cent) ;
impurity B : not more than 3 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.3 per cent) ;
unspecified impurities : for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.10 per cent) ;

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. impurity C

4. impurity F

7. atropine

2. impurity E

5. impurity B

8. impurity G

3. impurity D

6. impurity H

9. impurity A

Figure 2056.-1. Chromatogram for the test for related substances of atropine
(12) Aquasil C18, 100 x 4.6 mm is suitable.

396

PHARMEUROPA Vol. 18, No. 3, July 2006

Atropine sulphate

total: not more than the area of the principal peak in

the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
disregard limit : 0.1 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.2 per cent, determined G. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl
(2RS)-2-hydroxy-3-phenylpropanoate (littorine),
on 1.000 g by drying in an oven at 100-105 C for 2 h.
ASSAY
Dissolve 0.250 g in 40 ml of anhydrous acetic acid R,
warming if necessary. Allow the solution to cool. Titrate
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1 ml of 0.1 M perchloric acid is equivalent to 28.94 mg
of C17H23NO3.
STORAGE
Protected from light.
IMPURITIES
Specified impurities : A, B, C, D, E, F, G, H.

H. unknown structure.

Reference: PA/PH/Exp. 11/T (04) 97 ANP 1R

NOTE ON THE MONOGRAPH
Related substances : this monograph was published in
Pharmeuropa 17.2 to replace the TLC by an LC, but because
the column that was used is no longer available at a
suitable quality, a new method that shows better specificity
and improved peak symmetry has been developed.
Impurities : names of impurities have been harmonised
with the current monograph for Atropine (2056).
XXXX:0068

ATROPINE SULPHATE
Atropini sulfas
A. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl
2-phenylpropenoate (apoatropine),

C34H48N2O10S,H2O
B. (1R,3r,5S)-8-azabicyclo[3.2.1]oct-3-yl (2RS)-3-hydroxy-2phenylpropanoate (noratropine),

C. (2RS)-3-hydroxy-2-phenylpropanoic acid (tropic acid),

D. R1 = OH, R2 = H : (1R,3S,5R,6RS)-6-hydroxy-8methyl-8-azabicyclo[3.2.1]oct-3-yl (2S)-3-hydroxy-2phenylpropanoate (6-hydroxyhyoscyamine),

E. R1 = H, R2 = OH : (1S,3R,5S,6RS)-6-hydroxy-8methyl-8-azabicyclo[3.2.1]oct-3-yl (2S)-3-hydroxy-2phenylpropanoate (7-hydroxyhyoscyamine),
F. hyoscine,
PHARMEUROPA Vol. 18, No. 3, July 2006

Mr 695

DEFINITION
Bis[(1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl
(2RS)-3-hydroxy-2-phenylpropanoate] sulphate monohydrate.
Content : 99.0 per cent to 101.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or
colourless crystals.
Solubility : very soluble in water, freely soluble in ethanol
(96 per cent).
It melts at about 190 C with decomposition, determined on
the substance dried at 135 C for 15 min.
IDENTIFICATION
First identification : A, B, E.
Second identification : C, D, E, F.
A. An aqueous solution shows almost no optical rotation
(see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : atropine sulphate CRS.
C. Dissolve about 50 mg in 5 ml of water R and add 5 ml
of picric acid solution R. The precipitate, washed with
water R and dried at 100-105 C for 2 h, melts (2.2.14)
at 174 C to 179 C.
397

Atropine sulphate

D. To about 1 mg add 0.2 ml of fuming nitric acid R and

Mobile phase :
evaporate to dryness in a water-bath. Dissolve the residue mobile phase A : dissolve 3.5 g of sodium dodecyl
in 2 ml of acetone R and add 0.1 ml of a 30 g/l solution
sulphate R in 606 ml of a 7.0 g/l solution of potassium
of potassium hydroxide R in methanol R. A violet colour
dihydrogen phosphate R previously adjusted to pH 3.3
develops.
with 0.05 M phosphoric acid, and mix with 320 ml of
E. It gives the reactions of sulphates (2.3.1).
acetonitrile R ;
F. It gives the reaction of alkaloids (2.3.1).
mobile phase B : acetonitrile R ;
TESTS
pH (2.2.3) : 4.5 to 6.2.
Dissolve 0.6 g in carbon dioxide-free water R and dilute to
30 ml with the same solvent.
Optical rotation (2.2.7) : 0.50 to + 0.05 (measured in
a 2 dm tube).
Dissolve 2.50 g in water R and dilute to 25.0 ml with the
same solvent.
Foreign alkaloids and decomposition products. Examine by
thin-layer chromatography (2.2.27), using silica gel G R as
the coating substance.
Test solution. Dissolve 0.2 g of the substance to be examined
in methanol R and dilute to 10 ml with the same solvent.
Reference solution (a). Dilute 1 ml of the test solution to
100 ml with methanol R.
Reference solution (b). Dilute 5 ml of reference solution (a)
to 10 ml with methanol R.
Apply separately to the plate 10 l of each solution. Develop
over a path of 10 cm using a mixture of 90 volumes
of acetone R, 7 volumes of water R and 3 volumes of
concentrated ammonia R. Dry the plate at 100 C to 105 C
for 15 min. Allow to cool and spray with dilute potassium
iodobismuthate solution R until the spots appear. Any spot
in the chromatogram obtained with the test solution, apart
from the principal spot, is not more intense than the spot
in the chromatogram obtained with reference solution (a)
(1.0 per cent) and not more than one such spot is more
intense than the spot in the chromatogram obtained with
reference solution (b) (0.5 per cent).
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 60.0 mg of the substance to be
examined in mobile phase A and dilute to 50.0 ml with
mobile phase A. Dilute 10.0 ml of this solution to 50.0 ml
with mobile phase A.
Reference solution (a). Dilute 5.0 ml of the test solution to
100.0 ml with mobile phase A. Dilute 5.0 ml of this solution
to 50.0 ml with mobile phase A.
Reference solution (b). Dilute 5.0 ml of reference solution (a)
to 25.0 ml with mobile phase A.
Reference solution (c). Dissolve 5.0 mg of noratropine
sulphate CRS (impurity B) in the test solution and dilute to
20.0 ml with the test solution. Dilute 5.0 ml of this solution
to 25.0 ml with mobile phase A.
Reference solution (d). Dissolve 5.0 mg of atropine for
peak identification CRS (containing impurities A, B, C, D,
E, F, G and H) in mobile phase A and dilute to 20.0 ml with
mobile phase A.
Column :
size : l = 0.10 m, = 4.6 mm ;
stationary phase : octadecylsilyl silica gel for
chromatography R (3 m)(13) ;
temperature : 25 1 C.

Time
(min)
0-2

Mobile phase A
(per cent V/V)
95

Mobile phase B
(per cent V/V)
5

2 - 20

95 70

5 30

20 - 20.1

70 95

30 5

20.1 - 25

95

Flow rate : 1 ml/min.

Detection : spectrophotometer at 210 nm.
Injection : 10 l.
Relative retention with reference to atropine (retention
time = about 11 min) : impurity C = about 0.2 ;
impurity E = about 0.67 ; impurity D = about 0.73 ;
impurity F = about 0.8 ; impurity B = about 0.89 ;
impurity H = about 0.93 ; impurity G = about 1.1 ;
impurity A = about 1.7.
System suitability : reference solution (c) :
resolution : minimum 2.5 between the peaks due to
impurity B and atropine ;
symmetry factor : minimum 2.5 for the peak due to
atropine.
Limits :
correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity A = 0.6 ;
impurity C = 0.6 ;
impurities A, C, D, E, F, G, H : for each impurity, not
more than twice the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.2 per cent) ;
impurity B : not more than 3 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.3 per cent) ;
unspecified impurities : for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.10 per cent) ;
total : not more than the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
disregard limit : 0.1 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Apoatropine. Dissolve 0.10 g in 0.01 M hydrochloric acid
and dilute to 100.0 ml with the same acid. Determine the
absorbance (2.2.25) at 245 nm. The specific absorbance
is not greater than 4.0, calculated with reference to the
anhydrous substance (about 0.5 per cent).
Water (2.5.12) : 2.0 per cent to 4.0 per cent, determined on
0.50 g.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.

(13) Aquasil C18, 100 4.6 mm is suitable.

398

PHARMEUROPA Vol. 18, No. 3, July 2006

Atropine sulphate

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. impurity C

4. impurity F

7. atropine

2. impurity E

5. impurity B

8. impurity G

3. impurity D

6. impurity H

9. impurity A

Figure 0068.-1. Chromatogram for the test for related substances of atropine sulphate

ASSAY
Dissolve 0.500 g in 30 ml of anhydrous acetic acid R,
warming if necessary. Cool the solution. Titrate with
0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1 ml of 0.1 M perchloric acid is equivalent to 67.68 mg
of C34H48N2O10S.

C. (2RS)-3-hydroxy-2-phenylpropanoic acid (tropic acid),

STORAGE
Protected from light.
IMPURITIES
Specified impurities : A, B, C, D, E, F, G, H.
D. R1 = OH, R2 = H : (1R,3S,5R,6RS)-6-hydroxy-8methyl-8-azabicyclo[3.2.1]oct-3-yl (2S)-3-hydroxy-2phenylpropanoate (6-hydroxyhyoscyamine),
E. R1 = H, R2 = OH : (1S,3R,5S,6RS)-6-hydroxy-8methyl-8-azabicyclo[3.2.1]oct-3-yl (2S)-3-hydroxy-2phenylpropanoate (7-hydroxyhyoscyamine),
A. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl
2-phenylpropenoate (apoatropine),

F. hyoscine,

G. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl
(2RS)-2-hydroxy-3-phenylpropanoate (littorine).
B. (1R,3r,5S)-8-azabicyclo[3.2.1]oct-3-yl (2RS)-3-hydroxy-2phenylpropanoate (noratropine),
PHARMEUROPA Vol. 18, No. 3, July 2006

H. unknown structure.
399

Avian infectious bronchitis vaccine (live)

Reference: PA/PH/Exp. 15V/T (06) 9 ANP

NOTE ON THE MONOGRAPH
It is proposed below to replace the requirement for the
average ciliostasis score in the safety test for the respiratory
tract and kidneys, which should not be more than 25, by
a risk/benefit analysis of the average ciliostasis scores.
Indeed, since some vaccines cannot comply with the
requirement of 25, but can be useful as a booster injection
in a number of epidemiological situations, it is proposed to
maintain the test, since it provides useful information, but
not to specify a limit anymore.
XXXX:0442

AVIAN INFECTIOUS BRONCHITIS

VACCINE (LIVE)
Vaccinum bronchitidis infectivae
aviariae vivum
1. DEFINITION
Avian infectious bronchitis vaccine (live) is a preparation
of one or more suitable strains of different types of avian
infectious bronchitis virus. This monograph applies to
vaccines intended for administration to chickens for active
immunisation against respiratory disease caused by avian
infectious bronchitis virus.
2. PRODUCTION
2-1. PREPARATION OF THE VACCINE
The vaccine virus is grown in embryonated hens eggs or in
cell cultures.
2-2. SUBSTRATE FOR VIRUS PROPAGATION
2-2-1. Embryonated hens eggs. If the vaccine virus is grown
in embryonated hens eggs, they are obtained from flocks
free from specified pathogens (SPF) (5.2.2).
2-2-2. Cell cultures. If the vaccine virus is grown in cell
cultures, they comply with the requirements for cell cultures
for production of veterinary vaccines (5.2.4).
2-3. SEED LOTS
2-3-1. Extraneous agents. The master seed lot complies with
the tests for extraneous agents in seed lots (2.6.24). In these
tests on the master seed lot, the organisms used are not
more that 5 passages from the master seed lot at the start
of the test.
2-4. CHOICE OF VACCINE VIRUS
The vaccine virus shall be shown to be satisfactory with
respect to safety (5.2.6) and efficacy (5.2.7) for the chickens
for which it is intended.
The following tests for safety (section 2-4-1), increase in
virulence (section 2-4-2) and immunogenicity (section
2-4-3) may be used during the demonstration of safety and
immunogenicity.
2-4-1. Safety
2-4-1-1. Safety for the respiratory tract and kidneys. Carry
out the test in chickens not older than the minimum age to
be recommended for vaccination. Use vaccine virus at the
least attenuated passage level that will be present between
the master seed lot and a batch of the vaccine. Use not fewer
than 15 chickens from an SPF flock (5.2.2) and from the
same origin. Administer to each chicken by the oculonasal
route a quantity of the vaccine virus equivalent to not less
than 10 times the maximum virus titre likely to be contained
in 1 dose of the vaccine. On each of days 5, 7 and 10 after
administration of the virus, euthanise not fewer than 5 of
400

the chickens and take samples of trachea and kidney. Fix

kidney samples for histological examination. Remove the
tracheas and cut 10 rings from each trachea (3 from the top,
4 from the mid-part and 3 from the bottom) ; examine each
ring under low magnification and score for ciliostasis on a
scale from 0 (100 per cent ciliary activity) to 4 (no activity,
complete ciliostasis) ; calculate the mean ciliostasis score
(the maximum for each trachea being 40) for the 5 chickens
euthanised on each of days 5, 7 and 10. The test is not valid
if more than 10 per cent of the chickens die from causes not
attributable to the vaccine virus. The vaccine virus complies
with the test if :
no chicken shows notable clinical signs of avian infectious
bronchitis or dies from causes attributable to the vaccine
virus ;
the average ciliostasis score is not more than 25 ;
any inflammatory lesions seen during the kidney
histological examination are, at most, moderate.
A risk/benefit analysis is carried out on the average
ciliostasis scores obtained.
2-4-1-2. Safety for the reproductive tract. If the
recommendations for use state or imply that the vaccine may
be used in females less than 3 weeks old that are subsequently
kept to sexual maturity, it shall be demonstrated that there
is no damage to the development of the reproductive tract
when the vaccine is given to chickens of the minimum age
to be recommended for vaccination. The following test may
be carried out : use not fewer than 40 female chickens from
an SPF flock (5.2.2) that are not older than the minimum
age recommended for vaccination ; use the vaccine virus
at the least attenuated passage level that will be present
in a batch of vaccine ; administer to each chicken by a
recommended route a quantity of virus equivalent to not
less than the maximum titre likely to be present in 1 dose of
vaccine ; at least 10 weeks after administration of the vaccine
virus, euthanise the chickens and carry out a macroscopic
examination of the oviducts. The vaccine virus complies
with the test if abnormalities are present in not more than
5 per cent of the oviducts.
2-4-2. Increase in virulence. The test for increase in
virulence consists of : the administration of the vaccine
virus, at the least attenuated virus passage level that will
be present between the master seed lot and a batch of
the vaccine, to a group of 5 two-week-old chickens from
an SPF flock (5.2.2) ; sequential passages, 5 times where
possible, to further similar groups ; and testing of the final
recovered virus for increase in virulence. If the properties
of the vaccine virus allow sequential passage to 5 groups
via natural spreading, this method may be used, otherwise,
passage as described below is carried out and the maximally
passaged virus that has been recovered is tested for increase
in virulence. Care must be taken to avoid contamination
by virus from previous passages. Administer by eye-drop
a quantity of the vaccine virus that will allow recovery
of virus for the passages described below. 2-4 days after
administration of the vaccine virus, prepare a suspension
from the mucosa of the trachea of each chicken and pool
these samples. Administer 0.05 ml of the pooled samples
by eye-drop to each of 5 other two-week-old chickens from
an SPF flock (5.2.2). Carry out this passage operation not
fewer than 5 times ; verify the presence of the virus at each
passage. If the virus is not found at a passage level, carry
out a second series of passages. Carry out the test for safety
for the respiratory tract and kidney (section 2-4-1-1) and,
where applicable, the test for safety for the reproductive
tract (section 2-4-1-2) using the unpassaged vaccine virus
and the maximally passaged virus that has been recovered.
Administer the virus by the route to be recommended for
PHARMEUROPA Vol. 18, No. 3, July 2006

Avian infectious bronchitis vaccine (live)

vaccination that is likely to be the least safe. The vaccine

virus complies with the test if no indication of an increase
in virulence of the maximally passaged virus compared with
the unpassaged virus is observed. If virus is not recovered
at any passage level in the 1st and 2nd series of passages, the
vaccine virus also complies with the test.
2-4-3. Immunogenicity. Immunogenicity is demonstrated for
each strain of virus to be included in the vaccine. A test is
carried out for each route and method of administration to
be recommended using in each case chickens from an SPF
flock (5.2.2) that are not older than the minimum age to be
recommended for vaccination. The quantity of the vaccine
virus administered to each chicken is not greater than the
minimum virus titre to be stated on the label and the virus is
at the most attenuated passage level that will be present in a
batch of the vaccine. Either or both of the tests below may
be used during the demonstration of immunogenicity.
2-4-3-1. Ciliary activity of tracheal explants. Use not fewer
than 25 chickens of the same origin and from an SPF flock
(5.2.2). Vaccinate by a recommended route not fewer than
20 chickens. Maintain not fewer than 5 chickens as controls.
Challenge each chicken after 21 days by eye-drop with a
sufficient quantity of virulent avian infectious bronchitis
virus of the same type as the vaccine virus to be tested.
Euthanise the chickens 4-7 days after challenge and prepare
3 transverse sections from the upper part, 4 from the middle
part, and 3 from the lower part of the trachea of each
chicken. Examine all explants as soon as possible and at the
latest 2 h after sampling by low-magnification microscopy for
ciliary activity. For a given tracheal section, ciliary activity
is considered as normal when at least 50 per cent of the
internal ring shows vigorous ciliary movement. A chicken is
considered not affected if not fewer than 9 out of 10 rings
show normal ciliary activity.
The test is not valid if :
fewer than 80 per cent of the control chickens show
cessation or extreme loss of vigour of ciliary activity ;
and/or during the period between the vaccination and
challenge more than 10 per cent of vaccinated or control
chickens show abnormal clinical signs or die from causes
not attributable to the vaccine.
The vaccine virus complies with the test if not fewer than
80 per cent of the vaccinated chickens show normal ciliary
activity.
2-4-3-2. Virus recovery from tracheal swabs. Use not fewer
than 30 chickens of the same origin and from an SPF flock
(5.2.2). Vaccinate by a recommended route not fewer than
20 chickens. Maintain not fewer than 10 chickens as controls.
Challenge each chicken after 21 days by eye-drop with a
sufficient quantity of virulent avian infectious bronchitis
virus of the same type as the vaccine virus to be tested.
Euthanise the chickens 4-7 days after challenge and prepare
a suspension from swabs of the tracheal mucosa of each
chicken. Inoculate 0.2 ml of the suspension into the allantoic
cavity of each of 5 embryonated hens eggs, 9-11 days old,
from an SPF flock (5.2.2). Incubate the eggs for 6-8 days
after inoculation. Eggs that after 1 day of incubation do
not contain a live embryo are eliminated and considered as
non-specific deaths. Record the other eggs containing a dead
embryo and after 6-8 days incubation examine each egg
containing a live embryo for lesions characteristic of avian
infectious bronchitis. Make successively 3 such passages.
If 1 embryo of a series of eggs dies or shows characteristic
lesions, the inoculum is considered to be a carrier of avian
infectious bronchitis virus. The examination of a series of
eggs is considered to be definitely negative if no inoculum
concerned is a carrier. The test is not valid if :
PHARMEUROPA Vol. 18, No. 3, July 2006

the challenge virus is re-isolated from fewer than 80 per

cent of the control chickens ;
and/or during the period between vaccination and
challenge more than 10 per cent of the vaccinated or
control chickens show abnormal clinical signs or die from
causes not attributable to the vaccine ;
and/or more than 1 egg in any group is eliminated
because of non-specific embryo death.
The vaccine virus complies with the test if the challenge
virus is re-isolated from not more than 20 per cent of the
vaccinated chickens.
3. BATCH TESTS
3-1. Identification
3-1-1. Vaccines containing one type of virus. The vaccine,
diluted if necessary and mixed with avian infectious
bronchitis virus antiserum specific for the virus type, no
longer infects embryonated hens eggs from an SPF flock
(5.2.2) or susceptible cell cultures (5.2.4) into which it is
inoculated.
3-1-2. Vaccines containing more than one type of virus.
The vaccine, diluted if necessary and mixed with type-specific
antisera against each strain present in the vaccine except
that to be identified, infects embryonated hens eggs from
an SPF flock (5.2.2) or susceptible cell cultures (5.2.4) into
which it is inoculated, whereas after further admixture with
type-specific antiserum against the strain to be identified it
no longer produces such infection.
3-2. Bacteria and fungi
Vaccines intended for administration by injection comply
with the test for sterility prescribed in the monograph
Vaccines for veterinary use (0062).
Vaccines not intended for administration by injection
comply either with the test for sterility prescribed in the
monograph Vaccines for veterinary use (0062) or with the
following test : carry out a quantitative test for bacterial
and fungal contamination ; carry out identification tests for
micro-organisms detected in the vaccine ; the vaccine does
not contain pathogenic micro-organisms and contains not
more than 1 non-pathogenic micro-organism per dose.
Any liquid supplied with the vaccine complies with the
test for sterility prescribed in the monograph Vaccines for
veterinary use (0062).
3-3. Mycoplasmas. The vaccine complies with the test for
mycoplasmas (2.6.7).
3-4. Extraneous agents. The vaccine complies with the tests
for extraneous agents in batches of finished product (2.6.25).
3-5. Safety. Use not fewer than 10 chickens from an SPF
flock (5.2.2) and of the minimum age recommended for
vaccination. Administer by a recommended route to each
chicken 10 doses of the vaccine. Observe the chickens at
least daily for 21 days. The test is not valid if more than
20 per cent of the chickens show abnormal clinical signs
or die from causes not attributable to the vaccine. The
vaccine complies with the test if no chicken shows notable
clinical signs of disease or dies from causes attributable to
the vaccine.
3-6. Virus titre. Titrate the vaccine virus by inoculation
into embryonated hens eggs from an SPF flock (5.2.2) or
into suitable cell cultures (5.2.4). If the vaccine contains
more than 1 strain of virus, titrate each strain after having
neutralised the others with type-specific avian infectious
bronchitis antisera. The vaccine complies with the test if
1 dose contains for each vaccine virus not less than the
minimum titre stated on the label.
401

Boldo leaf dry extract

3-7. Potency. The vaccine complies with the requirements

of 1 of the tests prescribed under Immunogenicity
(section 2-4-3) when administered according to the
recommended schedule by a recommended route and
method. It is not necessary to carry out the potency test
for each batch of the vaccine if it has been carried out on a
representative batch using a vaccinating dose containing not
more than the minimum virus titre stated on the label.

Development : over a path of 15 cm [or 6 cm].

Drying : in air.
Detection : spray with potassium iodobismuthate
solution R2, allow to dry in air for 5 min and spray with
sodium nitrite solution R ; examine in daylight after 30 min.
Results : see below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.
Top of the plate

Reference: PA/PH/Exp. 13A/T (04) 44 ANP 1R

NOTE ON THE MONOGRAPH
Following the comments received after publication in
Pharmeuropa 17.1, the preparation of the test solutions
used for identification by TLC and for assay by LC have
been improved.
XXXX:1816

BOLDO LEAF DRY EXTRACT

Boldi folii extractum siccum
DEFINITION
Extract produced from Boldo leaf (1396).
Content :
for aqueous extracts : minimum 0.5 per cent 0.50 per
cent of total alkaloids, expressed as boldine (C19H21NO4 ;
Mr 327.4) (dried extract),
for hydroalcoholic extracts : minimum 1.0 per cent
1.00 per cent of total alkaloids, expressed as boldine
(C19H21NO4 ; Mr 327.4) (dried extract).

_______

_______
A yellowish-brown zone
An orange-yellow zone

Hyoscine : a pale brown zone

An yellow orange zone
An brown orange zone
A brown zone
_______

_______

Boldine : a brown zone

A brown zone (boldine)

Several orange zones

Reference solution

Test solution

TESTS
Loss on drying (2.8.17) : maximum 5.0 per cent.

ASSAY
Liquid chromatography (2.2.29).
Test solution. Dissolve 0.1000 g of the extract to be
examined in the mobile phase and dilute to 10.0 ml with
the mobile phase. To 1.000 g of the extract to be examined
PRODUCTION
add 50 ml of dilute hydrochloric acid R and sonicate for
The extract is produced from the drug by a suitable
10 min. Transfer to a separating funnel and wash with 10 ml
procedure using either hot water or a hydroalcoholic solvent of a mixture of equal volumes of ethyl acetate R and of
equivalent in strength to a minimum of between 45 per
hexane R. Adjust the aqueous phase to pH 9.5 with dilute
cent V/V and 75 per cent V/V ethanol.
ammonia R1. After cooling, shake successively with 100 ml,
50 ml and 50 ml of methylene chloride R, taking care not
CHARACTERS
to form an emulsion. Evaporate the combined lower layers
Appearance : brown or greenish-brown, hygroscopic powder. to dryness under reduced pressure. Dissolve the residue in
the mobile phase ; transfer the solution to a volumetric flask,
IDENTIFICATION
rinse and dilute to 10.0 ml with the mobile phase.
Thin-layer chromatography (2.2.27).
Reference solution. Dissolve 12 mg of boldine CRS in
Test solution. Mix 0.15 g of the extract to be examined and 100.0 ml of mobile phase. Dilute 1.0 ml of this solution to
5 ml de methanol R. Filter on a 3 cm 0.5 cm column of
10.0 ml with the mobile phase.
cellulose for chromatography R1. Use the first millilitre
Reference solution. Dissolve 12 mg of boldine CRS in the
of eluate. To 0.5 g of the extract to be examined add 1 ml
mobile phase and dilute to 100.0 ml with the mobile phase.
of hydrochloric acid R and 20 ml of water R. Sonicate
Solution A. Mix 0.2 ml of diethylamine R with 99.8 ml of
for 10 min. Transfer the liquid to a separating funnel and
acetonitrile R.
make alkaline with 2 ml of dilute ammonia R1. Shake
with 2 quantities, each of 20 ml, of methylene chloride R.
Solution B. Mix 0.2 ml of diethylamine R with 99.8 ml of
Evaporate the combined organic layers to dryness. Dissolve water R and adjust to pH 3 with anhydrous formic acid R.
the residue in 1 ml of methanol R.
Column :
Reference solution. Dissolve 2 mg of boldine R and 10 mg
size : l = 0.25 m, = 4.6 mm ;
of hyoscine hydrobromide R in 5 ml of methanol R.
stationary phase : octadecylsilyl silica gel for
Plate : TLC silica gel plate R (5-40 m) [or TLC silica gel
chromatography R (5 m)(14).
plate R (2-10 m)].
Mobile phase : solution A, solution B (16:84 V/V).
Mobile phase : diethylamine R, methanol R, toluene R
Flow rate : 1.5 ml/min.
(10:10:80 V/V/V).
Detection : spectrophotometer at 304 nm.
Application : 40 l [or 6 l] as bands of 15 mm [or 8 mm]
20 l [or 3 l] as bands of 15 mm [or 8 mm].
Injection : 20 l.
(14) Inertsil ODS3 is suitable.

402

PHARMEUROPA Vol. 18, No. 3, July 2006

Caffeine

Relative retention with reference to boldine (retention

time = about 6 min) : isoboldine = about 0.9 ; isocorydine
N-oxide = about 1.8 ; laurotetanine = about 2.2 ;
isocorydine = about 2.8 ; N-methyllaurotetanine = about 3.2.
Additional peaks may be present.
System suitability : test solution :
resolution : minimum 2 1 between the peaks due to
isoboldine and boldine.
Calculate the percentage content of total alkaloids, expressed
as boldine, using the following expression :

m1

mass of the extract to be examined, in grams ;

m2

mass of boldine CRS in the reference solution,

in grams ;
sum of the areas of the peaks due to the
6 alkaloids identified in the chromatogram
obtained with the test solution ;
area of the peak due to boldine in the
chromatogram obtained with the reference
solution.

A1 =
A2

IDENTIFICATION
First identification : A, B, E.
Second identification : A, C, D, E, F.
A. Melting point (2.2.14) : 234 C to 239 C.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : caffeine CRS.
C. To 2 ml of a saturated solution add 0.05 ml of iodinated
potassium iodide solution R. The solution remains
clear. Add 0.1 ml of dilute hydrochloric acid R. A brown
precipitate is formed. Neutralise with dilute sodium
hydroxide solution R. The precipitate dissolves.
D. In a ground-glass-stoppered tube, dissolve about 10 mg
in 0.25 ml of a mixture of 0.5 ml of acetylacetone R and
5 ml of dilute sodium hydroxide solution R. Heat in a
water-bath at 80 C for 7 min. Cool and add 0.5 ml of
dimethylaminobenzaldehyde solution R2. Heat again in
a water-bath at 80 C for 7 min. Allow to cool and add
10 ml of water R. An intense blue colour develops.
E. Loss on drying (see Tests).
F. It gives the reaction of xanthines (2.3.1).

TESTS
Solution S. Dissolve 0.5 g with heating in 50 ml of carbon
dioxide-free water R prepared from distilled water R, cool
and dilute to 50 ml with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Reference: PA/PH/Exp. 10A/T (06) 15 ANP
Acidity. To 10 ml of solution S add 0.05 ml of bromothymol
blue solution R1. The solution is green or yellow. Not more
NOTE ON THE MONOGRAPH
than 0.2 ml of 0.01 M sodium hydroxide is required to
change the colour of the indicator to blue.
Test for related substances : the TLC method has been
replaced by an LC method.
Related substances. Examine by thin-layer chromatography
(2.2.27) using silica gel GF254 R as the coating substance.
Only comments on the test for related substances are
requested.
Test solution. Dissolve 0.2 g of the substance to be examined
XXXX:0267 in a mixture of 4 volumes of methanol R and 6 volumes of
methylene chloride R and dilute to 10 ml with the same
mixture of solvents.
CAFFEINE
Reference solution. Dilute 0.5 ml of the test solution to
100 ml with a mixture of 4 volumes of methanol R and
Coffeinum
6 volumes of methylene chloride R.
Apply to the plate 10 l of each solution. Develop over a
path of 15 cm using a mixture of 10 volumes of concentrated
ammonia R, 30 volumes of acetone R, 30 volumes of
methylene chloride R and 40 volumes of butanol R. Allow
the plate to dry in air and examine in ultraviolet light at
254 nm. Any spot in the chromatogram obtained with the
test solution, apart from the principal spot, is not more
intense than the spot in the chromatogram obtained with
C8H10N4O2
Mr 194.2 the reference solution (0.5 per cent).
Liquid chromatography (2.2.29).
DEFINITION
Test solution. Dissolve 100.0 mg of the substance to be
1,3,7-Trimethyl-3,7-dihydro-1H-purine-2,6-dione.
examined in the mobile phase and dilute to 50.0 ml with the
Content : 98.5 per cent to 101.5 per cent (dried substance).
mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with
the mobile phase.
CHARACTERS
Reference solution (a). Dilute 1.0 ml of the test solution
Appearance : white or almost white, crystalline powder or
to 100.0 ml with the mobile phase. Dilute 1.0 ml of this
silky, white or almost white crystals.
solution to 10.0 ml with the mobile phase.
Solubility : sparingly soluble in water, freely soluble in
Reference solution (b). Dissolve 10.0 mg of caffeine for
boiling water, slightly soluble in ethanol (96 per cent). It
system suitability CRS (containing impurities A, C, D and F)
dissolves in concentrated solutions of alkali benzoates or
in the mobile phase and dilute to 10.0 ml with the mobile
salicylates.
phase. Dilute 1.0 ml of this solution to 100.0 ml with the
mobile phase.
It sublimes readily.
PHARMEUROPA Vol. 18, No. 3, July 2006

403

Caffeine

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. impurity B

3. impurity C

5. impurity F

2. impurity E

4. impurity D

6. impurity A

7. caffeine

Figure 0267.-1. Chromatogram for the test for related substances of caffeine : solution of caffeine spiked with
impurities A, B, C, D, E and F
Column :
size : l = 0.15 m, = 4.6 mm ;
stationary phase : base-deactivated end-capped
octadecylsilyl silica gel for chromatography R (5 m)(15).
Mobile phase : dissolve 1.64 g of anhydrous sodium
acetate R in water R and dilute to 2000 ml with the same
solvent. Mix 1910 ml of this solution, 50 ml of acetonitrile R
and 40 ml of tetrahydrofuran R. Adjust to pH 4.5 with
glacial acetic acid R.
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 275 nm.
Injection : 10 l.
Run time : 1.5 times the retention time of caffeine.
Identification of impurities : use the chromatogram
supplied with caffeine for system suitability CRS and the
chromatogram obtained with reference solution (b) to
identify the peaks due to impurities A, C, D and F.
Retention time : caffeine = about 8 min.
System suitability : reference solution (b) :
resolution : minimum 2.5 between the peaks due to
impurities C and D and minimum 2.5 between the peaks
due to impurities F and A.
Limits :
unspecified impurities : for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.10 per cent) ;
total: not more than the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.1 per cent) ;

disregard limit : 0.5 times the area of the principal peak

in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Sulphates (2.4.13) : maximum 500 ppm, determined on
15 ml of solution S.
Prepare the standard using a mixture of 7.5 ml of sulphate
standard solution (10 ppm SO4) R and 7.5 ml of distilled
water R.
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test C. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 100-105 C for 1 h.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Dissolve 0.170 g with heating in 5 ml of anhydrous acetic
acid R. Allow to cool, add 10 ml of acetic anhydride R and
20 ml of toluene R. Titrate with 0.1 M perchloric acid,
determining the end-point potentiometrically (2.2.20).
1 ml of 0.1 M perchloric acid is equivalent to 19.42 mg
of C8H10N4O2.
IMPURITIES
Specified impurities : A.
Other detectable impurities (the following substances
would, if present at a sufficient level, be detected by one
or other of the tests in the monograph. They are limited

(15) Supelcosil LC-18-DB is suitable.

404

PHARMEUROPA Vol. 18, No. 3, July 2006

Calcium carbonate

by the general acceptance criterion for other/unspecified

impurities and/or by the general monograph Substances
for pharmaceutical use (2034). It is therefore not
necessary to identify these impurities for demonstration
of compliance. See also 5.10. Control of impurities in
substances for pharmaceutical use): A, B, C, D, E, F.

FRCs are to be dealt with in a specific non-mandatory

section of the monograph, and the particular use or uses
for which a given FRC is relevant are to be specified.
FRCs are very diverse in nature, and their treatment in
monographs will have to be adapted to each type.
XXXX:0014

A. theophylline,

CALCIUM CARBONATE
Calcii carbonas
CaCO3

Mr 100.1

DEFINITION
Content : 98.5 per cent to 100.5 per cent (dried substance).
B. N-[6-amino-1,3-dimethyl-2,4(1H,3H)-dioxopyrimidin-5yl]formamide,

CHARACTERS
Appearance : white or almost white powder.
Solubility : practically insoluble in water.
IDENTIFICATION
A. It gives the reaction of carbonates (2.3.1).
B. 0.2 ml of solution S (see Tests) gives the reactions of
calcium (2.3.1).

C. 1,3,9-trimethyl-3,9-dihydro-1H-purine-2,6-dione
(isocaffeine),

D. 3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione
(theobromine),

E. N,1-dimethyl-4-(methylamino)-1H-imidazole-5carboxamide (caffeidine),

F. 1,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione.

Reference: PA/PH/Exp. INC/T (06) 13 ANP

NOTE ON THE MONOGRAPH
The European Pharmacopoeia Commission has decided
that more attention will be paid to functionality-related
characteristics (FRCs) in monographs on excipients.
While these characteristics are of importance for the
intended use of many excipients, they are essentially a
matter for agreement between the excipient supplier and
the manufacturer of a medicinal product. For this reason,
PHARMEUROPA Vol. 18, No. 3, July 2006

TESTS
Solution S. Dissolve 5.0 g in 80 ml of dilute acetic acid R.
When the effervescence has ceased, boil for 2 min, allow to
cool, and dilute to 100 ml with dilute acetic acid R. Filter, if
necessary, through a sintered-glass filter.
Substances insoluble in acetic acid : maximum 0.2 per cent.
Wash any residue obtained during the preparation of
solution S with 4 quantities, each of 5 ml, of hot water R and
dry at 100-105 C for 1 h. The residue weighs a maximum
of 10 mg.
Chlorides (2.4.4) : maximum 330 ppm.
Dilute 3 ml of solution S to 15 ml with water R.
Sulphates (2.4.13) : maximum 0.25 per cent.
Dilute 1.2 ml of solution S to 15 ml with distilled water R.
Arsenic (2.4.2, Method A) : maximum 4 ppm, determined
on 5 ml of solution S.
Barium. To 10 ml of solution S add 10 ml of calcium
sulphate solution R. After at least 15 min, any opalescence
in the solution is not more intense than that in a mixture of
10 ml of solution S and 10 ml of distilled water R.
Iron (2.4.9) : maximum 200 ppm.
Dissolve 50 mg in 5 ml of dilute hydrochloric acid R and
dilute to 10 ml with water R.
Magnesium and alkali metals : maximum 1.5 per cent.
Dissolve 1.0 g in 12 ml of dilute hydrochloric acid R. Boil
the solution for about 2 min and add 20 ml of water R, 1 g of
ammonium chloride R and 0.1 ml of methyl red solution R.
Add dilute ammonia R1 until the colour of the indicator
changes, then 2 ml in excess. Heat to boiling and add 50 ml
of hot ammonium oxalate solution R. Allow to stand for 4 h,
dilute to 100 ml with water R and filter through a suitable
filter. To 50 ml of the filtrate add 0.25 ml of sulphuric acid R.
Evaporate to dryness on a water-bath and ignite to constant
mass at 600 C. The residue weighs a maximum of 7.5 mg.
Heavy metals (2.4.8) : maximum 20 ppm.
12 ml of solution S complies with test A. Prepare the
reference solution using lead standard solution (1 ppm
Pb) R.
405

Calcium gluconate, anhydrous

Loss on drying (2.2.32) : maximum 2.0 per cent, determined

on 1.000 g by drying in an oven at 200 10 C.

Test solution. Dissolve 20 mg of the substance to be

examined in 1 ml of water R, heating if necessary in a
water-bath at 60 C.
ASSAY
Reference solution. Dissolve 20 mg of calcium
Dissolve 0.150 g in a mixture of 3 ml of dilute hydrochloric
gluconate CRS in 1 ml of water R, heating if necessary in
acid R and 20 ml of water R. Boil for 2 min, allow to cool, and
a water-bath at 60 C.
dilute to 50 ml with water R. Carry out the complexometric
Plate : TLC silica gel plate R (5-40 m) [or TLC silica gel
titration of calcium (2.5.11).
plate R (2-10 m)].
1 ml of 0.1 M sodium edetate is equivalent to 10.01 mg
Mobile phase : concentrated ammonia R, ethyl acetate R,
of CaCO3.
water R, ethanol (96 per cent) R (10:10:30:50 V/V/V/V).
Application
: 1 l.
FUNCTIONALITY-RELATED CHARACTERISTICS
Development
: over 2/3 of the plate.
This section provides information on characteristics that
Drying
:
at
100
C for 20 min, then allow to cool.
are recognised as being relevant control parameters for
one or more functions of the substance when used as an
Detection : spray with a solution containing 25 g/l of
excipient (see general chapter 5.15)(16). This section is
ammonium molybdate R and 10 g/l of cerium sulphate R
a non-mandatory part of the monograph and it is not
in dilute sulphuric acid R, and heat at 100-105 C for
necessary to verify the characteristics to demonstrate
about 10 min.
compliance. Control of these characteristics can however
Results : the principal spot in the chromatogram obtained
contribute to the quality of a medicinal product by
with the test solution is similar in position, colour and
improving the consistency of the manufacturing process.
size to the principal spot in the chromatogram obtained
Where control methods are cited, they are recognised as
with the reference solution.
being suitable for the purpose, but other methods can also B. Solution S (see Tests) gives the reactions of calcium
be used. Wherever results for a particular characteristic
(2.3.1).
are reported, the control method must be indicated.
C. Loss on drying (see Tests).
The following characteristics may be relevant for calcium
carbonate used as a filler in tablets and capsules.
TESTS
Particle-size distribution (2.9.38) or (2.9.31).
Solution S. Dissolve 1.0 g in water R heated to 60 C and
dilute to 50 ml with the same solvent.
(17)
Bulk density and tapped density (2.9.34) .
Appearance of solution. At 60 C, solution S is not
more intensely coloured than reference solution Y6 (2.2.2,
Method II). After cooling, it is not more opalescent than
reference suspension II (2.2.1).
Reference: PA/PH/Exp. INC/T (06) 7 ANP
Organic impurities and boric acid. Place 0.5 g in a porcelain
dish previously rinsed with sulphuric acid R and placed in
NOTE ON THE MONOGRAPH
a bath of iced water. Add 2 ml of cooled sulphuric acid R
This monograph is based on the current monograph for
and mix. No yellow or brown colour develops. Add 1 ml of
Calcium gluconate (0172).
chromotrope II B solution R. A violet colour develops and
XXXX:2364
does not become dark blue. Compare the colour obtained
with that of a mixture of 1 ml of chromotrope II B solution R
CALCIUM GLUCONATE, ANHYDROUS and 2 ml of cooled sulphuric acid R.
Sucrose and reducing sugars. Dissolve 0.5 g in a mixture
of 2 ml of hydrochloric acid R1 and 10 ml of water R. Boil
Calcii gluconas anhydricus
for 5 min, allow to cool, add 10 ml of sodium carbonate
solution R and allow to stand for 10 min. Dilute to 25 ml
with water R and filter. To 5 ml of the filtrate add 2 ml of
cupri-tartaric solution R and boil for 1 min. Allow to stand
for 2 min. No red precipitate is formed.
Chlorides (2.4.4) : maximum 200 ppm.
Dilute 12.5 ml of solution S to 15 ml with water R.
C12H22CaO14
Mr 430.4 Sulphates (2.4.13) : maximum 100 ppm.
Dissolve 10.0 g with heating in a mixture of 10 ml of acetic
DEFINITION
acid R and 90 ml of distilled water R.
Anhydrous calcium D-gluconate.
Magnesium and alkali metals : maximum 0.4 per cent.
Content : 98.0 per cent to 102.0 per cent (dried substance).
Dissolve 1.00 g in 100 ml of boiling water R, add 10 ml of
CHARACTERS
ammonium chloride solution R, 1 ml of ammonia R and,
Appearance : white or almost white, crystalline or granular dropwise, 50 ml of hot ammonium oxalate solution R. Allow
to stand for 4 h, dilute to 200 ml with water R and filter.
powder.
Evaporate 100 ml of the filtrate to dryness and ignite. The
Solubility : sparingly soluble in water, freely soluble in
residue weighs a maximum of 2 mg.
boiling water.
Heavy metals (2.4.8) : maximum 10 ppm.
IDENTIFICATION
2.0 g complies with test D. Heat the substance to be
examined gradually and with care until it is almost
A. Thin-layer chromatography (2.2.27).
(16) See draft in this issue.
(17) See draft in Pharmeuropa 16.2.

406

PHARMEUROPA Vol. 18, No. 3, July 2006

Carbomers

completely transformed into a white mass, and then ignite.

C. Add 2 ml of a 100 g/l solution of calcium chloride R with
Prepare the reference solution using 2 ml of lead standard
continuous stirring to 10 ml of the gel from identification
solution (10 ppm Pb) R.
test B. A white precipitate is immediately produced.
Loss on drying (2.2.32) : maximum 3.0 per cent, determined D. Add 0.5 ml of thymol blue solution R to 10 ml of a 10 g/l
on 1.000 g by drying in an oven at 105 C for 16 h.
dispersion. An orange colour is produced. Add 0.5 ml of
cresol red solution R to 10 ml of a 10 g/l dispersion. A
Microbial contamination. Total viable aerobic count (2.6.12)
yellow colour is produced.
not more than 103 micro-organisms per gram, determined
by plate count.
E. It complies with the nominal apparent viscosity indicated
on the label.
ASSAY
Dissolve 0.350 g in 20 ml of hot water R, allow to cool and
dilute to 300 ml with water R. Carry out the complexometric TESTS
titration of calcium (2.5.11).
Apparent viscosity : the nominal apparent viscosity is
1 ml of 0.1 M sodium edetate is equivalent to 43.04 mg
between 300 mPas and 115 000 mPas. For a product with
of C12H22CaO14.
a nominal apparent viscosity of 20 000 mPas or greater,
the apparent viscosity is 70.0 per cent to 130.0 per cent of
the value stated on the label ; for a product with a nominal
apparent viscosity of less than 20 000 mPas, the apparent
viscosity is 50.0 per cent to 150.0 per cent of the value stated
on the label.
Dry the substance to be examined in vacuo at 80 C for 1 h.
Carefully add 2.50 g of the previously dried substance to be
examined to 500 ml of water R in a 1000 ml beaker while
NOTE ON THE MONOGRAPH
stirring continuously at 1000 50 r/min, with the stirrer
Identification by infrared absorption spectrophotometry :
shaft set at an angle of 60 to one side of the beaker. Add
a revision is proposed to more accurately reflect the IR
the previously dried substance over a period of 45-90 s, at
absorption bands observed with the products on the market. a uniform rate, ensuring that loose aggregates of powder
The bands listed are flexible enough to account for any
are broken up, and continue stirring at 1000 50 r/min
accuracy and precision variability in methodology and
for 15 min. Remove the stirrer, and place the beaker
instrumentation.
containing the dispersion in a water-bath at 25 0.2 C for
XXXX:1299 30 min. Insert the stirrer to a depth necessary to ensure
that air is not drawn into the dispersion, and while stirring
at 300 25 r/min, titrate with a glass-calomel electrode
CARBOMERS
system to pH 7.3-7.8 by adding a 180 g/l solution of sodium
hydroxide R below the surface, determining the end-point
potentiometrically (2.2.20). The total volume of the 180 g/l
Carbomera
solution of sodium hydroxide R used is about 6.2 ml. Allow
2-3 min before the final pH determination. If the final pH
DEFINITION
exceeds 7.8, discard the preparation, and prepare another
using a smaller amount of sodium hydroxide for titration.
High-molecular-mass polymers of acrylic acid cross-linked
Return the neutralised preparation to the water-bath at 25 C
with polyalkenyl ethers of sugars or polyalcohols.
for 1 h, then perform the viscosity determination without
Content : 56.0 per cent to 68.0 per cent of carboxylic
delay to avoid slight viscosity changes that occur 75 min
acid (-CO2H) groups (dried substance).
after neutralisation. Determine the viscosity (2.2.10) using
a rotating viscometer with a spindle rotating at 20 r/min,
CHARACTERS
using a spindle suitable for the expected apparent viscosity.
Appearance : white or almost white, fluffy hygroscopic,
Free acrylic acid. Liquid chromatography (2.2.29).
powder.
Test solution. Mix 0.125 g of the substance to be examined
Solubility : swells in water and in other polar solvents
with a 25 g/l solution of aluminium potassium sulphate R
after dispersion and neutralisation with sodium hydroxide
and dilute to 25.0 ml with the same solution. Heat the
solution.
suspension at 50 C for 20 min with shaking. Then shake
the suspension at room temperature for 60 min. Centrifuge
IDENTIFICATION
and use the clear supernatant solution as the test solution.
First identification : A, E.
Reference solution. Dissolve 62.5 mg of acrylic acid R in a
Reference: PA/PH/Exp. 11/T (06) 39 ANP

Second identification : B, C, D, E.

A. Infrared absorption spectrophotometry (2.2.24).

Main bands : at 2960 cm 1, 1720 cm 1, 1710 5 cm 1,
1455 cm 1, 1454 5 cm 1, 1415 cm 1, 1414 5 cm 1,
1250 cm 1, 1245 5 cm 1, 1175 cm 1, 1172 5 cm 1,
800 cm 1, 1115 5 cm 1 and 801 5 cm 1, with the
strongest band at 1720 cm 1, 1710 5 cm 1.
B. Adjust a 10 g/l dispersion to about pH 7.5 with
1 M sodium hydroxide. A highly viscous gel is formed.
PHARMEUROPA Vol. 18, No. 3, July 2006

25 g/l solution of aluminium potassium sulphate R and

dilute to 100.0 ml with the same solution. Dilute 1.0 ml of
this solution to 50.0 ml with a 25 g/l solution of aluminium
potassium sulphate R.
Column :
size : l = 0.12 m, = 4.6 mm ;
stationary phase : octadecylsilyl silica gel for
chromatography R (5 m).

407

Carprofen for veterinary use

Mobile phase :
mobile phase A : 1.361 g/l solution of potassium
dihydrogen phosphate R, adjusted to pH 2.5 using dilute
phosphoric acid R ;
mobile phase B : a mixture of equal volumes of a 1.361 g/l
solution of potassium dihydrogen phosphate R and
acetonitrile for chromatography R ;
Time
(min)
0-8

Mobile phase A
(per cent V/V)
100

Mobile phase B
(per cent V/V)
0

8-9

100 0

0 100

9 - 20

100

20 - 21

0 100

100 0

21 - 30

100

Flow rate : 1 ml/min.

Detection : spectrophotometer at 205 nm.
Injection: 20 l.
Retention time : acrylic acid = about 6.0 min.
Limit :
acrylic acid : not more than the area of the corresponding
peak in the chromatogram obtained with the reference
solution (0.25 per cent).
Benzene. Gas chromatography (2.4.24, System A).
Solution A. Dissolve 0.100 g of benzene R in dimethyl
sulphoxide R and dilute to 100.0 ml with the same solvent.
Dilute 1.0 ml of the solution to 100.0 ml with water R. Dilute
1.0 ml of this solution to 100.0 ml with water R.
Test solution. Weigh 50.0 mg of the substance to be
examined into an injection vial and add 5.0 ml of water R
and 1.0 ml of dimethyl sulphoxide R.
Reference solution. Weigh 50.0 mg of the substance to be
examined into an injection vial and add 4.0 ml of water R,
1.0 ml of dimethyl sulphoxide R and 1.0 ml of solution A.
Close the vials with a tight rubber membrane stopper
coated with polytetrafluoroethylene and secure with an
aluminium crimped cap. Shake to obtain a homogeneous
dispersion.
Static head-space conditions that may be used :
equilibration temperature : 80 C ;
equilibration time : 60 min ;
transfer line temperature : 90 C.
Injection: 1 ml of the gaseous phase of the test solution and
1 ml of the gaseous phase of the reference solution ; repeat
these injections twice more.
System suitability :
repeatability : maximum relative standard deviation of the
differences in area between the analyte peaks obtained
from the 3 replicate pair injections of the reference
solution and the test solution is 15 per cent.
Limit :
benzene : the mean area of the peak due to benzene in
the chromatograms obtained with the test solution is
not greater than 0.5 times the mean area of the peak
due to benzene in the chromatograms obtained with the
reference solution (2 ppm).
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test C. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 3.0 per cent, determined
on 1.000 g by drying in vacuo at 80 C for 60 min.
408

Sulphated ash (2.4.14) : maximum 4.0 per cent, determined

on 1.0 g.
ASSAY
Slowly add 50 ml of water R to 0.120 g whilst stirring and
heating at 60 C for 15 min. Stop heating, add 150 ml
of water R and continue stirring for 30 min. Add 2 g
of potassium chloride R and titrate with 0.2 M sodium
hydroxide, determining the end-point potentiometrically
(2.2.20).
1 ml of 0.2 M sodium hydroxide is equivalent to 9.0 mg of
carboxylic acid (-CO2H) groups.
STORAGE
In an airtight container.
LABELLING
The label states the nominal apparent viscosity.

Reference: PA/PH/Exp. 10A/T (04) 34 ANP

XXXX:2201

CARPROFEN FOR VETERINARY USE

Carprofenum ad usum veterinarium

C15H12ClNO2

Mr 273.7

DEFINITION
(2RS)-2-(6-Chloro-9H-carbazol-2-yl)propanoic acid.
Content : 98.5 per cent to 101.5 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, freely soluble in
acetone, soluble in methanol, slightly soluble in 2-propanol.
It shows polymorphism.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : carprofen CRS.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
substance separately in acetone R, evaporate to dryness and
record new spectra using the residues.
TESTS
Solution S. Dissolve 1.0 g in methanol R and dilute to
25.0 ml with the same solvent.
Appearance of solution. Solution S is clear and not more
intensely coloured than reference solution BY5 (2.2.2,
Method II).
PHARMEUROPA Vol. 18, No. 3, July 2006

Carprofen for veterinary use

Related substances. Liquid chromatography (2.2.29). Use

brown-glass flasks for preparing the solutions.
Test solution. Dissolve 50 mg of the substance to be
examined in the mobile phase and dilute to 100.0 ml with
the mobile phase.
Reference solution (a). Dissolve 5 mg of carprofen
impurity C CRS in the mobile phase, add 10 ml of the test
solution and dilute to 20 ml with the mobile phase. Dilute
2.0 ml of this solution to 100.0 ml with the mobile phase.
Reference solution (b). Dilute 1.0 ml of the test solution
to 100.0 ml with the mobile phase. Dilute 1.0 ml of this
solution to 10.0 ml with the mobile phase.
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase: end-capped polar-embedded
octadecylsilyl amorphous organosilica polymer R
(5 m)(18).
Mobile phase : mix 30 volumes of a 1.36 g/l solution of
potassium dihydrogen phosphate R adjusted to pH 3.0 and
70 volumes of methanol R.
Flow rate : 1.3 ml/min.
Detection : spectrophotometer at 235 nm.
Injection: 20 l.
Run time : 3.5 times the retention time of carprofen.
Retention time : carprofen = about 10 min.
System suitability : reference solution (a) :
resolution : minimum 1.5 between the peaks due to
carprofen and impurity C.
Limits :
unspecified impurities : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.2 per cent) ;
total: not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.5 per cent) ;
disregard limit : the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.1 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
Dissolve 2.0 g in ethanol (96 per cent) R and dilute to 20 ml
with the same solvent. 12 ml of the solution complies with
test B. Prepare the reference solution using lead standard
solution (2 ppm Pb) R.
Water (2.5.12) : maximum 0.5 per cent, determined on
0.500 g.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.

IMPURITIES
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical
use) : A, B, C, D, E, F, G, H.

A. (6-chloro-9H-carbazol-2-yl)(methyl)malonic acid,

B. (2RS)-2-(9H-carbazol-2-yl)propanoic acid,

C. (1RS)-1-(6-chloro-9H-carbazol-2-yl)ethanol,

D. 1-(6-chloro-9H-carbazol-2-yl)ethanone,

ASSAY
E. 3-chloro-9H-carbazole,
Dissolve 0.250 g in 50 ml of ethanol (96 per cent) R. Add
1.0 ml of 0.1 M hydrochloric acid. Titrate with 0.1 M sodium
hydroxide, determining the end-point potentiometrically
(2.2.20). Read the volume added between the 2 points of
inflexion.
1 ml of 0.1 M sodium hydroxide is equivalent to 27.37 mg of
C15H12ClNO2.
STORAGE
Protected from light.

F. diethyl (6-chloro-9H-carbazol-2-yl)(methyl)malonate,

(18) Waters C18 X Terra is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

409

2.2.46. Chromatographic separation techniques

G. ethyl (2RS)-2-(6-chloro-9H-carbazol-2-yl)propanoate,

on adsorption, mass distribution (partition), ion exchange,

etc., or may be based on differences in the physico-chemical
properties of the molecules, such as size, mass, volume, etc.
This chapter contains definitions and calculations of
common parameters and generally applicable requirements
for system suitability. Principles of separation, apparatus
and methods are given in the following general methods :
paper chromatography (2.2.26) ;
thin-layer chromatography (2.2.27) ;
gas chromatography (2.2.28) ;
liquid chromatography (2.2.29) ;
size-exclusion chromatography (2.2.30) ;
supercritical fluid chromatography (2.2.45).

DEFINITIONS
The following definitions have been used to calculate the
limits in monographs.
With some equipment, certain parameters, such as the
signal-to-noise ratio, can be calculated using software
Reference: PA/PH/SG (06) 28 ANP
provided by the manufacturer. It is the responsibility
of the user to ensure that the calculation methods used
NOTE ON THE GENERAL CHAPTER
in the software are compatible with the requirements
of the European Pharmacopoeia. If not, the necessary
Revision of the chapter on chromatographic separation
corrections must be made.
techniques has been undertaken in light of several years
experience. Changes are outlined below.
Chromatogram
Peak symmetry : changed to clarify that the requirement A chromatogram is a graphical or other representation
for peak symmetry of 0.8-1.5 applies to peaks in the
of detector response, effluent concentration or other
chromatogram obtained with a reference solution used quantity used as a measure of effluent concentration, versus
for quantification.
time, volume or distance. Idealised chromatograms are
Repeatability in impurities (related substances) tests :
represented as a sequence of gaussian peaks on a baseline.
this requirement has been added to take account of
RETENTION DATA
current good analytical practice ; the requirement refers
Retention time and retention volume
to peaks used for quantification and is related to the
peak size ( 0.2 per cent/> 0.2 per cent).
Retention measurements in elution chromatography may
be given as the retention time (tR), directly defined by the
Limit of detection : this requirement has been deleted ;
it is redundant for monographs, since compliance with position of the maximum of the peak in the chromatogram.
From the retention time, the retention volume (VR ml) may
the limit of quantitation is always required.
be calculated :
A recommendation has been added concerning the time
points at which injections used for verification of system
suitability should be made during the chromatographic
procedure.
For adjustment of chromatographic conditions, isocratic
and gradient elutions are now dealt with separately ; for
both, a formula has been added for adjustment of flow
rate where a column with different dimensions from
tR
= retention time, in minutes or distance along
those prescribed is used ; for isocratic conditions, the
the baseline from the point of injection to the
permitted adjustment for column temperature is now
perpendicular dropped from the maximum of the
more meaningfully expressed in absolute terms ( 5 C)
peak corresponding to the component ;
rather than as a percentage ; for gradient elutions, the
f
flow rate of the mobile phase, in millilitres per
v
=
only adjustments permitted in the revision proposal are
minute.
the flow rate (to allow for different column dimensions)
H. 6-chloro-2-ethyl-9H-carbazole.

and a correction for dwell volume.

2.2.46. CHROMATOGRAPHIC
SEPARATION TECHNIQUES

Mass distribution ratio

XXXX:20246 The mass distribution ratio (D ), also known as the retention
m
factor k (or capacity factor k), is defined as :

Chromatographic separation techniques are multi-stage

separation methods in which the components of a sample
are distributed between 2 phases, one of which is stationary,
KC
while the other is mobile. The stationary phase may be a
solid or a liquid supported on a solid or a gel. The stationary
phase may be packed in a column, spread as a layer, or
VS
distributed as a film, etc. The mobile phase may be gaseous
or liquid or a supercritical fluid. The separation may be based VM
410

equilibrium distribution coefficient (also known

as distribution constant) ;
volume of the stationary phase ;

volume of the mobile phase.

PHARMEUROPA Vol. 18, No. 3, July 2006

2.2.46. Chromatographic separation techniques

The mass distribution ratio of a component may be

determined from the chromatogram using the expression :

tR

tM

CHROMATOGRAPHIC DATA
The peak may be defined by the peak area (A) or the peak
height (h) and the peak width at half-height (wh) or the
peak height (h) and the peak width between the points of
inflection (wi). In gaussian peaks (Figure 2.2.46.-1) there is
the relationship :

retention time (or volume) or distance along

the baseline from the point of injection to the
perpendicular dropped from the maximum of the
peak corresponding to the component ;
hold-up time (or volume) : time (or volume) or
distance along the baseline from the point of
injection to the perpendicular dropped from
the maximum of the peak corresponding to an
unretained component.

Distribution coefficient
The elution characteristics of a component in a particular
column, in size-exclusion chromatography, may be given by
the distribution coefficient (Ko), which is calculated from the
expression :
Figure 2.2.46.-1.
Symmetry factor
The symmetry factor (or tailing factor) (As) of a peak
(Figure 2.2.46.-2) is calculated from the expression :

tR

to

tt

retention time (or volume) or distance along

the baseline from the point of injection to the
perpendicular dropped from the maximum of the
peak corresponding to the component ;
hold-up time (or volume) : time (or volume) or
distance along the baseline from the point of
injection to the perpendicular dropped from
the maximum of the peak corresponding to an
unretained component ;
retention time (or volume) or distance along
the baseline from the point of injection to the
perpendicular dropped from the maximum of the
peak corresponding to a component that has full
access to the pores of the stationary phase.

w0.05 =
d
=

width of the peak at 1/20 of the peak height ;

distance between the perpendicular dropped from
the peak maximum and the leading edge of the
peak at 1/20 of the peak height.

A value of 1.0 signifies complete (ideal) symmetry.

Retardation factor
The retardation factor (RF) (also known as retention
factor Rf), used in planar chromatography, is the ratio of the
distance from the point of application to the centre of the
spot and the distance travelled by the solvent front from the
point of application.

Figure 2.2.46.-2.
Column performance and apparent number of theoretical
plates
b
a

migration distance of the analyte ;

migration distance of the solvent front.

PHARMEUROPA Vol. 18, No. 3, July 2006

The column performance (apparent efficiency) may be

calculated from data obtained under either isothermal,
isocratic or isodense conditions, depending on the technique,
as the apparent number of theoretical plates (N), from the
411

2.2.46. Chromatographic separation techniques

following expression, where the values of tR and wh must be

expressed in the same units (time, volume or distance) :

tR

wh

retention time (or volume) or distance along

the baseline from the point of injection to the
perpendicular dropped from the maximum of the
peak corresponding to the component ;
width of the peak at half-height.

substances when baseline separation between 2 peaks is not

reached (Figure 2.2.46.-3).

Hp

Hv

height above the extrapolated baseline of the

minor peak ;
height above the extrapolated baseline at the
lowest point of the curve separating the minor
and major peaks.

The apparent number of theoretical plates varies with

the component as well as with the column, the column
temperature, the mobile phase and the retention time.
SEPARATION DATA
Resolution
The resolution (Rs) between peaks of 2 components may be
calculated from the expression :

tR1and tR2

wh1 and wh2

= retention times or distances along the

baseline from the point of injection to the
perpendiculars dropped from the maxima
of 2 adjacent peaks ;
= peak widths at half-height.

A resolution of greater than 1.5 corresponds to baseline

separation of peaks with ideal symmetry and of similar
height.

Figure 2.2.46.-3.
Relative retention
The relative retention (r) is calculated as an estimate from
the expression :

The expression given above may not be applicable if the

peaks are not baseline separated.
In quantitative planar chromatography, the migration
distances are used instead of retention times and the
resolution may be calculated using the expression :

tR2

retention time of the peak of interest ;

tR1

tM

retention time of the reference peak (usually

the peak corresponding to the substance to be
examined) ;
hold-up time : time or distance along the baseline
from the point of injection to the perpendicular
dropped from the maximum of the peak
corresponding to an unretained component.

= ratios of the distances from the point of

application to the centres of the spots and
the distance travelled by the solvent front
from the point of application (retardation
factor) ;
= peak widths at half-height ;

The unadjusted relative retention (rG) is calculated from the

expression :

= migration distance of the solvent front.

Peak-to-valley ratio

Unless otherwise indicated, values for relative retention

stated in monographs correspond to unadjusted relative
retention.

The peak-to-valley ratio (p/v) may be employed as a system

suitability requirement criterion in a test for related

In planar chromatography, the retardation factors RF2 and

RF1 are used instead of tR2 and tR1.

RF1 and RF2

wh1 and wh2

a

412

PHARMEUROPA Vol. 18, No. 3, July 2006

2.2.46. Chromatographic separation techniques

PRECISION OF QUANTIFICATION
Signal-to-noise ratio
The signal-to-noise ratio (S/N) influences the precision of
quantification and is calculated from the equation :

The For an assay, the maximum permitted relative standard

deviation (RSDmax) is calculated for a series of injections of
the reference solution for defined limits using the following
expression :

height of the peak (Figure 2.2.46.-4) corresponding

to the component concerned, in the chromatogram
obtained with the prescribed reference solution,
measured from the maximum of the peak to
the extrapolated baseline of the signal observed
over a distance equal to 20 times the width at
half-height ;
range of the background noise in a chromatogram
obtained after injection or application of a blank,
observed over a distance equal to 20 times
the width at half-height of the peak in the
chromatogram obtained with the prescribed
reference solution and, if possible, situated
equally around the place where this peak would
be found.

Figure 2.2.46.-4.
Repeatability
The repeatability of response is expressed as an estimated
percentage relative standard deviation (RSD%) of a
consecutive series of measurements of for not fewer than
3 injections or applications of a reference solution and is
calculated from the expression :

yi

individual values expressed as peak area,

peak height, or ratio of areas by the internal
standardisation method ;
mean of individual values ;

number of individual values.

PHARMEUROPA Vol. 18, No. 3, July 2006

constant (0.349), obtained from the expression

in which
represents the
required RSD after 6 injections for B = 1.0 ;
B
= upper limit given in the definition of the
individual monograph minus 100 per cent ;
n
= number of replicate injections of the reference
solution (3 n 6) ;
t90%,n1 = Students t at the 90 per cent probability level
(double sided) with n 1 degrees of freedom.
Repeatability requirements (maximum permitted relative
standard deviation) for assays are shown in Table 2.2.46.-1.
SYSTEM SUITABILITY
The system suitability tests represent an integral part of
the method and are used to ensure adequate performance
of the chromatographic system. Apparent efficiency,
mass distribution ratio, resolution, relative retention and
the symmetry factor are the parameters that are usually
employed in assessing the performance of the column.
Factors that may affect the chromatographic behaviour
include :
the composition, ionic strength, temperature and
apparent pH of the mobile phase ;
flow rate, column length dimensions, temperature and
pressure ;
stationary phase characteristics including type of
chromatographic support (particle-based or monolithic),
particle or macropore size, porosity, particle size, type of
particles, specific surface area ; and, in the case of
reversed-phase and other surface-modified supports
stationary phases, the extent of chemical modification (as
expressed by end-capping, carbon loading etc.).
The various components of the equipment employed must be
qualified and be capable of achieving the precision required
to conduct the test or assay.
The following requirements are to be fulfilled unless
otherwise stated in the monograph prescribed.
for a peak in the chromatogram obtained with a reference
solution used for quantification in a test or assay, the The
symmetry factor of the principal peak is to be between 0.8
and 1.5 unless otherwise stated in the monograph
prescribed ; This requirement has general applicability to
tests or assays described in the monographs.
in a test for related substances, the relative standard
deviation of the response for a peak in the chromatogram
obtained with a reference solution used for quantification
of impurities is:
not greater than 5.0 per cent for a peak corresponding
to a component whose concentration is greater than
0.2 per cent of the concentration of the substance to be
examined in the test solution (for example, if the test
solution is a 0.2 per cent solution of the substance to
be examined and a 250-fold dilution is used to quantify
an impurity, then the requirement is 5.0 per cent) ;
not greater than 10.0 per cent for a peak corresponding
to a component whose concentration is less than
or equal to 0.2 per cent of the concentration of the
=

413

2.2.46. Chromatographic separation techniques

substance to be examined in the test solution (for

example, if the test solution is a 0.2 per cent solution of
the substance to be examined and a 500-fold dilution
is used to quantify an impurity, then the requirement
is 10.0 per cent).
in an assay, the maximum permitted relative standard
deviation for the peak in the chromatogram obtained with
the prescribed reference solution used for quantitation,
after replicate injections, does not exceed the values
appropriate value given in Table 2.2.46.-1. This
requirement is applicable to assays for content only and
does not apply to the tests for related substances.
The limit of detection of the peak (corresponding to a
signal-to-noise ratio of 3) is below the disregard limit of
the test for related substances.
The limit of quantitation of the peak (corresponding to
a signal-to-noise ratio of 10) is equal to or less than the
disregard limit of the test for related substances.
Compliance with the system suitability criteria is required
throughout the chromatographic procedure. Depending on
various factors, such as the frequency of use of the procedure
and experience with the chromatographic system, the analyst
should choose appropriate stages for injection of the system
suitability solution(s), for example, at the beginning and/or
during the procedure.
Table 2.2.46.-1. Repeatability requirements
Number of individual injections
3

B
(per cent)
2.0

0.41

0.59

0.73

0.85

2.5

0.52

0.74

0.92

1.06

3.0

0.62

0.89

1.10

1.27

Maximum permitted relative standard deviation

ADJUSTMENT OF CHROMATOGRAPHIC CONDITIONS

The extent to which the various parameters of a
chromatographic test may be adjusted to satisfy the system
suitability criteria without fundamentally modifying the
methods are listed below for information for isocratic and
gradient elutions. Adjustment of conditions with gradient
elutions is more critical than with isocratic elutions, since
it may lead to shifts in peaks to a different step of the
gradient, thus leading to the incorrect assignment of the
peak, and to masking of peaks or a shift such that elution
occurs beyond the prescribed elution time. Changes other
than those indicated require revalidation of the system.
The chromatographic conditions described have been
validated during the elaboration of the monograph. The
system suitability tests are included to ensure verify that the
separation required for satisfactory performance of the test
or assay is achieved. Nonetheless, since the stationary phases
are described in a general way and there is such a variety
available commercially, with differences in chromatographic
behaviour, some adjustments of the chromatographic
conditions may be necessary to achieve the prescribed
system suitability requirements. With reversed-phase liquid
chromatographic methods, in particular, adjustment of the
various parameters will not always result in satisfactory
chromatography. In that case, it may be necessary to replace
the column with another of the same type (e.g. octadecylsilyl
silica gel), which exhibits the desired chromatographic
behaviour.
For critical parameters the adjustments are defined clearly
in the monograph to ensure the system suitability.
Multiple adjustments which may have a cumulative effect in
the performance of the system are to be avoided.
414

Thin-layer chromatography and paper chromatography

Composition of the mobile phase : the amount of the minor
solvent component may be adjusted by 30 per cent relative
or 2 per cent absolute, whichever is the larger ; for a minor
component at 10 per cent of the mobile phase, a 30 per cent
relative adjustment allows a range of 7-13 per cent whereas
a 2 per cent absolute adjustment allows a range of 8-12 per
cent, the relative value therefore being the larger ; for a
minor component at 5 per cent of the mobile phase, a 30 per
cent relative adjustment allows a range of 3.5-6.5 per cent
whereas a 2 per cent absolute adjustment allows a range of
3-7 per cent, the absolute value being the larger in this case.
No other component is altered by more than 10 per cent
absolute.
pH of the aqueous component of the mobile phase : 0.2 pH,
unless otherwise stated in the monograph prescribed, or
1.0 pH when neutral substances are to be examined.
Concentration of salts in the buffer component of a mobile
phase : 10 per cent.
Application volume : 10-20 per cent of the prescribed volume
if using fine particle size plates (2-10 m).
Migration distance of the solvent front is to be not less than
50 mm or 30 mm on high-performance plates.
Liquid chromatography : isocratic elution
Composition of the mobile phase : the amount of the minor
solvent component may be adjusted by 30 per cent relative
or 2 per cent absolute, whichever is the larger (see example
above). No other component is altered by more than 10 per
cent absolute.
pH of the aqueous component of the mobile phase : 0.2 pH,
unless otherwise stated in the monograph prescribed, or
1.0 pH when neutral substances are to be examined.
Concentration of salts in the buffer component of a mobile
phase : 10 per cent.
Detector wavelength : no adjustment permitted.
Stationary phase :
column length : 70 per cent ;
column internal diameter: 25 per cent ;
particle size : maximum reduction of 50 per cent, no
increase permitted.
Flow rate : 50 per cent. When in a monograph the retention
time of the principal peak is indicated, the flow rate is
adjusted if the column internal diameter has dimensions
have been changed. The flow rate is adjusted using the
following equation :

f1

f2

l1

l2

d1

d2

flow rate indicated in the monograph, in millilitres

per minute ;
adjusted flow rate, in millilitres per minute ;
length of the column indicated in the monograph,
in millimetres ;
length of the column used, in millimetres ;
column internal diameter indicated in the
monograph, in millimetres ;
internal diameter of the column used, in
millimetres.
PHARMEUROPA Vol. 18, No. 3, July 2006

2.2.46. Chromatographic separation techniques

No decrease of flow rate is permitted if If the monograph

uses indicates apparent number of theoretical plates in the
qualification section as a system suitability criterion, the flow
rate may be adjusted by 50 per cent to approach optimum
separation conditions.
Temperature : 5 C, where the operating temperature is
specified 10 per cent, to a maximum of 60 C.
Injection volume : may be decreased, provided detection and
repeatability of the peak(s) to be determined are satisfactory.
Gradient elution : the configuration of the equipment
employed may significantly alter the resolution, retention
time and relative retentions described in the method. Should
this occur, it may be due to excessive dwell volume which is
the volume between the point at which the 2 eluants meet
and the top of the column.
Liquid chromatography : gradient elution
Adjustment of chromatographic conditions for gradient
systems requires greater caution than for isocratic systems.
Adjustment of the composition of the mobile phase is not
recommended and, where compliance with the system
suitability requirements cannot be achieved, it will often be
preferable to consider the dwell volume or to change the
column.
Dwell volume. The configuration of the equipment employed
may significantly alter the resolution, retention time and
relative retentions described. Should this occur, it may
be due to excessive dwell volume which is the volume
between the point at which the 2 eluants meet and the top
of the column. Several monographs include an isocratic
step before the start of the gradient programme so that a
correction can be made to the gradient times to take account
of differences in dwell volume between the system used for
method development and that actually used.
Unless otherwise stated, gradient programmes described in
monographs are suitable for dwell volumes not greater than
1 ml. Where the system used has a larger dwell volume, the
gradient times (t min) stated may be replaced by corrected
gradient times (tc min), calculated using the following
equation :

D
D0
f

= dwell volume, in millilitres ;

= dwell volume, in millilitres, used for development
of the method (taken as 1 ml unless otherwise
stated) ;
= flow rate, in millilitres per minute.

Flow rate : where exactly the same stationary phase as

prescribed is used but the column dimensions differ, the
flow rate is adjusted to maintain Q constant in the following
equation :

tg

total analytical chromatographic run time, in

minutes ;
flow rate, in millilitres per minute ;

column length, in millimetres ;

column internal diameter, in millimetres.

PHARMEUROPA Vol. 18, No. 3, July 2006

Gas chromatography
Stationary phase :
column length : 70 per cent ;
column internal diameter: 50 per cent ;
particle size : maximum reduction of 50 per cent, no
increase permitted ;
film thickness : 50 per cent to + 100 per cent.
Flow rate : 50 per cent.
Temperature : 10 per cent no adjustment.
Injection volume : may be decreased, provided detection and
repeatability are satisfactory.
Supercritical fluid chromatography
Composition of the mobile phase : for packed columns, the
amount of the minor solvent component may be adjusted by
30 per cent relative or 2 per cent absolute, whichever is
the larger. No adjustment is permitted for a capillary column
system.
Detector wavelength : no adjustment permitted.
Stationary phase :
column length : 70 per cent ;
column internal diameter:
25 per cent (packed columns) ;
50 per cent (capillary columns) ;
particle size : maximum reduction of 50 per cent, no
increase permitted (packed columns).
Flow rate : 50 per cent.
Temperature : 5 C, where the operating temperature is
specified 10 per cent.
Injection volume : may be decreased, provided detection and
repeatability are satisfactory.
QUANTIFICATION
Peaks due to solvents and reagents or arising from the
mobile phase or the sample matrix are disregarded during
quantification.
Detector response. The detector sensitivity is the signal
output per unit concentration or unit mass of a substance
in the mobile phase entering the detector. The relative
detector response factor, commonly referred to as
response factor, expresses the sensitivity of a detector
relative to a standard substance. The correction factor is
the reciprocal of the response factor.
External standard method. The concentration of the
component(s) to be analysed is determined by comparing
the response(s) (peak(s)) obtained with the test solution
to the response(s) (peak(s)) obtained with a reference
solution.
Internal standard method. Equal amounts of a
component that is will be resolved from the substance to
be examined (the internal standard) are introduced into
the test solution and a reference solution. The internal
standard should is chosen such that it does not react
with the substance to be examined ; it must be, is stable
and must does not contain impurities with a the same
retention time similar to as that of the substance to be
examined. The concentration of the substance to be
examined is determined by comparing the ratio of the
peak areas or peak heights due to the substance to be
examined and the internal standard in the test solution
with the ratio of the peak areas or peak heights due to
the substance to be examined and the internal standard
in the reference solution.
415

Chromium (51Cr) edetate injection

Normalisation procedure. The percentage content of a

one or more component of the substance to be examined
is calculated by determining the area of the corresponding
peak or peaks as a percentage of the total area of all the
peaks, excluding those due to solvents or any added
reagents, and those at or below the disregard limit.
Calibration procedure. The relationship between the
measured or evaluated signal (y) and the amount quantity
(concentration, mass, etc.) of substance (x) is determined
and the calibration function is calculated. The analytical
results are calculated from the measured signal or
evaluated signal of the analyte by means of the inverse
function.
For assays and for quantitative determination of components,
the external standard method, the internal standard method
or the calibration procedure may be described in the
monograph, and the normalisation procedure is not normally
applied. In tests for related substances, either the external
standard method with a single reference solution or the
normalisation procedure is generally applied. However, with
For both the normalisation procedure or and the external
standard method, when a dilution of the test solution is
used for comparison, the correction factors indicated in
the monograph are applied if the responses of the related
substances are similar to different from that of the substance
itself (response factor of outside the range 0.8-1.2), otherwise
correction factors are included in the text.
When the related substances test prescribes the summation
of impurities or there is a quantitative determination of an
impurity, it is important to choose an appropriate threshold
setting and appropriate conditions for the integration of the
peak areas. In such tests the disregard limit, i.e. the limit at
or below which the area of a peak is not taken into account,
is generally 0.05 per cent. Thus, the threshold setting of the
data collection system corresponds to, at least, half of the
disregard limit. Integration of the any peak areas of the an
impurities impurity that are is not completely separated from
the main peak are is preferably performed by valley-to-valley
extrapolation (tangential skim). Peaks due to the solvent(s)
used to dissolve the sample are also to be disregarded.

DEFINITION
Sterile solution containing chromium-51 in the form of a
complex of chromium (III) with ethylenediaminetetraacetic
acid, the latter being present in excess. It may be made
isotonic by the addition of sodium chloride and may contain
a suitable antimicrobial preservative such as benzyl alcohol.
Content :
chromium-51 : 90.0 per cent to 110.0 per cent of the
declared chromium-51 radioactivity at the date and hour
stated on the label ;
chromium : maximum 1 mg/ml.
PRODUCTION
Chromium-51 is a radioactive isotope of chromium and may
be prepared by neutron irradiation of chromium, either of
natural isotopic composition or enriched in chromium-50.
CHARACTERS
Appearance : clear, violet solution.
Half-life and nature of radiation of chromium-51: see Table
of physical characteristics of radionuclides (5.7).
IDENTIFICATION
A. Radionuclidic purity (see Tests).
B. Examine the electropherogram obtained in the test for
radiochemical purity. The distribution of radioactivity
contributes to the identification of the preparation.
B. Examine the chromatograms obtained in the test for
radiochemical purity.
Results : the principal peak in the radiochromatogram
obtained with the test solution is similar in retention time
to the principal peak in the chromatogram obtained with
the reference solution.

TESTS
pH (2.2.3) : 3.5 to 6.5.
Chromium. Ultraviolet and visible absorption
spectrophotometry (2.2.25).
Test solution. The preparation to be examined.
Reference solution : dissolve 0.96 g of chromic potassium
sulphate R and 2.87 g of sodium edetate R in 50 ml of
water R, boil for 10 min, cool, adjust to pH 3.5-6.5 with
Reference: PA/PH/Exp. 14/T (05) 9 ANP
dilute sodium hydroxide solution R and dilute to 100.0 ml
with water R.
NOTE ON THE MONOGRAPH
Measure the absorbance of the test solution and the
It is proposed to revise the monograph in order to replace reference solution at the absorption maximum at 560 nm.
the test for radiochemical purity performed by zone
The absorbance of the test solution is not greater than that
electrophoresis by a test applying paper chromatography, of the reference solution (1 mg/ml).
since the high voltage electrophoresis equipment described
Sterility. It complies with the test for sterility
is no longer available on the market.
XXXX:0266 prescribed in the monograph on Radiopharmaceutical
preparations (0125). The injection may be released for use
before completion of the test.
CHROMIUM (51Cr) EDETATE
RADIONUCLIDIC PURITY
INJECTION
Chromium-51 : minimum 99.9 per cent of the total
radioactivity.
Chromii (51Cr) edetatis solutio iniectabilis Gamma-ray spectrometry.
Results : the only gamma photons have an energy of
0.320 MeV.
RADIOCHEMICAL PURITY
Examine by zone electrophoresis (2.2.31), using a paper strip
as the support and a solution containing 0.2 g/l of barbital
sodium R and 10 g/l of sodium nitrate R as the electrolyte
416

PHARMEUROPA Vol. 18, No. 3, July 2006

Cyclizine hydrochloride

solution. A paper with the following characteristics is

suitable : mass per unit area 120 g/m2 ; thickness 0.22 mm ;
capillary rise 105 mm to 115 mm per 30 min.
Apply to the paper 10 l of the injection as a 3 mm band at
a position 10 cm from the cathode. Apply an electric field
of about 30 V per centimetre for 30 min using a stabilised
current. [51Cr]chromium edetate moves about 5 cm towards
the anode. [51Cr]Chromate moves about 10 cm towards the
anode and [51Cr]chromic ion moves about 7 cm towards the
cathode. Determine the distribution of the radioactivity
using a suitable detector. Not less than 95 per cent of the
total radioactivity is found in the band corresponding to
[51Cr]chromium edetate.
[51Cr]chromium edetate. Descending paper chromatography
(2.2.26).
Test solution. The preparation to be examined.
Reference solution. Use the reference solution from the test
for chromium.
Chromate carrier solution : dissolve 0.1 g of potassium
chromate R in 1 ml of concentrated ammonia R1 and dilute
to 100 ml with water R.
Paper : paper for chromatography R(19).
Mobile phase : concentrated ammonia R1, ethanol (96 per
cent) R, water R (12.5:25:62.5 V/V/V).
Application : apply a band of a 50 g/l solution of lead
acetate R to the paper at about 4 cm from the origin and dry
in hot air. Apply 10 l of the chromate carrier solution at the
origin, followed by 10 l of the test solution on the same spot.
On a separate sheet, repeat the above procedure, applying
10 l of the reference solution instead of the test solution.
Development : immediately, over a path of 14 cm.
Drying : in air.
Detection : determine the distribution of radioactivity using
a suitable radioactivity detector.
Retention factors : impurity A = 0.0 ; impurity B = 0.2 to 0.4 ;
[51Cr]chromium edetate = 0.8 to 0.9.
System suitability. The band of lead acetate turns yellow due
to reaction with the chromate carrier solution. The retention
factor of the radioactive spot due to [51Cr]chromium edetate
in the radiochromatogram obtained with the test solution
is similar to that of the violet spot in the chromatogram
obtained with the reference solution.
Limit :
[51Cr]chromium edetate : minimum 97.0 per cent of the
total radioactivity due to chromium-51.
RADIOACTIVITY
Determine the radioactivity using a calibrated instrument.
IMPURITIES
A. [51Cr]chromium (III) ion,
B. [51Cr]chromate ion.

Reference: PA/PH/Exp. 10C/T (06) 21 ANP

NOTE ON THE MONOGRAPH
Identification : the TLC test formerly used for both the
identification and the related substances test is now
described under Identification.
Related substances : the TLC method has been replaced
by a GC method.

Impurities : introduction of an additional impurity covered

by the GC method.
XXXX:1092

CYCLIZINE HYDROCHLORIDE
Cyclizini hydrochloridum

C18H23ClN2

Mr 302.8

DEFINITION
1-(diphenylmethyl)-4-methylpiperazine hydrochloride.
Content : 98.5 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : slightly soluble in water and in ethanol 96 per
cent.
IDENTIFICATION
First identification : B, E.
Second identification : A, C, D, E.
A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution (a). Dissolve 20.0 mg in a 5 g/l solution of
sulphuric acid R and dilute to 100.0 ml with the same
acid solution.
Test solution (b). Dilute 10.0 ml of test solution (a) to
100.0 ml with a 5 g/l solution of sulphuric acid R.
Spectral range : 240-350 nm for test solution (a) ;
210-240 nm for test solution (b).
Resolution (2.2.25) : minimum 1.7.
Absorption maxima : at 258 nm and 262 nm for test
solution (a) ; at 225 nm for test solution (b).
Absorbance ratio : A262/A258 = 1.0 to 1.1.
Specific absorbance at the absorption maximum at
225 nm : 370 to 410.
B. Infrared absorption spectrophotometry (2.2.24),
Preparation : discs of potassium chloride R.
Comparison : cyclizine hydrochloride CRS.
C. Examine the chromatograms obtained in the test
for related substances. The principal spot in the
chromatogram obtained with test solution (b) is similar
in position, colour and size to the principal spot in the
chromatogram obtained with reference solution (a).
Thin-layer chromatography (2.2.27).
Test solution. Dissolve 10 mg of the substance to be
examined in methanol R and dilute to 10 ml with the
same solvent.
Reference solution. Dissolve 10 mg of cyclizine
hydrochloride CRS in methanol R and dilute to 10 ml
with the same solvent.
Plate : TLC silica gel plate R.
Mobile phase : concentrated ammonia R, methanol R,
methylene chloride R (2:13:85 V/V/V).

(19) Whatman No. 1 is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

417

Cyclizine hydrochloride

Application : 20 l.
Development : over a path of 15 cm.
Drying : in air for 30 min.
Detection : expose the plate to iodine vapour for 10 min.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with the reference solution.
D. Dissolve 0.5 g in 10 ml of ethanol (60 per cent) R using
heat, if necessary. Cool in iced water. Add 1 ml of dilute
sodium hydroxide solution R and 10 ml of water R.
Filter, wash the precipitate with water R and dry it at
60 C at a pressure not exceeding 0.7 kPa for 2 h. The
melting point (2.2.14) is 105 C to 108 C.
E. It gives reaction (a) of chlorides (2.3.1).

Reference solution (b). Dissolve 5 mg of methylpiperazine R

in methanol R and dilute to 50 ml with the same solvent.
Reference solution (c). Dilute 1 ml of test solution (b) to
10 ml with methanol R.
Reference solution (d). Dissolve 10 mg of cyclizine
hydrochloride CRS and 10 mg of hydroxyzine
hydrochloride CRS in methanol R and dilute to 10 ml with
the same solvent.
Apply to the plate 20 l of each solution. Develop over a
path of 15 cm using a mixture of 2 volumes of concentrated
ammonia R, 13 volumes of methanol R and 85 volumes
of methylene chloride R. Allow the plate to dry in air for
30 min. Expose the plate to iodine vapour for 10 min. In
the chromatogram obtained with test solution (a) : any spot
corresponding to 1-methylpiperazine is not more intense
than the spot in the chromatogram obtained with reference
solution (b) (0.5 per cent) ; any spot, apart from the principal
TESTS
spot and any spot corresponding to 1-methylpiperazine, is not
more intense than the principal spot in the chromatogram
pH (2.2.3) : 4.5 to 5.5.
obtained with reference solution (c) (0.5 per cent). The test is
Dissolve 0.5 g in a mixture of 40 volumes of ethanol (96 per not valid unless the chromatogram obtained with reference
cent) R and 60 volumes of carbon dioxide-free water R and solution (d) shows two clearly separated spots.
dilute to 25 ml with the same mixture of solvents.
Gas chromatography (2.2.28). Prepare the solutions
Related substances. Examine by thin-layer chromatography immediately before use.
(2.2.27), using a TLC silica gel plate R. Prepare the solutions Test solution. Dissolve 0.250 g of the substance to be
immediately before use.
examined in 4.0 ml of methanol R. Dilute to 5.0 ml with
Test solution (a). Dissolve 0.20 g of the substance to be
1 M sodium hydroxide.
examined in methanol R and dilute to 10 ml with the same Reference solution (a). Dissolve 25.0 mg of the substance to
solvent.
be examined in 10.0 ml of methanol R. Dilute 5.0 ml of this
Test solution (b). Dilute 5 ml of test solution (a) to 100 ml
solution to 50.0 ml with methanol R.
with methanol R.
Reference solution (b). Dissolve 5 mg of the substance to be
Reference solution (a). Dissolve 10 mg of cyclizine
examined, 5.0 mg of cyclizine impurity A CRS and 5.0 mg
hydrochloride CRS in methanol R and dilute to 10 ml with of cyclizine impurity B CRS in methanol R and dilute to
the same solvent.
2.0 ml with the same solvent.
The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. impurity A

2. impurity B

3. cyclizine

Figure 1092.-1. Chromatogram for the test for related substances of cyclizine hydrochloride : reference solution (b)
418

PHARMEUROPA Vol. 18, No. 3, July 2006

Desflurane

Column :
material : fused silica ;
size : l = 25 m, = 0.33 mm ;
stationary phase : poly(dimethyl)(diphenyl)siloxane R(20) A. 1-methylpiperazine,
(film thickness 0.50 m).
Carrier gas : helium for chromatography R.
Flow rate : 1.0 ml/min.
Temperature :

Column

Time
(min)
0 - 14

Temperature
(C)
100 240

14 - 16

240 270

16 - 30

270

Injection port

250

Detector

290

B. diphenylmethanol (benzhydrol).

Reference: PA/PH/Exp. 10D/T (03) 1 ANP

XXXX:1666

DESFLURANE

Detection : flame ionisation.

Desfluranum

Injection : 1 l.
Relative retention with reference to cyclizine (retention
time = about 15 min) : impurity A = about 0.17 ;
impurity B = about 0.72.
System suitability : reference solution (b) :
resolution : minimum 18 between the peaks due to
impurity B and cyclizine.
Limits :
impurities A, B : for each impurity, not more than
0.1 times the area of the corresponding peak in the
chromatogram obtained with reference solution (b)
(0.5 per cent) ;
unspecified impurities : for each impurity, not more
than 0.2 times the area of the principal peak in the
chromatogram obtained with reference solution (a)
(0.10 per cent) ;

C 3H 2F 6O

Mr 168.0

DEFINITION
(2RS)-2-(Difluoromethoxy)-1,1,1,2-tetrafluoroethane.
CHARACTERS
Appearance : clear, colourless, mobile, heavy liquid.
Solubility : practically insoluble in water, miscible with
anhydrous ethanol and with trichloroethylene.
Relative density : 1.47, determined at 15 C.
bp : about 22 C.
It is non-flammable.

total: not more than twice the area of the principal peak IDENTIFICATION
in the chromatogram obtained with reference solution (a)
Infrared absorption spectrophotometry (2.2.24).
(1.0 per cent) ;
Preparation : examine the substance in the gaseous state.
disregard limit : 0.1 times the area of the principal peak
in the chromatogram obtained with reference solution (a) Comparison : Ph. Eur. reference spectrum of desflurane.
(0.05 per cent).
Loss on drying (2.2.32) : maximum 1.0 per cent, determined
on 1.000 g by drying in an oven at 130 C.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.

TESTS
The substance to be examined must be cooled to a
temperature below 10 C and the tests must be carried out
at a temperature below 20 C.
Acidity or alkalinity. To 20 ml add 20 ml of carbon
dioxide-free water R, shake for 3 min and allow to stand.
ASSAY
Collect the upper layer and add 0.2 ml of bromocresol
Dissolve 0.200 g in 15 ml of anhydrous formic acid R and add purple solution R. Not more than 0.1 ml of 0.01 M sodium
hydroxide or 0.1 ml of 0.01 M hydrochloric acid is required
40 ml of acetic anhydride R. Titrate with 0.1 M perchloric
acid, determining the end-point potentiometrically (2.2.20). to change the colour of the indicator.
Related substances. Gas chromatography (2.2.28).
1 ml of 0.1 M perchloric acid is equivalent to 15.14 mg of
Test solution. The substance to be examined cooled to below
C18H23ClN2.
10 C.
Reference solution (a). To 25 ml of the substance to be
STORAGE
examined cooled to below 10 C in a 50 ml volumetric flask
Protected from light.
add 0.5 ml of desflurane impurity A CRS and 1.0 ml of
isoflurane CRS. To this solution add 50 l of acetone R
IMPURITIES
(impurity H), 10 l of chloroform R (impurity F) and 50 l of
methylene chloride R (impurity E), using an airtight syringe,
Specified impurities : A, B.
(20) SE 52 Ultra 2 is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

419

Desflurane

and dilute to 50 ml with the substance to be examined.

Dilute 5.0 ml of this solution to 50 ml with the substance to
be examined. Store at a temperature below 10 C.
Reference solution (b). Dilute 5.0 ml of reference solution (a)
to 50 ml with the substance to be examined. Store at a
temperature below 10 C.
Column :
material : fused silica ;
size : l = 105 m, = 0.32 mm ;
stationary phase : poly(trifluoropropylmethyl)siloxane R
(film thickness 1.5 m)(21).
Carrier gas : helium for chromatography R.
Flow rate : 2.0 ml/min.
Split ratio : 1:25.
Temperature :
column : 30 C ;
injection port : 150 C ;
detector : 200 C.
Detection : flame ionisation.
Injection: 2.0 l.
Run time : 35 min.
Relative retention with reference to desflurane (retention
time = about 11.5 min) : impurity C = about 1.06 ;
impurity D = about 1.09 ; impurity A = about 1.14 ;
impurity G = about 1.39 ; impurity E = about 1.5 ;
impurity B = about 1.7 ; impurity F = about 2.2 ;
impurity H = about 2.6.
System suitability : reference solution (a) :
number of theoretical plates: minimum 20 000,
calculated for the peak due to impurity A ;
symmetry factor : maximum 2.0 for the peak due to
impurity B.
Limits :
impurity A : not more than the difference between the
area of the corresponding peak in the chromatogram
obtained with reference solution (a) and the area of the
corresponding peak in the chromatogram obtained with
the test solution (0.1 per cent V/V) ;
impurity B : not more than the difference between the
area of the corresponding peak in the chromatogram
obtained with reference solution (a) and the area of the
corresponding peak in the chromatogram obtained with
the test solution (0.2 per cent V/V) ;
impurities C, D, G : for each impurity, not more than
the difference between the area of the peak due to
impurity A in the chromatogram obtained with reference
solution (b) and the area of the peak due to impurity A
in the chromatogram obtained with the test solution
(0.01 per cent V/V) ;
impurities E, H : for each impurity, not more than
the difference between the area of the corresponding
peak in the chromatogram obtained with reference
solution (a) and the area of the corresponding peak in the
chromatogram obtained with the test solution (0.01 per
cent V/V) ;
impurity F : not more than the difference between the
area of the corresponding peak in the chromatogram
obtained with reference solution (a) and the area of the
corresponding peak in the chromatogram obtained with
the test solution (0.002 per cent V/V) ;

sum of impurities other than A, B, C, D, E, F, G and H :

not more than the difference between the area of the
peak due to impurity A in the chromatogram obtained
with reference solution (b) and the area of the peak due
to impurity A in the chromatogram obtained with the test
solution (0.01 per cent V/V) ;
unspecified impurities : for each impurity, not more than
0.5 times the difference between the area of the peak
due to impurity A in the chromatogram obtained with
reference solution (b) and the area of the peak due to
impurity A in the chromatogram obtained with the test
solution (0.005 per cent V/V) ;
disregard limit : the difference between the area of the
peak due to impurity A in the chromatogram obtained
with reference solution (c) and the area of the peak due
to impurity A in the chromatogram obtained with the test
solution (0.002 per cent V/V).
Fluorides: maximum 10 ppm.
Potentiometry (2.2.36, Method I).
Test solution. To 10.0 ml in a separating funnel, add 10 ml
of a mixture of 30.0 ml of dilute ammonia R2 and 70.0 ml
of distilled water R. Shake for 1 min and collect the upper
layer. Repeat this extraction procedure twice, collecting the
upper layer each time. Adjust the combined upper layers
to pH 5.2 with dilute hydrochloric acid R. Add 5.0 ml of
fluoride standard solution (1 ppm F) R and dilute to 50.0 ml
with distilled water R. To 20.0 ml of this solution add 20.0 ml
of total-ionic-strength-adjustment buffer R and dilute to
50.0 ml with distilled water R.
Reference solutions. To each of 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml
and 5.0 ml of fluoride standard solution (10 ppm F) R add
20.0 ml of total-ionic-strength-adjustment buffer R and
dilute to 50.0 ml with distilled water R.
Indicator electrode: fluoride selective.
Reference electrode : silver-silver chloride.
Carry out the measurements on 20 ml of each solution.
Calculate the concentration of fluorides using the calibration
curve, taking into account the addition of fluoride to the
test solution.
Antimony : maximum 3.0 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Transfer 10 g cooled to below 10 C to a tared
flask containing 20 ml of water R cooled to below 5 C.
Add 1 ml of a mixture of equal volumes of hydrochloric
acid R and nitric acid R. Leave at room temperature for a
sufficiently long time to allow the desflurane to evaporate
completely. Subsequently, reduce the volume to about 8 ml
on a hot plate. Cool to room temperature and transfer to
a 10 ml volumetric flask. Add 1 ml of a mixture of equal
volumes of hydrochloric acid R and nitric acid R. Adjust to
10.0 ml with water R.
Reference solutions. To each of 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml
and 5.0 ml of antimony standard solution (100 ppm Sb) R
add 20 ml of a mixture of equal volumes of hydrochloric
acid R and nitric acid R and dilute to 100.0 ml with water R.
Source : antimony hollow-cathode lamp using a transmission
band of 0.2 nm and operated at 75 per cent lamp current.
Wavelength : 217.6 nm.
Atomisation device : air-acetylene flame.
Non-volatile matter : maximum 100 mg/l.
Evaporate 20.0 ml to dryness with the aid of a stream of
nitrogen R. The residue weighs not more than 2.0 mg.

(21) Rtx-200 is suitable (available from Restek Corp.)

420

PHARMEUROPA Vol. 18, No. 3, July 2006

Devils claw root

STORAGE
In a glass bottle fitted with a polyethylene-lined cap. Before
opening the bottle, cool the contents to below 10 C.

CHARACTERS
Devils claw root is greyish-brown to dark brown.

IDENTIFICATION
A. It consists of thick, fan-shaped or rounded slices or
of roughly crushed discs. The darker outer surface is
traversed by tortuous longitudinal wrinkles. The paler
cut surface shows a dark cambial zone and xylem bundles
distinctly aligned in radial rows. The central cylinder
shows fine concentric striations. Seen under a lens, the
cut surface presents yellow to brownish-red granules.
A. 1,1-oxybis(1,2,2,2-tetrafluoroethane),
B. Reduce to a powder (355). The powder is brownish-yellow.
B. isofluorane,
Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic
characters : fragments of cork layer consisting of
yellowish-brown, thin-walled cells ; fragments of
cortical parenchyma consisting of large, thin-walled
C. R = H, R = F : dichlorofluoromethane,
cells, sometimes containing reddish-brown granular
inclusions and isolated yellow droplets ; fragments of
D. R = Cl, R = F : trichlorofluoromethane,
reticulately thickened vessels and tracheidal vessels
with associated lignified parenchyma from the central
E. R = R = H : dichloromethane (methylene chloride),
cylinder ; small needles and crystals of calcium oxalate
F. R = H, R = Cl : trichloromethane (chloroform),
are present in the parenchyma. The powder may show
rectangular or polygonal pitted sclereids with dark
reddish-brown contents. With a solution of phloroglucinol
in hydrochloric acid, the parenchyma turns green.
C. Thin-layer chromatography (2.2.27).
Test solution. Heat 1.0 g of the powdered drug (355) with
G. 1,1,2-trichloro-1,2,2-trifluoroethane,
10 ml of methanol R on a water-bath at 60 C for 10 min.
Filter and reduce the filtrate to about 2 ml under reduced
H. acetone.
pressure at a temperature not exceeding 40 C.
Reference solution. Dissolve 1 mg of harpagoside R in
Reagents
1 ml of methanol R.
Poly(trifluoropropylmethyl)siloxane. XXXXXXX.
Plate : TLC silica gel plate R (5-40 m) [or TLC silica gel
Stationary phase for gas chromatography.
plate R (2-10 m)].
Contains 50 per cent of trifluoropropyl groups and 50 per
Mobile phase : water R, methanol R, ethyl acetate R
cent of methyl groups.
(8:15:77 V/V/V).
Application : 20 l, as bands of 20 mm.
Development : over a path of 10 cm [or 7.5 cm].
Drying : in a current of warm air.
Reference: PA/PH/Exp. 13B/T (06) 19 ANP
Detection A : examine in ultraviolet light at 254 nm.
Results A : see below the sequence of the zones present in
NOTE ON THE MONOGRAPH
the chromatogram obtained with the reference solution
It is proposed to revise the monograph in order to harmonise
and the test solution. The chromatogram obtained with
the assay method with the monograph for Devils claw dry
the test solution shows other distinct bands, mainly above
extract (1871) published in Pharmeuropa 18.1.
the zone due to harpagoside. Furthermore, other zones
In the Characters section it has been decided generally to
may be present in the chromatogram obtained with the
delete the cross reference to identification tests A and B
test solution.
(macroscopic and microscopic description), since the
Top of the plate
Identification section is mandatory while the Characters
_______
_______
section is only informative.
The test for foreign matter (2.8.2) is now omitted in
Harpagoside : a quenching zone
A quenching zone : harpagoside
monographs where it is limited to 2 per cent, since this test
_______
_______
is covered by the general monograph for herbal drugs.
XXXX:1095
Reference solution
Test solution
IMPURITIES
Specified impurities : A, B, C, D, E, F, G, H.

DEVILS CLAW ROOT

Harpagophyti radix
DEFINITION
Devils claw root consists of the cut and dried tuberous,
secondary roots of Harpagophytum procumbens D.C.
and/or H. zeyheri L. Decne.
Content : minimum 1.2 per cent of harpagoside (C24H30O11 ;
Mr 494.5) (dried drug).
PHARMEUROPA Vol. 18, No. 3, July 2006

Detection B : spray with a 10 g/l solution of

phloroglucinol R in ethanol (96 per cent) R and then
with hydrochloric acid R ; heat at 80 C for 5-10 min and
examine in daylight.
Results B : see below the sequence of the zones present in
the chromatogram obtained with the reference solution
and the test solution. The chromatogram obtained with
the test solution also shows several yellow to brown zones
above the zone due to harpagoside. Furthermore, other
zones may be present in the chromatogram obtained with
the test solution.
421

Devils claw root

Top of the plate

_______
Harpagoside : a green zone

_______

Calculate the percentage content of harpagoside from the

expression :

A green zone (harpagoside)

_______

_______
A yellow zone

Reference solution

A light green zone

m1

A yellowish-grey zone may be

present
A brown zone

mass of the drug, in grams,

m2

F1

F2

mass of methyl cinnamate R, in grams in the

internal standard solution,
area of the peak corresponding to harpagoside in
the chromatogram obtained with the test solution,
area of the peak corresponding to methyl
cinnamate in the chromatogram obtained with
the test solution.

Test solution

TESTS
Starch. Examine the powdered drug (355) under a
microscope using water R. Add iodine solution R1. No blue
colour develops.

Liquid chromatography (2.2.29).

Loss on drying (2.2.32) : maximum 12.0 per cent, determined The following chromatogram is shown for information but
on 1.000 g of the powdered drug (355) by drying in an oven will not be published in the European Pharmacopoeia.
at 100-105 C.
Total ash (2.4.16) : maximum 10.0 per cent.
ASSAY
Liquid chromatography (2.2.29) using methyl cinnamate R
as the internal standard.
Internal standard solution. Dissolve 0.130 g of methyl
cinnamate R in 50 ml of methanol R and dilute to 100.0 ml
with the same solvent.
Test solution. To 0.500 g of the powdered drug (355) add
50 ml of methanol R. Shake for 1 h and filter. Transfer
the filter with the residue to a 100 ml flask, add 50 ml of
methanol R and heat under a reflux condenser for 1 h. Cool
and filter. Rinse the flask and the filter with 2 quantities,
each of 5 ml, of methanol R. Combine the filtrate and the
rinsing solution and evaporate to dryness under reduced
pressure at a temperature not exceeding 40 C. Take up
the residue with 3 quantities, each of 5 ml, of methanol R
and filter the extracts into a 25 ml volumetric flask. Whilst
washing the filter, dilute to 25.0 ml with methanol R. To
10.0 ml of this solution add 1.0 ml of the internal standard
solution and dilute to 25.0 ml with methanol R.
Reference solution. Dilute 0.5 ml of the reference solution
described in Identification test C to 2.0 ml with methanol R.
The chromatographic procedure may be carried out using :

1. harpagoside

Figure 1095.-1. Chromatogram for the assay of

harpagoside in devils claw root
Test solution. To 0.500 g of the powdered drug (355) add
100 ml of methanol R. Shake for 4 h and filter through a
0.45 m filter.

Reference solution. Dissolve 0.015 g of harpagoside CRS

a stainless steel column 0.10 m long and 4 mm in
internal diameter packed with octadecylsilyl silica gel for in 100.0 ml of methanol R. Dilute 5.0 ml of this solution to
10.0 ml with methanol R.
chromatography R (5 m),
Column :
as mobile phase at a flow rate of 1.5 ml/min a mixture of
size : l = 0.10 m, = 4.0 mm ;
equal volumes of methanol R and water R,
as detector a spectrophotometer set at 278 nm,

stationary phase : octadecylsilyl silica gel for

chromatography R (5 m).

a 10 l loop injector.

Mobile phase : methanol R, water R (50:50 V/V).

Inject the test solution. Adjust the sensitivity of the detector Flow rate : 1.5 ml/min.
so that the height of the peak due to methyl cinnamate is
Detection : spectrophotometer at 278 nm.
about 50 per cent of the full scale of the recorder.
Injection : 10 l.
Determine the retention time of harpagoside using 10 l of
the reference solution examined under the same conditions Run time : 3 times the retention time of harpagoside.
as the test solution.
Retention time : harpagoside = about 7 min.
422

PHARMEUROPA Vol. 18, No. 3, July 2006

Dihydroergotamine mesilate

Calculate the percentage content of harpagoside using the

following expression :

Second identification : A, C, D.
A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution. Dissolve 5.0 mg in methanol R and dilute
to 100.0 ml with the same solvent.
Spectral range : 250-350 nm.
F1 = area of the peak due to harpagoside in the
Absorption maxima : at 281 nm and 291 nm.
chromatogram obtained with the test solution ;
Shoulder : at 275 nm.
F2 = area of the peak due to harpagoside in the
Absorbance : negligible above 320 nm.
chromatogram obtained with the reference
Specific absorbance at the absorption maximum at
solution ;
281 nm : 95 to 105 (dried substance).
m1 = mass of the drug to be examined, in grams ;
B. Infrared absorption spectrophotometry (2.2.24).
m2 = mass of harpagoside CRS in the reference
Preparation : discs.
solution, in grams.
Comparison : dihydroergotamine mesilate CRS.
C. Thin-layer chromatography (2.2.27). Prepare the
reference solution and the test solution immediately
before use.
Reference: PA/PH/Exp. 11/T (06) 38 ANP
Test solution. Dissolve 5 mg of the substance to be
examined in a mixture of 1 volume of methanol R and
NOTE ON THE MONOGRAPH
9 volumes of methylene chloride R and dilute to 2.5 ml
Related substances : the TLC method has been replaced
with the same mixture of solvents.
by an LC method.
Reference solution. Dissolve 5 mg of dihydroergotamine
XXXX:0551
mesilate CRS in a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R and dilute to 2.5 ml
with the same mixture of solvents.
DIHYDROERGOTAMINE MESILATE
Plate : TLC silica gel G plate R.
Dihydroergotamini mesilas
Mobile phase : concentrated ammonia R,
methanol R, ethyl acetate R, methylene chloride R
(1:6:50:50 V/V/V/V).
Application : 5 l.
Development : protected from light over a path of 15 cm.
Dry the plate in a current of cold air for not longer than
1 min. Repeat the development protected from light over
a path of 15 cm using a freshly prepared amount of the
mobile phase.
Drying : in a current of cold air.
C34H41N5O8S
Mr 680
Detection : spray abundantly with dimethylaminobenzaldehyde solution R7 and dry in a current of hot air for
DEFINITION
about 2 min.
(6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-Benzyl-10bResults : the principal spot in the chromatogram obtained
hydroxy-2-methyl-3,6-dioxooctahydro-8H-oxazolo[3,2with the test solution is similar in position, colour and
a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10,10asize to the principal spot in the chromatogram obtained
octahydroindolo[4,3-fg]quinoline-9-carboxamide
with the reference solution.
methanesulphonate.
D. To 0.1 g add 5 ml of dilute hydrochloric acid R and shake
Content : 98.0 per cent to 101.0 per cent (dried substance).
for about 5 min. Filter and add 1 ml of barium chloride
solution R1. The filtrate remains clear. Mix 0.1 g with
PRODUCTION
0.4 g of powdered sodium hydroxide R, heat to fusion
The production method must be evaluated to determine
and continue to heat for 1 min. Cool, add 5 ml of water R,
the potential for formation of alkyl mesilates, which is
boil and filter. Acidify the filtrate by the addition of
particularly likely to occur if the reaction medium contains
hydrochloric acid R1 and filter again. The filtrate gives
lower alcohols. Where necessary, the production method
reaction (a) of sulphates (2.3.1).
is validated to demonstrate that alkyl mesilates are not
detectable in the final product.
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
CHARACTERS
more intensely coloured than reference solution Y7 or BY7
Appearance : white or almost white, crystalline powder or
(2.2.2, Method II).
colourless crystals.
Dissolve 0.10 g in a mixture of 0.1 ml of a 70 g/l solution of
Solubility : slightly soluble in water, sparingly soluble in
methanesulphonic acid R and 50 ml of water R.
methanol, slightly soluble in ethanol (96 per cent).
pH (2.2.3) : 4.4 to 5.4.
IDENTIFICATION
Dissolve 0.10 g in carbon dioxide-free water R and dilute to
First identification : B, C.
100 ml with the same solvent.
PHARMEUROPA Vol. 18, No. 3, July 2006

423

Dihydroergotamine mesilate

Specific optical rotation (2.2.7) : 42 to 47 (dried

substance).
Dissolve 0.250 g in anhydrous pyridine R and dilute to
25.0 ml with the same solvent.
Related substances. Thin-layer chromatography (2.2.27).
Prepare the reference solutions and the test solutions
immediately before use and in the order indicated below.
Reference solution (a). Dissolve 10.0 mg of
dihydroergotamine mesilate CRS in a mixture of
1 volume of methanol R and 9 volumes of methylene
chloride R and dilute to 5 ml with the same mixture of
solvents.
Reference solution (b). Dilute 2.5 ml of reference solution (a)
to 50 ml with a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R.
Reference solution (c). Dilute 2 ml of reference solution (b)
to 5 ml with a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R.
Test solution (a). Dissolve 0.10 g of the substance to be
examined in a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R and dilute to 5 ml with
the same mixture of solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml
with a mixture of 1 volume of methanol R and 9 volumes of
methylene chloride R.
Plate : TLC silica gel G plate R.
Mobile phase : concentrated ammonia R, methanol R, ethyl
acetate R, methylene chloride R (1:6:50:50 V/V/V/V).
Application : 5 l.
Development : protected from light over a path of 15 cm. Dry
the plate in a current of cold air for not longer than 1 min.
Repeat the development protected from light over a path of
15 cm using a freshly prepared amount of the mobile phase.
Drying : in a current of cold air.
Detection : spray abundantly with dimethylaminobenzaldehyde solution R7 and dry in a current of hot air for about
2 min.
Limits : test solution (a) :
any impurity : any spot, apart from the principal spot,
is not more intense than the principal spot in the
chromatogram obtained with reference solution (b)
(0.5 per cent) and not more than 2 such spots are more
intense than the principal spot in the chromatogram
obtained with reference solution (c) (0.2 per cent).
Liquid chromatography (2.2.29). Carry out the test protected
from light.
Solvent mixture : acetonitrile R, water R (50:50 V/V).
Test solution. Dissolve 70 mg of the substance to be
examined in the solvent mixture and dilute to 100.0 ml with
the solvent mixture.
Reference solution (a). Dilute 1.0 ml of the test solution
to 10.0 ml with the solvent mixture. Dilute 1.0 ml of this
solution to 100.0 ml with the solvent mixture.
Reference solution (b). Dissolve 7 mg of the substance to be
examined and 6.8 mg of ergotamine tartrate CRS (equivalent
to 7 mg of ergotamine mesilate) in the solvent mixture and
dilute to 100.0 ml with the solvent mixture. Dilute 5.0 ml of
this solution to 10.0 ml with the solvent mixture.
Reference solution (c). Dissolve 5 mg of dihydroergotamine
for peak identification CRS (containing impurities A, B, C, D
and E) in the solvent mixture, add 100 l of dilute sulphuric
acid R and dilute to 5 ml with the solvent mixture.

Column :
size : l = 0.15 m, = 4.6 mm ;
stationary phase : spherical octadecylsilyl silica gel for
chromatography R (5 m)(22) ;
temperature : 25 C.
Mobile phase :
mobile phase A : 3 g/l solution of sodium
heptanesulphonate monohydrate R adjusted to
pH 2.0 with phosphoric acid R ;
mobile phase B : mobile phase A, acetonitrile for
chromatography R (20:80 V/V) ;
Time
(min)
0 - 15

Mobile phase A
(per cent V/V)
58 40

Mobile phase B
(per cent V/V)
42 60

15

40

60

15 - 15.1

40 58

60 42

15.1 - 20

58

42

Flow rate : 1.5 ml/min

Detection : spectrophotometer at 220 nm.
Injection : 5 l.
Identification of impurities : use the chromatogram supplied
with dihydroergotamine for peak identification CRS and
the chromatogram obtained with reference solution (c) to
identify the peaks due to impurities A, B, C, D and E.
Relative retention with reference to dihydroergotamine
(retention time = about 6.5 min) : impurity D = about 0.7 ;
impurity C = about 0.86 ; impurity A = about 0.95 ;
impurity B = about 1.20.
System suitability : reference solution (b) :
resolution : minimum 1.5 between the peaks due to
ergotamine and dihydroergotamine.
Limits :
correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity A = 1.3 ;
impurity C = 1.3 ;
impurity A : not more than 1.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.15 per cent) ;
impurity B : not more than 5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent) ;
impurity C : not more than 3 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.3 per cent) ;
impurities D, E : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent) ;
unspecified impurities : for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.10 per cent) ;
total : not more than 10 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (1.0 per cent) ;
disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 4.0 per cent, determined
on 0.500 g by drying at 100-105 C at a pressure not
exceeding 0.1 kPa for 5 h.

(22) Inertsil ODS III is suitable.

424

PHARMEUROPA Vol. 18, No. 3, July 2006

Dihydroergotamine mesilate

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

Figure 0551.-1. Chromatogram for the test for related substances of dihydroergotamine mesilate : solution spiked
with impurities A, B, C, D and E
ASSAY
Dissolve 0.500 g in a mixture of 10 ml of anhydrous
acetic acid R and 70 ml of acetic anhydride R. Titrate
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1 ml of 0.1 M perchloric acid is equivalent to 68.00 mg of
C34H41N5O8S.
STORAGE
Protected from light.

C. (5,10)-5-benzyl-8,12-dihydroxy-2-methyl-3,6,18trioxo-9,10-dihydroergotaman,

IMPURITIES
Specified impurities : A, B, C, D, E.

D. (2,5,10)-5-benzyl-12-hydroxy-2-methyl-3,6,18trioxo-9,10-dihydroergotaman,
A. (5)-5-benzyl-12-hydroxy-2-methyl-3,6,18trioxoergotaman,

B. (5,10)-5-benzyl-2-ethyl-12-hydroxy-3,6,18-trioxo-9,
10-dihydroergotaman,
PHARMEUROPA Vol. 18, No. 3, July 2006

E. (5,10)-5-benzyl-12-hydroxy-2-isopropyl-3,6,18trioxo-9,10-dihydroergotaman.
425

Disodium phosphate dodecahydrate

Sulphates (2.4.13) : maximum 500 ppm.

To 3 ml of solution S add 2 ml of dilute hydrochloric acid R
NOTE ON THE MONOGRAPH
and dilute to 15 ml with distilled water R.
Loss on drying /water : comparative results from 56 batches
Arsenic (2.4.2, Method A) : maximum 2 ppm, determined
of this substance gave equivalent results using the current
on 5 ml of solution S.
method for the determination of water (2.5.12), or for
Iron (2.4.9) : maximum 20 ppm.
the loss on drying (2.2.32). The mean of the water
determination of all 56 batches was 0.07 per cent higher
Dilute 5 ml of solution S to 10 ml with water R.
than the mean of the loss on drying.
Heavy metals (2.4.8) : maximum 10 ppm.
On the other hand, by calculating the content using the
12 ml of solution S complies with test A. Prepare the
formula given in the assay, some batches were just at the
reference solution using lead standard solution (1 ppm
limit or outside of the specifications, regardless of the
Pb) R.
method used for the water determination. Indeed, a small
mistake in the calculation of the water content (for example Water (2.5.12) : 57.0 per cent to 61.0 per cent, determined
0.1 per cent) has serious repercussions on the calculations on 50.0 mg. Use a mixture of 10 volumes of anhydrous
methanol R and 40 volumes of formamide R as solvent.
in the assay (0.25 per cent in this case). Therefore, it is
proposed to give the limits of content of the substance as is, ASSAY
without allowing for the results of the test for water.
XXXX:0118 Dissolve 4.00 g (m) in 25 ml of water R and add 25.0 ml
of 1 M hydrochloric acid. Carry out a potentiometric
titration (2.2.20) using 1 M sodium hydroxide. Read the
DISODIUM PHOSPHATE
volume added at the 1st inflexion point (n1 ml). Continue
the titration to the 2nd inflexion point (total volume of 1 M
DODECAHYDRATE
sodium hydroxide required, n2 ml).
Calculate the percentage content of Na2HPO4 from the
Dinatrii phosphas dodecahydricus
following expression :
Reference: PA/PH/Exp. INC/T (06) 16 ANP

Na2HPO4,12H2O

Mr 358.1

DEFINITION
Content : 98.0 per cent 39.1 per cent to 101.0 per cent
40.6 per cent (anhydrous substance) (without allowing for
the results of the test for water).
CHARACTERS
Appearance : colourless, transparent crystals, very
efflorescent.
Solubility : very soluble in water, practically insoluble in
ethanol (96 per cent).
IDENTIFICATION
A. Solution S (see Tests) is slightly alkaline (2.2.4).
B. Water (see Tests).
C. Solution S gives reaction (b) of phosphates (2.3.1).
D. Solution S gives reaction (a) of sodium (2.3.1).
TESTS
Solution S. Dissolve 5.0 g in distilled water R and dilute to
50 ml with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Reducing substances. To 5 ml of solution S add 5 ml of
dilute sulphuric acid R and 0.25 ml of 0.02 M potassium
permanganate and heat on a water-bath for 5 min. The
solution retains a slight red colour.
Monosodium phosphate : maximum 2.5 per cent.
From the volume of 1 M hydrochloric acid (25 ml) and of
1 M sodium hydroxide (n1 ml and n2 ml) used in the assay,
calculate the following ratio :

This ratio is not greater than 0.025.

Chlorides (2.4.4) : maximum 200 ppm.
To 2.5 ml of solution S add 10 ml of dilute nitric acid R and
dilute to 15 ml with water R.
426

(deleted)

percentage content of water.

(added)

Reference: PA/PH/Exp. 10A/T (06) 16 ANP

NOTE ON THE MONOGRAPH
Related substances : the TLC method has been replaced
by an LC method.
Only comments on the test for related substances are
requested.
XXXX:1096

DOXEPIN HYDROCHLORIDE
Doxepini hydrochloridum

C19H22ClNO

Mr 315.8

DEFINITION
(E)-3-(Dibenzo[b,e]oxepin-11(6H)-ylidene)-N,Ndimethylpropan-1-amine hydrochloride.
PHARMEUROPA Vol. 18, No. 3, July 2006

Doxepin hydrochloride

Content : 98.0 per cent to 101.0 per cent of C19H22ClNO

(dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : freely soluble in water, in ethanol (96 per cent)
and in methylene chloride.
IDENTIFICATION
First identification : C, E.
Second identification : A, B, D, E.
A. Melting point (2.2.14) : 185 C to 191 C.
B. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution. Dissolve 50.0 mg in a 1 g/l solution of
hydrochloric acid R in methanol R and dilute to 100.0 ml
with the same acid solution. Dilute 5.0 ml to 50.0 ml with
a 1 g/l solution of hydrochloric acid R in methanol R.
Spectral range : 230-350 nm.
Absorption maximum : at 297 nm.
Specific absorbance at the absorption maximum : 128
to 142.
C. Infrared absorption spectrophotometry (2.2.24).
Comparison : Ph. Eur. reference spectrum of doxepin
hydrochloride.
D. Dissolve about 5 mg in 2 ml of sulphuric acid R. A dark
red colour is produced.
E. Solution S (see Tests) gives reaction (a) of chlorides
(2.3.1).
TESTS
Solution S. Dissolve 1.5 g in carbon dioxide-free water R
and dilute to 30 ml with the same solvent.
Appearance of solution. Dilute 10 ml of solution S to 25 ml
with water R. The solution is clear (2.2.1) and colourless
(2.2.2, Method II).
Acidity. To 10 ml of solution S add 0.1 ml of methyl red
solution R. Not more than 0.1 ml of 0.1 M sodium hydroxide
is required to change the colour of the indicator to yellow.
Related substances. Examine by thin-layer chromatography
(2.2.27), using a TLC silica gel F254 plate R (2-10 m) with
a concentration zone.
Test solution. Dissolve 0.10 g of the substance to be
examined in methanol R and dilute to 10 ml with the same
solvent.
Reference solution (a). Dissolve 10.0 mg of doxepin
impurity A CRS in methanol R, add 1 ml of the test solution
and dilute to 10 ml with methanol R. Dilute 1.0 ml of this
solution to 50 ml with methanol R.
Reference solution (b). Dissolve 10.0 mg of doxepin
impurity B CRS in methanol R, add 1 ml of the test solution
and dilute to 10 ml with methanol R. Dilute 1.0 ml of this
solution to 50 ml with methanol R.
Reference solution (c). Dissolve 10.0 mg of doxepin
impurity B CRS in methanol R and dilute to 200 ml with
the same solvent.
Apply to one plate (plate A) 2 l of the test solution and
2 l of reference solution (a). Develop over a path of 5 cm
using a mixture of 30 volumes of methyl ethyl ketone R and
60 volumes of heptane R. Doxepin remains on the starting
line.

Apply to another plate (plate B) 2 l of the test solution and

2 l of reference solutions (b) and (c). Develop over a path
of 5 cm using a mixture of 10 volumes of methanol R and
90 volumes of methylene chloride R.
Allow the plates to dry in a current of air. Spray with a
solution prepared as follows : dissolve 20 g of zinc chloride R
in 30 ml of glacial acetic acid R, add 3 ml of phosphoric
acid R and 0.80 ml of strong hydrogen peroxide solution R
and dilute to 60 ml with water R. Heat the plates at 120 C
for 15 min and examine immediately in ultraviolet light at
365 nm.
Plate A. In the chromatogram obtained with the test
solution : any spot corresponding to impurity A is not more
intense than the corresponding spot in the chromatogram
obtained with reference solution (a) (0.2 per cent) ; any spot,
apart from the principal spot and the spot corresponding
to impurity A, is not more intense than the spots in the
chromatogram obtained with reference solution (a) (0.2 per
cent).
Plate B. In the chromatogram obtained with the test
solution : any spot corresponding to impurity C (Rf about
0.12) is not more intense than the spot in the chromatogram
obtained with reference solution (c) (0.5 per cent) ; any spot
corresponding to impurity B is not more intense than the
corresponding spot in the chromatogram obtained with
reference solution (b) (0.2 per cent) ; any spot, apart from
the principal spot, the spot corresponding to impurity B or
the spot corresponding to impurity C, is not more intense
than the spots in the chromatogram obtained with reference
solution (b) (0.2 per cent).
The test is not valid unless the chromatograms obtained
with reference solutions (a) and (b) show clearly visible and
separated principal spots.
Liquid chromatography (2.2.29). Prepare the solutions
immediately before use.
Phosphate buffer solution. Dissolve 1.42 g of anhydrous
disodium hydrogen phosphate R in water R and dilute to
1000 ml with the same solvent. Adjust to pH 7.7 with dilute
phosphoric acid R.
Solvent mixture. Mix 1 volume of 1 M sodium hydroxide
and 250 volumes of the mobile phase.
Test solution. Dissolve 50.0 mg of the substance to be
examined in the solvent mixture and dilute to 50.0 ml with
the solvent mixture.
Reference solution (a). Dilute 1.0 ml of the test solution
to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this
solution to 10.0 ml with the solvent mixture.
Reference solution (b). Dissolve 20.0 mg of doxepin
hydrochloride for system suitability CRS (containing
impurities A, B and C) in the solvent mixture and dilute
to 50.0 ml with the solvent mixture. Dilute 1.0 ml of this
solution to 20.0 ml with the solvent mixture.
Column:
size : l = 0.25 m, = 4.6 mm ;
stationary phase : end-capped octadecylsilyl silica gel
for chromatography R (5 m)(23) ;
temperature : 30 C.
Mobile phase : acetonitrile R1, phosphate buffer solution,
methanol R1 (20:30:50 V/V/V).
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 215 nm.
Injection : 20 l.
Run time : 1.5 times the retention time of doxepin.

(23) Luna C18 is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

427

Doxepin hydrochloride

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. impurity A

2. impurity C

3. impurity B

4. doxepin

Figure 1096.-1. Chromatogram for the test for related substances of doxepin hydrochloride : solution of doxepin
hydrochloride spiked with impurities A, B and C
Identification of impurities : use the chromatogram supplied
with doxepin hydrochloride for system suitability CRS and
the chromatogram obtained with reference solution (b) to
identify the peaks due to impurities A, B and C. Doubling of
the peak due to doxepin may be observed.
Relative retention with reference to doxepin (retention
time = about 18 min) : impurity A = about 0.5 ;
impurity C = about 0.6 ; impurity B = about 0.7.
System suitability : reference solution (b) :
resolution : minimum 1.5 between the peaks due to
impurities A and C, and minimum 1.5 between the peaks
due to impurities C and B ;
symmetry factor : maximum 1.6 for the peak due to
doxepin ;
the chromatogram obtained is similar to the
chromatogram supplied with doxepin hydrochloride for
system suitability CRS.
Limits :
correction factor : for the calculation of content, multiply
the peak area of impurity B by 1.7 ;
impurities A, B : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent) ;
impurity C : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.2 per cent) ;
unspecified impurities : for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.10 per cent) ;
total: not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.3 per cent) ;
428

disregard limit : 0.5 times the area of the principal peak

in the chromatogram obtained with reference solution (a)
(0.05 per cent) ; disregard any peak with a relative
retention less than 0.25.
(Z)-Isomer. Liquid chromatography (2.2.29).
Test solution. Dissolve 20.0 mg of the substance to be
examined in the mobile phase and dilute to 20.0 ml with the
mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with
the mobile phase.
Column :
size : l = 0.12 m, = 4 mm ;
stationary phase : spherical octylsilyl silica gel for
chromatography R (5 m) with a specific surface area of
220 m2/g and a pore size of 80 nm ;
temperature : 50 C.
Mobile phase : mix 30 volumes of methanol R and
70 volumes of a 30 g/l solution of sodium dihydrogen
phosphate R previously adjusted to pH 2.5 with phosphoric
acid R.
Flow rate : 1 ml/min.
Detection : spectrophotometer at 254 nm.
Injection : 20 l.
System suitability :
resolution : minimum 1.5 between the peaks due to the
(E)-isomer (1st peak) and to the (Z)-isomer (2nd peak).
Results :
calculate the ratio of the area of the peak due to the
(E)-isomer to the area of the peak due to the (Z)-isomer :
this ratio is 4.4 to 6.7 (13.0 per cent to 18.5 per cent of
the (Z)-isomer).
PHARMEUROPA Vol. 18, No. 3, July 2006

5.2.7. Evaluation of efficacy of veterinary vaccines and immunosera

Heavy metals (2.4.8) : maximum 20 ppm.

1.0 g complies with test D. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 100-105 C.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.

Reference: PA/PH/Exp. 15V/T (05) 62 ANP

NOTE ON THE GENERAL CHAPTER
In order to clarify interpretation of the test, this chapter
has been revised to include a statement on duration of
immunity, and notably to indicate that the test model
described under Immunogenicity is not necessarily used
as such to determine the claimed duration of immunity.
XXXX:50207

ASSAY
Dissolve 0.250 g in a mixture of 5 ml of anhydrous acetic
5.2.7. EVALUATION OF EFFICACY
acid R and 35 ml of acetic anhydride R. Titrate with 0.1 M OF VETERINARY VACCINES AND
perchloric acid until the colour changes from blue to green,
IMMUNOSERA
using 0.2 ml of crystal violet solution R as indicator.
1 ml of 0.1 M perchloric acid is equivalent to 31.58 mg of
The term product means either a vaccine or an
C19H22ClNO.
immunoserum throughout the text.
During development of the product, tests are carried
STORAGE
out to demonstrate that the product is efficacious when
Protected from light.
administered by each of the recommended routes and
methods of administration and using the recommended
IMPURITIES
schedule to animals of each species and category for which
use of the product is to be recommended. The type of efficacy
Specified impurities : A, B, C, D.
testing to be carried out varies considerably depending on
the particular type of product.
As part of tests carried out during development to establish
efficacy, the tests described in the Production section of a
monograph may be carried out ; the following must be taken
into account.
The dose to be used is that quantity of the product to be
recommended for use and containing the minimum titre or
potency expected at the end of the period of validity.
For live vaccines, use vaccine containing virus/bacteria at
A. dibenzo[b,e]oxepin-11(6H)-one (doxepinone),
the most attenuated passage level that will be present in a
batch of vaccine.
For immunosera, if appropriate, the dose tested also contains
minimum quantities of immunoglobulin or gammaglobulin
and/or total protein.
The efficacy evidence must support all the claims being
made. For example, claims for protection against respiratory
disease must be supported by at least evidence of protection
from clinical signs of respiratory disease. Where it is
claimed that there is protection from infection this must be
demonstrated using re-isolation techniques. If more than one
B. (11RS)-11-[3-(dimethylamino)propyl]-6,11claim is made, supporting evidence for each claim is required.
dihydrodibenzo[b,e]oxepin-11-ol (doxepinol),
Claims related to duration of immunity are supported by
evidence of protection. The test model described under
Immunogenicity is not necessarily used to support claims
regarding the duration of immunity afforded by a vaccine.
Vaccines. The influence of passively acquired and maternally
derived antibodies on the efficacy of a vaccine is adequately
evaluated. Any claims, stated or implied, regarding onset and
duration of protection shall be supported by data from trials.
The efficacy of each of the components of multivalent
C. (E)-3-(dibenzo[b,e]oxepin-11(6H)-ylidene)-Nand combined vaccines shall be demonstrated using the
methylpropan-1-amine (desmethyldoxepin),
combined vaccine.
Immunosera. Particular attention must be paid to providing
supporting data for the efficacy of the regime that is to
be recommended. For example, if it is recommended that
the immunoserum needs only to be administered once to
achieve a prophylactic or therapeutic effect then this must
be demonstrated. Any claims, stated or implied, regarding
onset and duration of protection or therapeutic effect must
be supported by data from trials. For example, the duration
of the protection afforded by a prophylactic dose of an
antiserum must be studied so that appropriate guidance for
D. (Z)-3-(dibenzo[b,e]oxepin-11(6H)-ylidene)-N,Nthe user can be given on the label.
dimethylpropan-1-amine.
PHARMEUROPA Vol. 18, No. 3, July 2006

429

Extracts

Studies of immunological compatibility are undertaken when

simultaneous administration is recommended or where it is a
part of a usual administration schedule. Wherever a product
is recommended as part of an administration scheme, the
priming or booster effect or the contribution of the product
to the efficacy of the scheme as a whole is demonstrated.
LABORATORY TESTS
In principle, demonstration of efficacy is undertaken under
well-controlled laboratory conditions by challenge of the
target animal under the recommended conditions of use.
In so far as possible, the conditions under which the
challenge is carried out shall mimic the natural conditions for
infection, for example with regard to the amount of challenge
organism and the route of administration of the challenge.
Vaccines. Unless otherwise justified, challenge is carried out
using a strain different from the one used in the production
of the vaccine.
If possible, the immune mechanism (cell-mediated/humoral,
local/general, classes of immunoglobulin) that is initiated
after the administration of the vaccine to target animals shall
be determined.
Immunosera. Data are provided from measurements of
the antibody levels achieved in the target species after
administration of the product, as recommended. Where
suitable published data exist, references are provided to
relevant published literature on protective antibody levels
and challenge studies are avoided.
Where challenges are required, these can be given before or
after administration of the product, in accordance with the
indications and specific claims to be made.

XXXX:0765

EXTRACTS
Extracta
DEFINITION
Extracts are preparations of liquid (liquid extracts and
tinctures), semi-solid (soft extracts and oleoresins) or solid
(dry extracts) consistency, obtained from herbal drugs or
animal matter, which are usually in a dry state.
Different types of extract may be distinguished. Standardised
extracts are adjusted within an acceptable tolerance to
a given content of constituents with known therapeutic
activity ; standardisation is achieved by adjustment of
the extract with inert material or by blending batches of
extracts. Quantified extracts are adjusted to a defined range
of constituents ; adjustments are made by blending batches
of extracts. Other extracts are essentially defined by their
production process (state of the herbal drug or animal
matter to be extracted, solvent, extraction conditions) and
their specifications.

PRODUCTION
Extracts are prepared by suitable methods using ethanol or
other suitable solvents. Different batches of the herbal drug
or animal matter may be blended prior to extraction. The
herbal drug or animal matter to be extracted may undergo a
preliminary treatment, for example, inactivation of enzymes,
grinding or defatting. In addition, unwanted matter may be
removed after extraction.
FIELD TRIALS
Herbal drugs, animal matter and organic solvents used
for the preparation of extracts comply with any relevant
In general, results from laboratory tests are supplemented
monograph of the Pharmacopoeia. For soft and dry extracts
with data from field trials, carried out, unless otherwise
where the organic solvent is removed by evaporation,
justified, with untreated control animals. Provided that
recovered or recycled solvent may be used, provided that the
laboratory tests have adequately assessed the safety and
recovery procedures are controlled and monitored to ensure
efficacy of a product under experimental conditions using
that solvents meet appropriate standards before re-use or
vaccines of maximum and minimum titre or potency
admixture with other approved materials. Water used for
respectively, a single batch of product could be used to
the preparation of extracts is of a suitable quality. Except
assess both safety and efficacy under field conditions. In
for the test for bacterial endotoxins, water complying with
these cases, a typical routine batch of intermediate titre or
the section on Purified water in bulk in the monograph on
potency may be used. Where laboratory trials cannot be
Purified water (0008) is suitable. Potable water may be
supportive of efficacy, the performance of field trials alone
suitable if it complies with a defined specification that allows
may be acceptable.
the consistent production of a suitable extract.
Where applicable, concentration to the intended consistency
is carried out using suitable methods, usually under reduced
pressure and at a temperature at which deterioration of the
constituents is reduced to a minimum. Essential oils that
Reference: PA/PH/Exp. 13B/T (06) 14 ANP
have been separated during processing may be restored to
the extracts at an appropriate stage in the manufacturing
NOTE ON THE GENERAL MONOGRAPH
process. Suitable excipients may be added at various stages
A section on oleoresins has been introduced, following the of the manufacturing process, for example to improve
adoption of the monograph on Capsicum oleoresin, refined technological qualities such as homogeneity or consistency.
Suitable stabilisers and antimicrobial preservatives may also
and quantified (2336).
be added.
The sections on soft extracts and dry extracts have been
modified to indicate that residual solvents are controlled as Extraction with a given solvent leads to typical proportions
described in general chapter 5.4 Residual solvents (which of characterised constituents in the extractable matter ;
is equivalent to ICH Guideline Q3C : Impurities : Residual during production of standardised and quantified extracts,
purification procedures may be applied that increase these
Solvents), unless a different limit is given in a specific
proportions with respect to the expected values ; such
monograph. This approach reflects the provisions of the
EMEA note for guidance on specifications for herbal drug extracts are referred to as refined.
preparations.
IDENTIFICATION
Please restrict comments to the section on oleoresins and
Extracts are identified using a suitable method.
the modifications regarding solvents.
430

PHARMEUROPA Vol. 18, No. 3, July 2006

Extracts

TESTS
Where applicable, as a result of analysis of the herbal drug
or animal matter used for production and in view of the
production process, tests for microbiological quality (5.1.4),
heavy metals, aflatoxins and pesticide residues (2.8.13) in
the extracts may be necessary.
ASSAY
Wherever possible, extracts are assayed by a suitable method.
LABELLING
The label states :
the herbal drug or animal matter used ;
whether the extract is liquid, soft or dry, or whether it
is a tincture ;
for standardised extracts, the content of constituents with
known therapeutic activity ;
for quantified extracts, the content of constituents
(markers) used for quantification ;
the ratio of the starting material to the genuine extract
(extract without excipients) (DER) ;
the solvent or solvents used for extraction ;
where applicable, that a fresh herbal drug or fresh animal
matter has been used ;
where applicable, that the extract is refined ;
the name and amount of any excipient used, including
stabilisers and antimicrobial preservatives ;
where applicable, the percentage of dry residue.

STORAGE
Protected from light.
LABELLING
The label states in addition to the requirements listed above :
where applicable, the ethanol content in per cent V/V in
the final extract.

Tinctures tincturae
DEFINITION
Tinctures are liquid preparations that are usually obtained
using either 1 part of herbal drug or animal matter and
10 parts of extraction solvent, or 1 part of herbal drug or
animal matter and 5 parts of extraction solvent.

PRODUCTION
Tinctures are prepared by maceration or percolation (outline
methodology is given below) using only ethanol of a suitable
concentration for extraction of the herbal drug or animal
matter, or by dissolving a soft or dry extract (which has been
produced using the same strength of extraction solvent, as
is used in preparing the tincture by direct extraction) of
the herbal drug or animal matter in ethanol of a suitable
concentration. Tinctures are filtered, if necessary.
Tinctures are usually clear. A slight sediment may form on
standing, which is acceptable as long as the composition of
the tincture is not changed significantly.
Production by maceration. Unless otherwise prescribed,
reduce the herbal drug or animal matter to be extracted to
pieces of suitable size, mix thoroughly with the prescribed
Liquid extracts extracta fluida
extraction solvent and allow to stand in a closed container
for an appropriate time. The residue is separated from the
DEFINITION
extraction solvent and, if necessary, pressed out. In the latter
Liquid extracts are liquid preparations of which, in general, case, the 2 liquids obtained are combined.
1 part by mass or volume is equivalent to 1 part by mass of the
dried herbal drug or animal matter. These preparations are Production by percolation. If necessary, reduce the herbal
adjusted, if necessary, so that they satisfy the requirements drug or animal matter to be extracted to pieces of suitable
for content of solvent, and, where applicable, for constituents. size. Mix thoroughly with a portion of the prescribed
extraction solvent and allow to stand for an appropriate time.
Transfer to a percolator and allow the percolate to flow at
PRODUCTION
room temperature slowly making sure that the herbal drug
Liquid extracts are prepared by using ethanol of a suitable
concentration or water to extract the herbal drug or animal or animal matter to be extracted is always covered with the
matter, or by dissolving a soft or dry extract (which has been remaining extraction solvent. The residue may be pressed
out and the expressed liquid combined with the percolate.
produced using the same strength of extraction solvent as
is used in preparing the liquid extract by direct extraction)
TESTS
of the herbal drug or animal matter in either ethanol of a
Relative density (2.2.5). Where applicable, the tincture
suitable concentration or water. Liquid extracts may be
complies with the limits prescribed in the monograph.
filtered, if necessary.
A slight sediment may form on standing, which is acceptable Ethanol (2.9.10). The ethanol content complies with that
prescribed.
as long as the composition of the liquid extract is not
changed significantly.
Methanol and 2-propanol (2.9.11) : maximum 0.05 per
cent V/V of methanol and maximum 0.05 per cent V/V of
TESTS
2-propanol, unless otherwise prescribed.
Relative density (2.2.5). Where applicable, the liquid extract
Dry residue (2.8.16). Where applicable, the tincture complies
complies with the limits prescribed in the monograph.
with the limits prescribed in the monograph, corrected if
Ethanol (2.9.10). For alcoholic liquid extracts, carry out
necessary, taking into account any excipient used.
the determination of ethanol content. The ethanol content
STORAGE
complies with that prescribed.
Protected from light.
Methanol and 2-propanol (2.9.11) : maximum 0.05 per
cent V/V of methanol and maximum 0.05 per cent V/V of
LABELLING
2-propanol for alcoholic liquid extracts, unless otherwise
The label states in addition to the requirements listed above :
prescribed.
for tinctures other than standardised and quantified
Dry residue (2.8.16). Where applicable, the liquid extract
tinctures, the ratio of starting material to extraction liquid
complies with the limits prescribed in the monograph,
or of starting material to final tincture ;
corrected if necessary, taking into account any excipient
used.
the ethanol content in per cent V/V in the final tincture.
PHARMEUROPA Vol. 18, No. 3, July 2006

431

Fluvoxamine maleate

Soft extracts extracta spissa

Reference: PA/PH/Exp. 10B/T (05) 4 ANP

XXXX:1977

DEFINITION
Soft extracts are semi-solid preparations obtained by
evaporation or partial evaporation of the solvent used for
extraction.

FLUVOXAMINE MALEATE
Fluvoxamini maleas

TESTS
Dry residue (2.8.16). The soft extract complies with the
limits prescribed in the monograph.
Solvents. Where applicable, a monograph on a soft extract
prescribes a limit test for the solvent used for extraction.
Residual solvents are controlled as described in chapter 5.4,
unless otherwise prescribed or justified and authorised.
STORAGE
Protected from light.

Oleoresins oleoresina
DEFINITION
Oleoresins are mobile semi-solid preparations composed
of a resin in solution in an essential and/or fixed oil and
are obtained by evaporation of the solvent(s) used for their
production.

C19H25F3N2O6

Mr 434.4

DEFINITION
5-Methoxy-1-[4-(trifluoromethyl)phenyl]pentan-1-one-(E)-O(2-aminoethyl)oxime (Z)-butenedioate.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : sparingly soluble in water, freely soluble in
ethanol (96 per cent) and in methanol.

IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
TESTS
Comparison : fluvoxamine maleate CRS.
B. Thin-layer chromatography (2.2.27).
Water (2.2.13). The oleoresin complies with the limits
prescribed in the monograph.
Test solution. Dissolve 50 mg of the substance to be
examined in methanol R and dilute to 5 ml with the same
Solvents. Residual solvents are controlled as described in
solvent.
chapter 5.4, unless otherwise prescribed or justified and
authorised.
Reference solution. Dissolve 50 mg of fluvoxamine
maleate CRS in methanol R and dilute to 5 ml with the
same solvent.
STORAGE
Plate : TLC silica gel F254 plate R.
In a well-filled, airtight container, protected from light at a
Mobile phase : concentrated ammonia R, ethanol (96 per
temperature not exceeding 25 C.
cent) R (5:95 V/V).
Application : 5 l.
Dry extracts extracta sicca
Development : over 2/3 of the plate.
Drying : in air.
DEFINITION
Detection : examine in ultraviolet light at 254 nm.
Dry extracts are solid preparations obtained by evaporation
Results : the 2 principal spots in the chromatogram
of the solvent used for their production. Dry extracts usually
obtained with the test solution are similar in position and
have a loss on drying or a water content of not greater than
size to the principal spots in the chromatogram obtained
5 per cent m/m.
with the reference solution.
TESTS
Water (2.2.13). Where applicable, the dry extract complies
with the limits prescribed in the monograph.
Loss on drying (2.8.17). Where applicable, the dry extract
complies with the limits prescribed in the monograph.
Solvents. Where applicable, a monograph on a dry extract
prescribes a limit test for the solvent used for extraction.
Residual solvents are controlled as described in chapter 5.4,
unless otherwise prescribed or justified and authorised.
STORAGE
In an airtight container, protected from light.
432

TESTS
Related substances. Liquid chromatography (2.2.29).
Prepare the test solution immediately before use.
Test solution. Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 25.0 ml with the
mobile phase.
Reference solution (a). Dilute 1.0 ml of the test solution to
10.0 ml with the mobile phase. Dilute 1.0 ml of this solution
to 100.0 ml with the mobile phase.
Reference solution (b). Dissolve 10 mg of fluvoxamine
maleate for system suitability CRS (containing impurities A,
B, C, D and F) in the mobile phase and dilute to 5.0 ml with
the mobile phase.
PHARMEUROPA Vol. 18, No. 3, July 2006

Fluvoxamine maleate

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. maleic acid

3. impurity C

5. fluvoxamine

2. impurities F and G

4. impurity B

6. impurity A

7. impurity D

Figure 1977.-1. Chromatogram for the test for related substances of fluvoxamine maleate : solution spiked with
impurities A, B, C, D, F and G
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase: octylsilyl silica gel for
chromatography R (5 m)(24).
Mobile phase : mix 370 volumes of acetonitrile R and
630 volumes of a pH 3 buffer solution prepared as follows :
dissolve 0.7 g of potassium dihydrogen phosphate R and
1.2 g of sodium pentanesulphonate R in 630 ml of water R
and adjust to pH 3 with phosphoric acid R.
Flow rate : 1.2 ml/min.
Detection : spectrophotometer at 234 nm.
Injection: 20 l.
Run time : 5.5 times the retention time of fluvoxamine.
Relative retention with reference to fluvoxamine (retention
time = about 15 min) : impurities F and G = about 0.5 ;
impurity C = about 0.6 ; impurity B = about 0.8 ;
impurity A = about 2.5 ; impurity D = about 5.4.
System suitability : reference solution (b) :
resolution : minimum 2.0 between the peaks due to
impurities F and C.
Limits :
correction factor : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity A = 0.7 ;
impurity D = 0.3 ;
impurities B, C : for each impurity, not more than 5 times
the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.5 per cent) ;
impurities A, D : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent) ;

sum of impurities F and G : not more than twice the area

of the principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent) ;
unspecified impurities : for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.10 per cent) ;
total : not more than 8 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.8 per cent) ;
disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent) ; disregard the peak due to maleic acid.
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test C. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in a desiccator over diphosphorus
pentoxide R at a pressure not exceeding 0.7 kPa for 2 h.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g in a platinum crucible.
ASSAY
Dissolve 0.350 g in 50 ml of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1 ml of 0.1 M perchloric acid is equivalent to 43.44 mg of
C19H25F3N2O6.
IMPURITIES
Specified impurities : A, B, C, D, F, G.
Other detectable impurities (the following substances
would, if present at a sufficient level, be detected by one
or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified

(24) Kromasil 100 C-8 is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

433

5.15. Functionality-related characteristics of excipients

impurities and/or by the general monograph Substances for

pharmaceutical use (2034). It is therefore not necessary to
identify these impurities for demonstration of compliance.
See also 5.10. Control of impurities in substances for
pharmaceutical use) : E, I.

A. R1 = R2 = H, R3 = CF3 : 1-[4-(trifluoromethyl)phenyl]pentan-1-one-(E)-O-(2-aminoethyl)oxime,
E. R1 = H, R2 = OCH3, R3 = CHF2 : 1-[4-(difluoromethyl)phenyl]-5-methoxypentan-1-one-(E)-O-(2aminoethyl)oxime,
F. R1 = CH2-CH2-NH2, R2 = OCH3, R3 = CF3 :
5-methoxy-1-[4-(trifluoromethyl)phenyl]pentan-1one-(E)-O-[2-[(2-aminoethyl)amino]ethyl]oxime,
G. R1 = H, R2 = OH, R3 = CF3 : 5-hydroxy-1-[4(trifluoromethyl)phenyl]pentan-1-one-(E)-O-(2aminoethyl)oxime,

B. R = CH2-CH2-NH2 : 5-methoxy-1-[4-(trifluoromethyl)phenyl]pentan-1-one-(Z)-O-(2-aminoethyl)oxime,
I. R = H : 5-methoxy-1-[4-(trifluoromethyl)phenyl]pentan-1-one-(E)-O-(2-hydroxy)oxime

C. (2RS)-2-[[2-[[[(E)-5-methoxy-1-[4-(trifluoromethyl)phenyl]pentylidene]amino]oxy]ethyl]amino]butanedioic
acid,

function of an excipient is to guarantee the required physical

and biopharmaceutical properties of the pharmaceutical
preparation.
The functionality of an excipient is determined by its physical
and chemical properties and, in some cases, also by its
content of by-products or of additives used to improve the
intended functionality. In addition, the functionality may
depend on complex interactions between the ingredients
of the formulation and processing stresses. Excipient
functionality can therefore be evaluated only in the context
of a particular formulation and manufacturing process,
frequently by the use of a multitude of analytical methods.
Enhanced knowledge of excipient functionalities facilitates
the application of Process Analytical Technology (PAT).
Certain excipient properties, such as the particle size of an
excipient intended for a solid dosage form or the molecular
mass of a polymeric material used as a viscosity-increasing
agent, may however relate to functionality in a more general
sense. Such functionality-related characteristics (FRCs) can
be controlled and may be subject to a product-specific quality
specification when the pharmaceutical development work
has demonstrated their critical role for the manufacturing
process and quality attributes of the final preparation.
Monographs of the European Pharmacopoeia on excipients
are designed to ensure acceptable quality for users.
Information on the appearance and characters of the
excipient, and requirements concerning identity, chemical
and microbiological purity and physical characteristics
associated with the chemical structure, such as optical
rotation, are given in specific monographs and in the general
monograph Substances for pharmaceutical use (2034).
FRCs are listed in excipient monographs to aid manufacturers
of pharmaceutical products in establishing specifications
based on standard analytical methods that have been
independently assessed. They provide manufacturers and
users of excipients with a common language to support
the supply of excipients with specified properties. FRCs
may be labelled (in the certificate of analysis, for example)
by the excipient manufacturer with a reference to the
Pharmacopoeia monograph, thus indicating the method
used to test a particular characteristic. The FRC section
in specific monographs contains FRCs that are known to
have an impact on the functionality of the excipient for
the stated uses. The uses and the FRCs listed may not be
exhaustive because of the multiple uses of many excipients
and development of new uses.

REGULATORY GUIDANCE
According to current regulatory guidelines, e.g. ICH Q8
Pharmaceutical Development, the marketing authorisation
application should discuss the excipients chosen and
their concentration, and demonstrate the characteristics
that can influence the final preparation performance and
D. 5-methoxy-1-[4-(trifluoromethyl)phenyl]pentan-1-one.
manufacturability relative to the respective function of each
excipient. The ability of excipients to provide their intended
functionality throughout the intended shelf-life of the
formulation should also be demonstrated. The information
Reference: PA/PH/Exp. FRC/T (05) 2 ANP
on excipient performance can be used as appropriate to
justify the choice and quality attributes of the excipient.
XXXX:51500
Excipients are normally produced by batch processes, so
there is a possibility of batch-to-batch variation from the
5.15. FUNCTIONALITY-RELATED
same manufacturer. Excipients from different sources may
not have identical properties with respect to their use in a
CHARACTERISTICS OF EXCIPIENTS
specific formulation. The inevitable variation in chemical
PREAMBLE
and physical properties is one of the most important input
Excipients that have been appropriately evaluated for safety variables that can impact on a pharmaceutical manufacturing
are used in the formulation of pharmaceutical preparations process, since excipients typically make up the major
proportion of a final preparation. Many excipients are of
to bring functionality to the formulation. The intended
434

PHARMEUROPA Vol. 18, No. 3, July 2006

5.15. Functionality-related characteristics of excipients

CHEMICAL GRADES
Excipients that are available in different chemical grades are
of natural, semi-synthetic or synthetic origin. The specific
monograph usually controls the chemical composition
of excipients that are composed of a mixture of related
compounds, for example, the composition of fatty acids
in vegetable oils or surfactants. There are, however,
specific monographs in the Pharmacopoeia each describing
a class of polymeric materials that may vary in their
The key to a successful, robust formulation is to understand composition with regard to the structure of homopolymers
and block-copolymers, the degree of polymerisation,
the chemical and physical nature of the active substance(s)
and thus the molecular mass and mass distribution, the
and the excipients alone and how their properties
degree of substitution and in some cases even different
interact with other constituents of the formulation and
substituents on the polymer backbone. They are combined
the manufacturing process. During pharmaceutical
into one monograph frequently referred to as a family
development, the ingredient properties that are critical to
monograph when there is no health risk associated with
the manufacturing process and performance of the final
the chemical property variation as such. This variation may,
preparation are identified. Having identified the critical
however, have a profound effect on the functionality of the
properties of the excipients, preferably on a risk-based
excipient and should be subject to investigations during the
approach, pharmaceutical development may establish the
pharmaceutical development, preferably to establish the
acceptable range of the critical characteristics including
both the physical and chemical property variation. The FRCs acceptable range of each characteristic being critical to the
concerned may not be properties controlled by the excipient manufacturing process and performance of the end-product.
While, in the past, the mandatory part of monographs on
manufacturer and are therefore variable. The design of a
robust process that limits the effect of the normal excipient polymeric excipients may have contained some tests for
physical or chemical characteristics, for example, a test
variability is preferable.
for viscosity including acceptance criteria, such tests will
gradually be moved to the non-mandatory FRC section,
PHYSICAL GRADES
unless the concerned characteristic is an indispensable
part of the identification tests. This development should
Excipients that are particulate solids can be available in
be seen in light of regulatory guidance on pharmaceutical
a variety of physical grades, for example with regard to
particle-size distribution, which is usually controlled by the development and the desired regulatory flexibility based
excipient supplier. However, FRCs for solids may concern a on establishing the acceptable range of material properties
wide range of properties, resulting from solid-state properties within the design space. Thus, evaluation of the chemical
grades and, when appropriate, the setting of a specification
and bulk properties of the particulate solid, which may not
for the critical characteristics, is a part of the pharmaceutical
be controlled by the excipient supplier.
development irrespective of the non-mandatory character of
Examples of solid-state properties to be considered
FRCs.
in the development of solid dosage forms include
FUNCTIONALITY-RELATED CHARACTERISTICS
polymorphism, pseudopolymorphism, crystallinity and
SECTION IN MONOGRAPHS
density. Complimentary techniques to study crystalline
forms and solvates are given in the general chapters :
Monographs on excipients may have a section entitled
Functionality-related characteristics. This section is
5.9. Polymorphism ;
included for information for the user and is not a mandatory
part of the monograph. The section gives a statement of
2.2.34. Thermal analysis ;
characteristics that are known to be relevant for certain
2.9.33. Characterisation of crystalline and partially
uses of the excipient. The use for which the characteristic
crystalline solids by XRPD ;
is relevant is stated. For other uses, the characteristic
may be irrelevant. For this reason, the section should not
2.2.42. Density of solids ;
be seen simply as a supplement to the monograph. It is
the responsibility of the pharmaceutical manufacturer to
2.9.23. Pycnometric density of solids.
decide how the information on FRCs will be applied in the
Examples of bulk properties of particulate solids include
manufacturing process in light of the use of the excipient
particle-size distribution, specific surface area, bulk density, and data from pharmaceutical development.
flowability, wettability and water sorption. Depending
The information on the functionality-related characteristics
on the size range, the particle size distribution can be
may be given in different ways :
determined by sieve analysis (see general chapter on
name of the FRC ;
2.9.38. Particle-size distribution estimation by analytical
name of the FRC and a recommended method for its
sieving) or instrumental methods, for example, 2.9.31.
determination, referring wherever possible to a general
Particle-size analysis by laser light diffraction. 2.9.26.
chapter of the Pharmacopoeia ;
Specific surface area by gas adsorption is based on the
BET-technique. Methods to characterise flowability and bulk name of the FRC with a recommended method for its
determination and typical acceptance criteria, which may
density of powders are described in the general chapters on
be in the form of tolerances for the nominal value.
2.9.36. Powder flow and 2.9.34. Bulk and tapped density.
A given characteristic may be the subject of a mandatory
Solid-state properties may impact on the wettability and
requirement in the monograph and also mentioned in
water-solid interactions of particulate solids. A range of
the FRC section. The degree of polymerisation is used in
instrumental methods is available for determining these
characteristics, for example, techniques to measure the static the mandatory Identification section of the monographs
on microcrystalline cellulose and powdered cellulose to
and dynamic contact angles and the gravimetric vapour
distinguish the two types. The degree of polymerisation
sorption.
natural origin and composed of a mixture of chemically
related compounds. Other excipients are made in chemical
plants primarily designed for producing chemicals for
industries other than the pharmaceutical industry. The
excipient manufacturers process capability therefore may be
focused on the chemical characteristics and some physical
properties addressing the manufacturers primary market.
In many cases, the excipient manufacturer has limited
knowledge of the pharmaceutical uses of the product.

PHARMEUROPA Vol. 18, No. 3, July 2006

435

Ginkgo dry extract, purified and standardised

of microcrystalline cellulose is not greater than 350,

whereas that of powdered cellulose is 440 to 2250. The
actual degree of polymerisation is relevant for certain uses
and it is therefore also cited as a relevant FRC that the
pharmaceutical manufacturer may choose to specify for the
grade used for a particular pharmaceutical preparation.

Reference: PA/PH/Exp. 13A/T (06) 13 ANP

NOTE ON THE MONOGRAPH

As decided by the European Pharmacopoeia Commission,
the monograph contains a determination of ginkgolic acids
with a limit of 5 ppm. The elaboration of a monograph on
Ginkgo dry extract, standardised, non-refined has been
The section on FRCs is intended to reflect current knowledge initiated as well.
related to the major uses of an excipient. In view of the
XXXX:1827
multiple uses of some excipients and the continuous
development of new uses, the section may not be complete.
GINKGO DRY EXTRACT, PURIFIED
In addition, the methods cited for the determination of a
AND STANDARDISED
particular characteristic are given as recommendations for
methods that are known to be satisfactory for the purpose,
but the use of other methods is not excluded.
Ginkgois extractum siccum purificatum et

normatum

INTERNATIONAL HARMONISATION
A number of excipient monographs are subject to
international harmonisation among the European, Japanese
and United States Pharmacopoeias ; see chapter 5.8.
Pharmacopoeial harmonisation. Introduction of the FRC
section in the monographs of the European Pharmacopoeia
means that the presentation of harmonised monographs
differs. Tests for physical and chemical characteristics
regarded as functionality-related in the European
Pharmacopoeia are, in the two other pharmacopoeias,
included in the body of the monograph. The different format
has no implications on the pharmaceutical manufacturers
specification of excipient characteristics. Current regulatory
guidance recommends the identification and specification of
only such critical properties that impact the manufacturing
process and performance of the end-product. The different
legal environments of the three pharmacopoeias allow for
different formats of the monographs without affecting the
international harmonisation status.

DEFINITION
Purified and standardised dry extract produced from Ginkgo
leaf (1828).
Content :
flavonoids, expressed as flavone glycosides (Mr 756.7) :
22.0 per cent to 27.0 per cent (dried extract) ;
bilobalide : 2.6 per cent to 3.2 per cent (dried extract) ;
ginkgolides A, B and C : 2.8 per cent to 3.4 per cent
(dried extract) ;
ginkgolic acids : maximum 5 ppm (dried extract).
PRODUCTION
Purified and standardised ginkgo dry extract is produced
from the herbal drug by an appropriate procedure using one
or more of the following solvents : acetone or its mixture
with water, butanol, hexane, dichloromethane.
CHARACTERS
Bright yellow-brown powder or friable mass.

IDENTIFICATION
Thin-layer chromatography (2.2.27).
Critical characteristic : any physical or chemical material
Test solution. Dissolve 20.0 mg of the extract to be examined
characteristic that has been demonstrated to impact
in 10 ml of a mixture of 2 volumes of water R and 8 volumes
significantly on the manufacturability and/or performance
of methanol R.
of the final preparation.
Reference solution. Dissolve 1.0 mg of chlorogenic acid R
Design space : the multidimensional combination and
and 3.0 mg of rutin R in 20 ml of methanol R.
interaction of input variables (e.g. material attributes) and
Plate
: TLC silica gel plate R.
process parameters that have been demonstrated to provide
Mobile
phase : anhydrous formic acid R, glacial acetic
assurance of quality.
acid R, water R, ethyl acetate R (7.5:7.5:17.5:67.5 V/V/V/V).
Functionality testing : the direct testing of the concerned
Application : 20 l, as bands.
function of an excipient in a particular formulation and
Development
: over a path of 17 cm.
manufacturing process to verify that the excipient provides
Drying : at 100-105 C.
the intended functionality.
Functionality-related characteristic : a controllable physical Detection : spray the warm plate with a 10 g/l solution of
diphenylboric acid aminoethyl ester R in methanol R,
or chemical characteristic of an excipient that is shown
then spray with a 50 g/l solution of macrogol 400 R in
to impact on its functionality, but in itself is without any
methanol R ; allow to dry in air for about 30 min and examine
associated health risk.
in ultraviolet light at 365 nm.
Performance tests: analytical tests on the critical properties Results : see below the sequence of the zones present in the
of a pharmaceutical preparation.
chromatograms obtained with the reference solution and
Process robustness : ability of a process to tolerate variability the test solution. Furthermore, other, weaker fluorescent
of materials and changes of the process and equipment
zones may be present in the chromatogram obtained with
without negative impact on quality.
the test solution.
GLOSSARY

436

PHARMEUROPA Vol. 18, No. 3, July 2006

Ginkgo dry extract, purified and standardised

Top of the plate

A blue fluorescent zone
Several faint coloured zones
_______

_______
A yellow-brown fluorescent zone
A green fluorescent zone
Two yellow-brown fluorescent
zones
An intense light blue fluorescent
zone sometimes overlapped by a
greenish-brown fluorescent zone

Chlorogenic acid : a light blue

fluorescent zone
A Two green fluorescent zones
Rutine : a yellowish-brown
fluorescent zone
_______

Two One or two yellowish-brown

fluorescent zones
_______
A green fluorescent zone Several
green and yellowish-brown
fluorescent zones

Reference solution

A yellowish-brown fluorescent
zone
Test solution

stopper and secure with an aluminium crimped cap. Heat on

a water-bath for 25 min. Allow to cool to 20 C.
Reference solution. Dissolve 10.0 mg of quercetin
dihydrate CRS in 20 ml of methanol R. Add 15.0 ml of
dilute hydrochloric acid R and 5 ml of water R and dilute
to 50.0 ml with methanol R.
Column :
size : l = 0.125 m, = 4 mm ;
stationary phase : octadecylsilyl silica gel for
chromatography R(25) (5 m) ;
temperature : 25 C.
Mobile phase :
mobile phase A : 0.3 g/l solution of phosphoric acid R
adjusted to pH 2.0 ;
mobile phase B : methanol R ;
Time
(min)
0-1

Mobile phase A
(per cent V/V)
60

Mobile phase B
(per cent V/V)
40

1 - 20

60 45

40 55

20 - 21

45 0

55 100

21 - 25

100

Flow rate : 1.0 ml/min.

Detector: spectrophotometer at 370 nm.
Injection : 10 l.
ASSAY
Relative retention with reference to quercetin
Flavonoids. Liquid chromatography (2.2.29).
(retention time = about 12.5 min) : peak A = about 0.4 ;
Test solution. Dissolve 0.200 g of the extract to be examined kaempferol = about 1.4 ; isorhamnetin = about 1.5.
in 20 ml of methanol R. Add 15.0 ml of dilute hydrochloric
System suitability : test solution :
acid R and 5 ml of water R and dilute to 50.0 ml with
methanol R. Transfer 10 ml of this solution into a 10 ml
resolution : minimum 1.5 between the peaks due to
brown-glass vial. Close the vial with a tight rubber membrane
kaempferol and isorhamnetin.
TESTS
Loss on drying (2.8.17) : maximum 5.0 per cent.

1. quercetin

2. kaempferol

3. isorhamnetin

Figure 1827.-1. Chromatogram for the assay of flavonoids in purified and standardised ginkgo dry extract
(25) Lichrospher 100 RP18 is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

437

Ginkgo dry extract, purified and standardised

each of 5 ml, of phosphate buffer solution pH 5.8 R and

transfer the washings to the chromatography column. Allow
to stand for 15 min. Elute with 100 ml of ethyl acetate R.
Evaporate the eluate to dryness at a pressure not exceeding
4 kPa in a water-bath at 50 C. The residue of solvent is
eliminated by an air-current. Dissolve the residue in 2.5 ml
Calculate the percentage content of flavonoids, expressed as of the mobile phase.
flavone glycosides, using the following expression :
Reference solution. Dissolve 30.0 mg of benzyl alcohol R
in the mobile phase and dilute to 100.0 ml with the same
solvent.
Determine the area of the peak due to quercetin in the
chromatogram obtained with the reference solution.
Determine the sum of the areas including all the peaks from
peak A to isorhamnetin in the chromatogram obtained with
the test solution (see Figure 1827.-1).

Column :
size : l = 0.25 m, = 4 mm ;
F1

F2

m1

m2

sum of the areas of all the considered peaks in the

chromatogram obtained with the test solution ;
area of the peak due to quercetin in the
chromatogram obtained with the reference
solution ;
mass of quercetin dihydrate CRS in the reference
solution, in grams ;
mass of the extract to be examined in the test
solution, in grams ;
percentage content of anhydrous quercetin in the
quercetin dihydrate CRS.

Terpene lactones. Liquid chromatography (2.2.29).

Test solution. Place 0.120 g of the extract to be examined
in a 25 ml beaker and dissolve it in 10 ml of phosphate
buffer solution pH 5.8 R by stirring. Transfer the solution
to a chromatography column, about 0.15 m long and about
30 mm in internal diameter, containing 15 g of kieselguhr
for chromatography R. Rinse the beaker with 2 quantities,

1. ginkgolide C

2. bilobalide

stationary phase : octylsilyl silica gel for

chromatography R(26) (5 m) ;
temperature : 20 C.
Mobile phase : tetrahydrofuran R, methanol R, water R
(10:20:75 V/V/V).
Flow-rate : 1.0 ml/min.
Detection : refractometer maintained at 35 C.
Injection volume : 100 l.
System suitability :
relative retention with reference to benzyl alcohol
(retention time between 5 min and 12 min) :
ginkgolide C = about 1.0 ; bilobalide = about 1.3 ;
ginkgolide A = about 1.8 ; ginkgolide B = about 2.3.
Determine the area of the peak due to benzyl alcohol in the
chromatogram obtained with the reference solution. Locate
the peaks due to bilobalide and ginkgolides A, B and C by
comparison with the chromatogram in Figure 1827.-2.

3. ginkgolide A

4. ginkgolide B

Figure 1827.-2 Chromatogram for the assay of terpene lactones in purified and standardised ginkgo dry extract

(26) Lichrospher 100 RP8 is suitable.

438

PHARMEUROPA Vol. 18, No. 3, July 2006

Ginkgo dry extract, purified and standardised

Calculate the percentage content of bilobalide, using the

following expression :

Calculate the percentage content of ginkgolide A, using the

following expression :

Calculate the percentage content of ginkgolide B, using the

following expression :

Calculate the percentage content of ginkgolide C, using the

following expression :

F1

F2

F3

F4

F5

area of the peak due to bilobalide in the

chromatogram obtained with the test solution ;
area of the peak due to ginkgolide A in the
chromatogram obtained with the test solution ;
area of the peak due to ginkgolide B in the
chromatogram obtained with the test solution ;
area of the peak due to ginkgolide C in the
chromatogram obtained with the test solution ;
area of the peak due to benzyl alcohol in the
chromatogram obtained with the reference
solution ;

m1

GA

percentage content of ginkgolide A ;

GB

percentage content of ginkgolide B ;

GC

percentage content of ginkgolide C.

mass of benzyl alcohol used to prepare the

reference solution, in grams ;
m2 = mass of the extract to be examined in the test
solution, in grams ;
p
= percentage content of benzyl alcohol in benzyl
alcohol R.
Calculate the percentage content of the sum of ginkgolides
A, B and C, using the following expression :

Ginkgolic acids. Liquid chromatography (2.2.29).

Test solution. Dissolve 500.0 mg of the powdered extract to
be examined in 8 ml of methanol R, sonicating if necessary,
and dilute to 10.0 ml with the same solvent. Centrifuge if
necessary.
Reference solution. Dissolve 10.0 mg of ginkgolic acids CRS
in 8 ml of methanol R, sonicating if necessary, and dilute to
10.0 ml with the same solvent. Dilute 2.0 ml of this solution
to 10.0 ml with methanol R.
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase : octylsilyl silica gel for
chromatography R (5 m)(27) ;
temperature : 35 C.
Mobile phase :
mobile phase A : dilute 0.1 ml of trifluoroacetic acid R
to 1000 ml with water R ;
mobile phase B : dilute 0.1 ml of trifluoroacetic acid R to
1000 ml with acetonitrile R ;

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. ginkgolic acid C13

2. ginkgolic acid C15

3. ginkgolic acid C17

Figure 1827.-3. Chromatogram for the assay of ginkgolic acids in purified and standardised ginkgo dry extract
(27) Zorbax Eclipse XDB-C8 or Phenomenex Kromasil C8 are suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

439

Ginseng dry extract

Time
(min)
0 - 30

Mobile phase A
(per cent V/V)
25 10

Mobile phase B
(per cent V/V)
75 90

30 - 35

10

90

35 - 36

10 25

90 75

36 - 45

25

75

Flow rate : 1.0 ml/min.

Detection : spectrophotometer at 210 nm.
Injection : 50 l.
Identification of components : use the chromatogram
supplied with ginkgolic acids CRS and the chromatogram
obtained with the test solution to identify the peaks due to
ginkgolic acids C13, C15 and C17.
System suitability : reference solution :

Calculate the total content of ginkgolic acids in purified

and standardised ginkgo dry extract, using the following
expression :

TC13 =
TC15 =
TC17 =

content of ginkgolic acid C13 in purified and

standardised ginkgo dry extract, in parts per
million ;
content of ginkgolic acid C15 in purified and
standardised ginkgo dry extract, in parts per
million ;
content of ginkgolic acid C17 in purified and
standardised ginkgo dry extract, in parts per
million.

Reference: PA/PH/Exp. 13A/T (04) 45 ANP 1R

NOTE ON THE MONOGRAPH

Definition : following the comments received after
publication in Pharmeuropa 17.1, the minimum content of
combined ginsenosides Rg1 and Rb1 has been lowered to
symmetry factor : 0.8 to 2.0 for the peaks due to ginkgolic 1.00 per cent to cover additional products on the European
acids C13, C15 and C17.
market.
Production : since it was shown that the extraction ratio
Calculate the mean response factor (RFi) of each ginkgolic
between ginsenoside Rb1 and ginsenoside Rg1 is almost
acid (i) (C13, C15 and C17) in the reference solution, using
independent of the strength of the hydroalcoholic solvent,
the following expression :
the allowed range of hydroalcoholic solvents has been
enlarged.
Assay : for the preparation of the test solution, a solid-phase
extraction has been introduced to eliminate interfering
peaks.
XXXX:2356
resolution : minimum 1.0 between the peaks due to
ginkgolic acid C13 and ginkgolic acid C15 ;

GINSENG DRY EXTRACT

Ai

Ci

area of the peak due to ginkgolic acid i in the

chromatogram obtained with the test solution ;
percentage content of ginkgolic acid i in the
reference solution ;
concentration of the reference solution, in
micrograms per millilitre.

Calculate the content in parts per million of each ginkgolic

acid (i) (C13, C15 and C17) in purified and standardised
ginkgo dry extract, using the following expression :

Ginseng extractum siccum

DEFINITION
Dry extract produced from Ginseng (1523).
Content : minimum 4.00 1.00 per cent of combined
ginsenosides Rg1 (C42H72O14,2H2O ; Mr 837) and Rb1
(C54H92O23,3H2O ; Mr 1163) (dried extract).
PRODUCTION
The dry extract is produced from the drug by a suitable
procedure using a hydroalcoholic solvent equivalent in
strength to between 45 35 per cent V/V and 65 90 per
cent V/V ethanol.
CHARACTERS
Appearance : light brownish-yellow, hygroscopic powder or
brittle mass.

= area of the peak due to ginkgolic acid i in

the chromatogram obtained with the test
solution ;
= response factor of ginkgolic acid i ;
RFi
= concentration of purified and standardised
C
ginkgo dry extract, in micrograms per
millilitre,
1000 000 = conversion factor to parts per million.
Ai

440

IDENTIFICATION
Thin-layer chromatography (2.2.27).
Test solution. Dissolve 10.0 mg 0.3 g of the extract to
be examined in 10 ml of a 70 per cent V/V solution of
methanol R.
Reference solution. Dissolve 5.0 mg 5 mg of arbutin R and
5.0 mg 5 mg of aescin R in 1 ml of methanol R.
Plate : TLC silica gel plate R (5-40 m) [or TLC silica gel
plate R (2-10 m)].
PHARMEUROPA Vol. 18, No. 3, July 2006

Ginseng dry extract

Mobile phase : ethyl acetate R, water R, butanol R

(25:50:100 V/V/V) ; allow the phases to stand separate for
10 min, and use the upper layer.
Application : 20 l [or 4 l] as bands.
Development : over a path of 10 cm [or 5 cm] in an
unsaturated tank.
Drying : in air.
Detection : spray with anisaldehyde solution R and heat at
105-110 C for 5-10 min. Examine in daylight.
Results : see below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution.
Top of the plate

Reference solution. Dissolve 3.0 1.0 mg of ginsenoside

Rg1 R, 3.0 1.0 mg of ginsenoside Re R, 3.0 1.0 mg of
ginsenoside Rf R and 3.0 1.0 mg of ginsenoside Rb1 R in
methanol R and dilute to 10.0 25.0 ml with the same solvent.
Column :
size : l = 0.125 m, = 4.6 mm ;
stationary phase : octadecylsilyl silica gel for
chromatography R (5 m)(29) ;
temperature : 35 C.
Mobile phase :
mobile phase A : water R adjusted to pH 2 with
phosphoric acid R ;
mobile phase B : acetonitrile R ;
Time
(min)
0-8

Arbutin : a brown zone

_______

_______

Mobile phase B
(per cent V/V)
20

A violet zone
(ginsenoside Rg1 + Rg2)
A pale violet zone (ginsenoside Rf)

8 - 40

80 60

20 40

40 - 45

60 40

40 60

45 - 47

40 0

60 100

A violet zone (ginsenoside Re)

47 - 52

100

52 - 55

0 80

100 20

A violet zone (ginsenoside Rd)

A pale violet zone
_______

_______
A violet zone (ginsenoside Rc)

Aescin : a grey zone

Mobile phase A
(per cent V/V)
80

A violet zone
(ginsenoside Rb1 + Rb2)

Reference solution

Test solution

TESTS
Loss on drying (2.8.17) : maximum 5.0 7.0 per cent.
ASSAY
Liquid chromatography (2.2.29).
Buffer solution. Dissolve 3.5 g of sodium dihydrogen
phosphate R and 7.2 g of potassium dihydrogen phosphate R
in water R and dilute to 1000 ml with the same solvent.
Test solution. In a 150 ml round-bottomed flask dissolve
10.0 mg of the extract to be examined in 20.0 ml of a
mixture of 20 volumes of acetonitrile R and 80 volumes
of water R. Dilute 2.0 ml of this solution to 10.0 ml with a
mixture of 20 volumes of acetonitrile R and 80 volumes
of water R. Filter through an appropriate membrane
filter (0.45 m). Dissolve 0.1000 g in the buffer solution
and dilute to 10.0 ml with the same solution. Prepare a
ready-to-use sample-preparation cartridge containing 500 mg
of octadecylsilyl silica gel (45 m)(28), using a mixture of 5 ml
of methanol R and 20 ml of water R. Apply 5.0 ml of the
solution to be analysed to the top of the cartridge. Wash the
cartridge with 20 ml of water R and then 15 ml of a 30 per
cent V/V solution of methanol R. Elute the cartridge with
20 ml of methanol R ; collect the filtrate. Under reduced
pressure, evaporate the filtrate to dryness. Dissolve the dry
residue in 2.0 ml of methanol R. Filter through a suitable
membrane filter (0.45 m) before injection.

Flow rate : 1.0 ml/min.

Detection : spectrophotometer at 203 nm.
Injection : 20 l.
Elution order : ginsenoside Rg1, ginsenoside Re,
ginsenoside Rf, ginsenoside Rb1.
System suitability : reference solution :
resolution : minimum 1.0 between the peaks due to
ginsenosides Rg1 and Re.
Locate the peaks due to ginsenosides Rb1 and Rg1.
Calculate the percentage content of ginsenosides Rb1
and Rg1, using the following expression :

A1

A2

A3

A4

m1

m2

m3

p1

mass of ginsenoside Rb1 in the reference solution,

in milligrams ;
mass of ginsenoside Rg1 in the reference solution,
in milligrams,
declared content of ginsenoside Rb1 R,

p2

declared content of ginsenoside Rg1 R.

area of the peak due to ginsenoside Rb1 in the

chromatogram obtained with the test solution ;
area of the peak due to ginsenoside Rg1 in the
chromatogram obtained with the test solution ;
area of the peak due to ginsenoside Rb1 in
the chromatogram obtained with the reference
solution ;
area of the peak due to ginsenoside Rg1 in
the chromatogram obtained with the reference
solution ;
mass of the dried test sample, in grams ;

(28) Supelco cartridge 57054 is suitable.

(29) LiChrosphere RP-18 and Hypersil Hypurity are suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

441

Ibuprofen

Reference: PA/PH/Exp. 10B/T (06) 21 ANP

NOTE ON THE MONOGRAPH
Related substances:
a CRS for system suitability and for the identification of
specified impurities J and N has been added ;
in view of the high purity of the batches on the market,
the limits have been tightened considerably ; given
the maximum daily dose of > 2 g/day, the limits for
unspecified impurities and the disregard limit were
adapted to the requirements of the general monograph
Substances for Pharmaceutical Use (2034) ;
impurities A to E have been classified as other detectable
impurities and are now covered by the limit for
unspecified impurities.
XXXX:0721

IBUPROFEN

Reference solution. Dissolve 50 mg of ibuprofen CRS in

methylene chloride R and dilute to 10 ml with the same
solvent.
Plate : TLC silica gel plate R.
Mobile phase : anhydrous acetic acid R, ethyl acetate R,
hexane R (5:24:71 V/V/V).
Application : 5 l.
Development : over a path of 10 cm.
Drying : at 120 C for 30 min.
Detection : lightly spray with a 10 g/l solution of
potassium permanganate R in dilute sulphuric acid R
and heat at 120 C for 20 min ; examine in ultraviolet
light at 365 nm.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with the reference solution.

TESTS
Solution S. Dissolve 2.0 g in methanol R and dilute to 20 ml
with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Optical rotation (2.2.7) : 0.05 to + 0.05.
Dissolve 0.50 g in methanol R and dilute to 20.0 ml with
C13H18O2
Mr 206.3 the same solvent.
Related substances. Liquid chromatography (2.2.29).
DEFINITION
Test solution. Dissolve 20 mg of the substance to be
(2RS)-2-[4-(2-Methylpropyl)phenyl]propanoic acid.
examined in 2 ml of acetonitrile R and dilute to 10.0 ml with
Content : 98.5 per cent to 101.0 per cent (dried substance).
mobile phase A.
CHARACTERS
Reference solution (a). Dilute 1.0 ml of the test solution to
Appearance : white or almost white, crystalline powder or
100.0 ml with mobile phase A. Dilute 1.0 ml of this solution
colourless crystals.
to 10.0 ml with mobile phase A.
Solubility : practically insoluble in water, freely soluble in
Reference solution (b). Dissolve 20 mg of ibuprofen CRS in
acetone, in methanol and in methylene chloride. It dissolves 2 ml of acetonitrile R, add 1.0 ml of a 0.06 g/l solution of
in dilute solutions of alkali hydroxides and carbonates.
ibuprofen impurity B CRS in acetonitrile R and dilute to
10.0 ml with mobile phase A.
IDENTIFICATION
Reference solution (b). Dissolve 5 mg of ibuprofen for
First identification : A, C.
system suitability CRS (containing impurities B, J and N) in
Second identification : A, B, D.
1 ml of acetonitrile R and dilute to 5 ml with mobile phase A.
A. Melting point (2.2.14) : 75 C to 78 C.
Column :
B. Ultraviolet and visible absorption spectrophotometry
size : l = 0.15 m, = 4.6 mm ;
(2.2.25).
stationary phase : octadecylsilyl silica gel for
Test solution. Dissolve 50.0 mg in a 4 g/l solution of
chromatography R (5 m)(30).
sodium hydroxide R and dilute to 100.0 ml with the same
Mobile phase :
alkaline solution.
Spectral range : 240-300 nm, using a spectrophotometer mobile phase A : mix 0.5 volumes of phosphoric acid R,
340 volumes of acetonitrile R and 600 volumes of
with a band width of 1.0 nm and a scan speed of not more
water
R ; allow to equilibrate and dilute to 1000 volumes
than 50 nm/min.
with water R ;
Absorption maxima: at 264 nm and 272 nm.
mobile phase B : acetonitrile R ;
Shoulder : at 258 nm.
Time
Mobile phase A
Mobile phase B
Absorbance ratio :
(min)
(per cent V/V)
(per cent V/V)
A264 / A258 = 1.20 to 1.30 ;
0
0 - 25
100
A272 / A258 = 1.00 to 1.10.
25 - 55
100 15
0 85
C. Infrared absorption spectrophotometry (2.2.24).
55 - 70
15
85
Preparation : discs.
Comparison : ibuprofen CRS.
70 - 75
15 100
85 0
D. Thin-layer chromatography (2.2.27).
Flow rate : 2 ml/min.
Test solution. Dissolve 50 mg of the substance to be
Detection : spectrophotometer at 214 nm.
examined in methylene chloride R and dilute to 10 ml
with the same solvent.
Equilibration : for about 45 min with mobile phase A.

Ibuprofenum

(30) Sperisorb ODS-2 is suitable.

442

PHARMEUROPA Vol. 18, No. 3, July 2006

Ibuprofen

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

Figure 0721.-1. Chromatogram for the test for related substances of ibuprofen : solution of ibuprofen spiked with
impurities A, B, C, D, E, J and N
Injection: 20 l.
Identification of impurities : use the chromatogram
supplied with ibuprofen for system suitability CRS and
the chromatogram obtained with reference solution (b) to
identify the peaks due to impurities B, J and N.
Relative retention with reference to ibuprofen (retention
time = about 16 min) : impurity J = about 0.2 ;
impurity N = about 0.3 ; impurity B = about 1.1.
System suitability : reference solution (b) :
peak-to-valley ratio : minimum 1.5, where Hp = height
above the baseline of the peak due to impurity B, and
Hv = height above the baseline of the lowest point of
the curve separating this peak from the peak due to
ibuprofen. If necessary, adjust the concentration of
acetonitrile in mobile phase A.
Limits :
impurity B : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (b) (0.3 per cent),
impurities J, N : for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
reference solution (a) (0.1 per cent) ;
any other impurity unspecified impurities : not more
than 0.3 0.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.3
0.05 per cent) ;
total of all impurities apart from impurity B : not more
than 0.7 times the area of the principal peak in the
chromatogram obtained with reference solution (a)
(0.7 per cent),
total: not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.2 per cent) ;

disregard limit : 0.05 0.3 times the area of the principal

peak in the chromatogram obtained with reference
solution (a) (0.05 0.03 per cent).
Impurity F. Gas chromatography (2.2.28) : use the
normalisation procedure.
Methylating solution. Dilute 1 ml of N,N-dimethylformamide
dimethyl acetal R and 1 ml of pyridine R to 10 ml with ethyl
acetate R.
Test solution. Weigh about 50.0 mg of the substance to be
examined into a sealable vial, dissolve in 1.0 ml of ethyl
acetate R, add 1 ml of the methylating solution, seal and
heat at 100 C in a block heater for 20 min. Allow to cool.
Remove the reagents under a stream of nitrogen at room
temperature. Dissolve the residue in 5 ml of ethyl acetate R.
Reference solution (a). Dissolve 0.5 mg of ibuprofen
impurity F CRS in ethyl acetate R and dilute to 10.0 ml with
the same solvent.
Reference solution (b). Weigh about 50.0 mg of
ibuprofen CRS into a sealable vial, dissolve in 1.0 ml of
reference solution (a), add 1 ml of the methylating solution,
seal and heat at 100 C in a block heater for 20 min. Allow
to cool. Remove the reagents under a stream of nitrogen
at room temperature. Dissolve the residue in 5 ml of ethyl
acetate R.
Column :
material : fused silica ;
size : l = 25 m, = 0.53 mm ;
stationary phase : macrogol 20 000 R (film thickness
2 m)(31).
Carrier gas : helium for chromatography R.
Flow rate : 5.0 ml/min.

(31) WCOT fused silica coating CP-WAX 52 CB is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

443

Ibuprofen

Temperature :
column : 150 C ;
injection port : 200 C ;
detector : 250 C.
Detection : flame-ionisation.
E. 1-[4-(2-methylpropyl)phenyl]ethanone,
Injection: 1 l of the test solution and reference solution (b).
Run time : twice the retention time of ibuprofen.
System suitability :
relative retention with reference to ibuprofen (retention
time = about 17 min) : impurity F = about 1.5.
F. 3-[4-(2-methylpropyl)phenyl]propanoic acid,
Limit :
impurity F : maximum 0.1 per cent.
Heavy metals (2.4.8) : maximum 10 ppm.
12 ml of solution S complies with test B. Prepare the
reference solution using lead standard solution (1 ppm Pb)
obtained by diluting lead standard solution (100 ppm Pb) R
with methanol R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in vacuo.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
G. cis-7-(2-methylpropyl)-1-[4-(2-methylpropyl)phenyl]-1,2,3,
4-tetrahydronaphthalene-1,4-dicarboxylic acid,
ASSAY
Dissolve 0.450 g in 50 ml of methanol R. Add 0.4 ml of
phenolphthalein solution R1. Titrate with 0.1 M sodium
hydroxide until a red colour is obtained. Carry out a blank
titration.
1 ml of 0.1 M sodium hydroxide is equivalent to 20.63 mg of
C13H18O2.
IMPURITIES
H. X = O : (3RS)-1,3-bis[4-(2-methylpropyl)phenyl]butan-1-one,
Specified impurities : F, J, N.
I. X = H2 : (3RS)-1,3-bis[4-(2-methylpropyl)phenyl]butane,
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
J. R = H, R4 = CO-CH(CH3)2 : (2RS)-2-[4-(2Control of impurities in substances for pharmaceutical
methylpropanoyl)phenyl]propanoic acid,
use) : A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R.
K. R = H, R4 = CHO : (2RS)-2-(4-formylphenyl)propanoic acid,
L. R = H, R4 = CHOH-CH(CH3)2 : 2-[4-(1-hydroxy-2methylpropyl)phenyl]propanoic acid,
M. R = OH, R4 = CH2-CH(CH3)2 : (2RS)-2-hydroxy-2-[4-(2methylpropyl)phenyl]propanoic acid,
A. R1 = OH, R2 = CH2-CH(CH3)2, R3 = H :
(2RS)-2-[3-(2-methylpropyl)phenyl]propanoic
acid,

N. R = H, R4 = C2H5 : (2RS)-2-(4-ethylphenyl)propanoic acid,

O. R = H, R4 = CH(CH3)-C2H5 : 2-[4-(1-methylpropyl)phenyl]propanoic acid,

B. R1 = OH, R2 = H, R3 = [CH2]3-CH3 : (2RS)-2-(4butylphenyl)propanoic acid,

C. R1 = NH2, R2 = H, R3 = CH2-CH(CH3)2 :
(2RS)-2-[4-(2-methylpropyl)phenyl]propanamide,
D. R1 = OH, R2 = H, R3 = CH3 : (2RS)-2-(4methylphenyl)propanoic acid,
444

P. R = CH3 : (2RS)-2-[4-(2-methylpropyl)phenyl]propan-1-ol,
Q. R = H : 2-[4-(2-methylpropyl)phenyl]ethanol,
PHARMEUROPA Vol. 18, No. 3, July 2006

Iopromide

Impurity A and related primary aromatic amines. Protect

the solutions from light throughout the test. All given times
are critical for the test results. The test solution, reference
solution and blank solution must be processed in parallel.
Test solution. Dissolve 0.500 g of the substance to be
examined in 20.0 ml of water R in a 25 ml volumetric flask.

R. 1,1-bis[4-(2-methylpropyl)phenyl]ethane.

Reference solution. Dissolve 2.5 mg of iopromide

impurity A CRS in water R and dilute to 100.0 ml with the
same solvent. Transfer 2.0 ml of this solution to a 25 ml
volumetric flask and add 18.0 ml of water R.
Blank solution. Place 20.0 ml of water R in a 25 ml
volumetric flask.

Reference: PA/PH/Exp. 10C/T (03) 28 ANP

NOTE ON THE MONOGRAPH
Related substances : it has not been possible to obtain a
good separation of the impurities when methylene chloride
is used instead of chloroform ; the spraying reagent is
necessary to obtain a satisfactory detection of the spots at
0.10 per cent.
Assay : the isomers are not correctly separated when
methylene chloride is used instead of chloroform.
XXXX:1753

IOPROMIDE
Iopromidum

C18H24I3N3O8

Cool the test solution, reference solution and blank solution

in a bath of iced water for 5 min. Add 1.0 ml of hydrochloric
acid R1 to each solution and cool again for 5 min in a bath
of iced water. Add 1.0 ml of a 20 g/l solution of sodium
nitrite R, shake vigorously and cool for another 5 min in
a bath of iced water. To each solution add 0.50 ml of an
80 g/l solution of sulphamic acid R. Over the next 5 min,
shake vigorously several times, raising the stoppers to vent
the gas that evolves. Afterwards, add to each solution
1.0 ml of a 1 g/l solution of naphthylethylenediamine
dihydrochloride R in a mixture of 300 volumes of water R
and 700 volumes of propylene glycol R, shake, allow to cool
to room temperature for 10 min and dilute to 25.0 ml with
water R. Degas the solutions in an ultrasonic bath for 1 min
and measure the absorbance (2.2.25) of the test solution and
the reference solution at 495 nm against the blank, within
5 min. The test is not valid unless the absorbance of the
reference solution is at least 0.20. The absorbance of the test
solution is not greater than the absorbance of the reference
solution (0.01 per cent).
Impurity B. Liquid chromatography (2.2.29) as described in
the assay with the following modifications.

Relative retention with reference to iopromide

isomer Z2 (retention time = about 34 min) : impurity B
Mr 791 isomer Y1 = about 0.28 ; impurity B isomer Y2 = about 0.31.

DEFINITION
N,N-bis(2,3-Dihydroxypropyl)-2,4,6-triiodo-5[(methoxyacetyl)amino]-N-methylisophthalamide.
Content : 97.0 per cent to 102.5 per cent (anhydrous
substance).

System suitability : reference solution (d):

the chromatogram obtained shows 2 principal peaks.
Limit :
sum of impurity B isomers Y1 and Y2 : not more than
1.5 times the sum of the areas of the 2 principal peaks in
the chromatogram obtained with reference solution (c)
(1.5 per cent).
Related substances. Thin-layer chromatography (2.2.27).

CHARACTERS
Appearance : white to slightly yellowish powder.
Solubility : freely soluble in water, freely soluble in dimethyl
sulphoxide, practically insoluble in ethanol (96 per cent) and Test solution. Dissolve 1.0 g of the substance to be examined
in a mixture of equal volumes of methanol R and water R
in acetone.
and dilute to 10.0 ml with the same mixture of solvents.
IDENTIFICATION
Reference solution (a). Dilute 1.0 ml of the test solution to
100.0 ml with a mixture of equal volumes of methanol R
Infrared absorption spectrophotometry (2.2.24).
and water R.
Comparison : iopromide CRS.
TESTS
Appearance of solution. The solution is clear (2.2.1) and
not more intensely coloured than reference solution BY6
(2.2.2, Method II).
Dissolve 16.5 g in 20 ml of carbon dioxide-free water R while
heating on a water-bath at a temperature not exceeding
70 C. Allow to cool to room temperature.
Conductivity (2.2.28) : maximum 50 Scm 1.
Dissolve 1.000 g in water R and dilute to 50.0 ml with the
same solvent.
PHARMEUROPA Vol. 18, No. 3, July 2006

Reference solution (b). Dilute 5.0 ml of reference solution (a)

to 10.0 ml with a mixture of equal volumes of methanol R
and water R.
Reference solution (c). Dilute 2.0 ml of reference solution (a)
to 10.0 ml with a mixture of equal volumes of methanol R
and water R.
Reference solution (d). Dilute 1.0 ml of reference solution (a)
to 10.0 ml with a mixture of equal volumes of methanol R
and water R.
Reference solution (e). Dissolve 1 mg of iopromide
impurity E CRS in 1 ml of the test solution.
445

Iopromide

Reference solution (f). Dissolve the contents of a vial of

iopromide for system suitability CRS (containing impurities
B, C, D and F) in 50 l of a mixture of equal volumes of
methanol R and water R.
Plates : TLC silica gel F254 plate R(32) (2 plates).
Mobile phase A : concentrated ammonia R, water R,
dioxan R (4:15:85 V/V/V).
Mobile phase B : anhydrous formic acid R, water R,
methanol R, chloroform R (2:6:32:62 V/V/V/V).
Application : 2 l, on 2 separate plates.
Development :
plate 1 over 3/4 of the plate ; with mobile phase A ;
plate 2 over 3/4 of the plate ; with mobile phase B.
Drying : in a current of air, until complete evaporation of
the solvents.
Detection : examine in ultraviolet light at 254 nm ; expose
the plates to ultraviolet light for 2-5 min until the principal
spots appear clearly as yellow spots, then spray with ferric
chloride-ferricyanide-arsenite reagent R and examine in
daylight.
Rf values :
plate 1 : impurity B = about 0.39 ; iopromide = about 0.43 ;
impurity E = about 0.47 ;
plate 2 : impurity C = about 0.11 ; impurity D = about 0.16 ;
impurity B = about 0.23 ; iopromide = about 0.30 ;
impurity F = about 0.53.
System suitability :
plate 1 : the chromatogram obtained with reference
solution (e) shows 2 clearly separated spots ;
plate 2 : the chromatogram obtained with reference
solution (f) shows 5 clearly separated spots.
Limits :
impurity D : on plate 2, any spot due to impurity D is not
more intense than the principal spot in the chromatogram
obtained with reference solution (a) (1 per cent) ;
impurity C : on plate 2, any spot due to impurity C is not
more intense than the principal spot in the chromatogram
obtained with reference solution (b) (0.5 per cent) ;
impurity E : on plate 1, any spot due to impurity E is not
more intense than the principal spot in the chromatogram
obtained with reference solution (b) (0.5 per cent) ;
impurity F : on plate 2, any spot due to impurity F is not
more intense than the principal spot in the chromatogram
obtained with reference solution (c) (0.2 per cent) ;
unspecified impurities : on plate 1 and plate 2, any
other spot is not more intense than the principal spot in
the chromatogram obtained with reference solution (d)
(0.10 per cent) ; disregard any spot due to impurity B.
Isomer distribution. Liquid chromatography (2.2.29) as
described in the assay with the following modifications.
Calculate the percentage content of the isomer groups
with reference to the total area of all the peaks due to the
4 isomers of iopromide, using the chromatogram obtained
with the test solution.
Limits :
sum of iopromide isomers E1 and Z1 : 40.0 per cent to
51.0 per cent ;
sum of iopromide isomers E2 and Z2 : 49.0 per cent to
60.0 per cent.

Free iodine. Dissolve 2.0 g in 20 ml of water R in a

glass-stoppered test tube. Add 2 ml of dilute sulphuric
acid R and 2 ml of toluene R, close and shake vigorously.
The upper layer remains colourless (2.2.2, Method II).
Iodide : maximum 20 ppm.
Dissolve 10.0 g in 50 ml of carbon dioxide-free water R.
Adjust to pH 3-4 adding about 0.15 ml of 0.1 M sulphuric
acid. Titrate with 0.001 M silver nitrate, determining the
end-point potentiometrically (2.2.20). Not more than 1.5 ml
of 0.001 M silver nitrate are required to reach the end-point.
Heavy metals (2.4.8) : maximum 10 ppm.
Dissolve 2.0 g in water R and dilute to 20 ml with the
same solvent. 12 ml of the solution complies with test A.
Prepare the reference solution using lead standard solution
(1 ppm Pb) R.
Water (2.5.12) : maximum 1.5 per cent, determined on
1.000 g.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
Bacterial endotoxins (2.6.14) : less than 0.6 IU/g.
ASSAY
Liquid chromatography (2.2.29).
Solvent mixture. Mix equal volumes of methanol R and
water R.
Test solution. Dissolve 40.0 mg of the substance to be
examined in the solvent mixture and dilute to 25.0 ml with
the solvent mixture.
Reference solution (a). Dissolve 40.0 mg of iopromide CRS
in the solvent mixture and dilute to 25.0 ml with the solvent
mixture.
Reference solution (b). Introduce several millilitres of
reference solution (a) into a vial sealed with a crimp-top.
Heat at 121 C for 15 min.
Reference solution (c). Dilute 1.0 ml of the test solution to
100.0 ml with the solvent mixture.
Reference solution (d). Dissolve 5 mg of iopromide
impurity B CRS in the solvent mixture and dilute to 200.0 ml
with the solvent mixture.
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase : end-capped octadecylsilyl silica gel
for chromatography R (5 m)(33) ;
temperature : 20 C.
Mobile phase : mix 6 g of chloroform R with 59 g of
methanol R. Add 900 g of water for chromatography R. Use
only degassed and filtered solvents and do not stir or pass
any gas through the mobile phase during use.
Flow rate : 1.2 ml/min.
Detection : spectrophotometer at 254 nm.
Injection : 10 l.
Run time : 40 min.
Identification of the isomers : the 2 principal peaks in the
chromatogram obtained with reference solution (a) are due
to iopromide isomers Z1 and Z2 . The 2 peaks that have an
increased size in the chromatogram obtained with reference
solution (b) in comparison to the chromatogram obtained
with reference solution (a), are due to iopromide isomers
E1 and E2.

(32) Merck KGaA Silica gel 60 F254 is suitable.

(33) Shandon ODS-Hypersil, 5 m is suitable.

446

PHARMEUROPA Vol. 18, No. 3, July 2006

Iopromide

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. impurity B isomer Y1

3. iopromide isomer E1

5. iopromide isomer Z1

2. impurity B isomer Y2

4. iopromide isomer E2

6. iopromide isomer Z2

Figure 1753.-1. Chromatogram for the assay of iopromide

Relative retention with reference to iopromide

isomer Z2 (retention time = about 34 min) : iopromide
isomer E1 = about 0.70 ; iopromide isomer E2 = about 0.75 ;
iopromide isomer Z1 = about 0.85.
System suitability : reference solution (a):
resolution : minimum 2.0 between the peaks due to
iopromide isomers Z1 and Z2.

A. 5-amino-N,N-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-Nmethylisophthalamide,

Calculate the percentage content of iopromide from the total

area of all of the peaks of the isomer groups E and Z and the
declared content of iopromide CRS.

STORAGE
Protected from light.

IMPURITIES

B. 5-acetamido-N,N-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-Nmethylisophthalamide,

Specified impurities : A, B, C, D, E, F.
Other detectable impurities (the following substances
would, if present at a sufficient level, be detected by one
or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified
impurities and/or by the general monograph Substances for
pharmaceutical use (2034). It is therefore not necessary to
identify these impurities for demonstration of compliance.
C. N,N-bis(2,3-dihydroxypropyl)-5-(glycoloylamino)-2,4,6See also 5.10. Control of impurities in substances for
triiodo-N-methylisophthalamide,
pharmaceutical use) : G, H.
PHARMEUROPA Vol. 18, No. 3, July 2006

447

Lidocaine

H. 3-[(2,3-dihydroxypropyl)carbamoyl]-2,4,6-triiodo-5[(methoxyacetyl)amino]benzoic acid.
Reagents
Sodium arsenite. NaAsO2. (Mr 129.9). XXXXXXX.
[7784-46-5].

D. N,N-bis(2,3-dihydroxypropyl)-5-([3-((3-[(2,3dihydroxypropyl)(methyl)carbamoyl]-2,4,6triiodo-5-[(methoxyacetyl)amino]benzoyl)amino)-2hydroxypropyl](methoxyacetyl)amino)-2,4,6-triiodo-Nmethylisophthalamide,

Sodium arsenite solution. XXXXXXX.

Dissolve 5.0 g of sodium arsenite R in 30 ml of 1 M
sodium hydroxide. Cool to 0 C and add, while stirring,
65 ml of dilute hydrochloric acid R.
Ferric chloride-ferricyanide-arsenite reagent. XXXXXXX.
Immediately before use, mix 10 ml of a 27 g/l solution of
ferric chloride R in dilute hydrochloric acid R, 7 ml of
potassium ferricyanide solution R, 3 ml of water R and
10 ml of sodium arsenite solution R.

Reference: PA/PH/Exp. 10A/T (06) 36 ANP

E. 3-[(3-[(2,3-dihydroxypropyl)carbamoyl]-2,4,6-triiodo5-[(methoxyacetyl)amino]benzoyl)(methyl)amino]-2hydroxypropyl 3-[(2,3-dihydroxypropyl)carbamoyl]-2,4,6triiodo-5-[(methoxyacetyl)amino]benzoate,

NOTE ON THE MONOGRAPH

Related substances : introduction of an LC method to
control the impurities, including 2,6-dimethylaniline. The
revision is identical to the one proposed for the monograph
on Lidocaine hydrochloride (0227) in Pharmeuropa 18.2.
XXXX:0727

LIDOCAINE
Lidocainum

C14H22N2O

F. N-(2,3-dihydroxypropyl)-N-([2-(hydroxymethyl)2-methyl-1,3-dioxolan-4-yl]methyl)-2,4,6-triiodo-5[(methoxyacetyl)amino]-N-methylisophthalamide,

Mr 234.3

DEFINITION
2-(Diethylamino)-N-(2,6-dimethylphenyl)acetamide.
Content : 99.0 per cent to 101.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, very soluble in
ethanol (96 per cent) and in methylene chloride.

G. N-(2-chloro-3-hydroxypropyl)-N-(2,3-dihydroxypropyl)2,4,6-triiodo-5-[(methoxyacetyl)amino]-Nmethylisophthalamide,
448

IDENTIFICATION
First identification : A, B.
Second identification : A, C, D, E.
A. Melting point (2.2.14) : 66 C to 70 C, determined
without previous drying.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : lidocaine CRS.
PHARMEUROPA Vol. 18, No. 3, July 2006

Lidocaine

C. Dissolve 0.20 g with warming, in a mixture of 0.5 ml of

dilute hydrochloric acid R and 10 ml of water R and add
10 ml of picric acid solution R. The precipitate, washed
with water R and dried, melts (2.2.14) at about 230 C,
with decomposition.
D. To about 5 mg add 0.5 ml of fuming nitric acid R.
Evaporate to dryness on a water-bath, cool, and dissolve
the residue in 5 ml of acetone R. Add 0.2 ml of alcoholic
potassium hydroxide solution R. A green colour is
produced.
E. Dissolve about 0.1 g in 1 ml of ethanol (96 per cent) R
and add 0.5 ml of a 100 g/l solution of cobalt nitrate R.
A bluish-green precipitate is formed.
TESTS
Appearance of solution. The solution is clear (2.2.1) and
colourless (2.2.2, Method II).
Dissolve 1.0 g in 3 ml of dilute hydrochloric acid R and
dilute to 10 ml with water R.
2,6-Dimethylaniline : maximum 100 ppm.
Dissolve 0.25 g in methanol R and dilute to 10 ml with the
same solvent. To 2 ml of the solution add 1 ml of a freshly
prepared 10 g/l solution of dimethylaminobenzaldehyde R
in methanol R and 2 ml of glacial acetic acid R and allow
to stand for 10 min. Any yellow colour in the solution is

not more intense than that in a standard prepared at the

same time and in the same manner using 2 ml of a 2.5 mg/l
solution of 2,6-dimethylaniline R in methanol R.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 10.0 ml with the
mobile phase.
Reference solution (a). Dissolve 10.0 mg of lidocaine
impurity A CRS in the mobile phase and dilute to 200.0 ml
with the mobile phase.
Reference solution (b). Dissolve 5 mg of
2-chloro-N-(2,6-dimethylphenyl)acetamide R (impurity H) in
the mobile phase and dilute to 10.0 ml with the mobile phase.
Reference solution (c). Dilute 1.0 ml of the test solution to
10.0 ml with the mobile phase.
Reference solution (d). Mix 1.0 ml of reference solution (a),
1.0 ml of reference solution (b) and 1.0 ml of reference
solution (c) and dilute to 100.0 ml with the mobile phase.
Column :
size : l = 0.15 m, = 3.9 mm ;
stationary phase : end-capped polar-embedded
octadecylsilyl amorphous organosilica polymer R
(5 m)(34) ;
temperature : 30 C.

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. impurity C

3. impurity G

5. impurity A

7. lidocaine

9. impurity I

2. impurity D

4. impurity H

6. impurity E

8. impurity F

10. impurity J

Figure 0727.-1. Chromatogram for the test for related substances of lidocaine
(34) XTerra RP C18 is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

449

Lidocaine

Mobile phase : mix 30 volumes of acetonitrile for

chromatography R and 70 volumes of a 4.85 g/l solution of
potassium dihydrogen phosphate R adjusted to pH 8.0 with
strong sodium hydroxide solution R.
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 230 nm.

A. 2,6-dimethylaniline.

l(35).

Injection : 20
Relative retention with reference to lidocaine (retention
time = about 17 min) : impurity H = about 0.37 ;
impurity A = about 0.40.
System suitability : reference solution (d) :
resolution : minimum 1.5 between the peaks due to
impurities A and H.
Limits :

B. 2-(diethylnitroryl)-N-(2,6-dimethylphenyl)acetamide
(lidocaine N-oxide),

impurity A : not more than the area of the corresponding

peak in the chromatogram obtained with reference
solution (d) (0.01 per cent) ;
unspecified impurities : for each impurity, not more than
the area of the peak due to lidocaine in the chromatogram C. N-(2,6-dimethylphenyl)acetamide,
obtained with reference solution (d) (0.10 per cent) ;
total: not more than 5 times the area of the peak due to
lidocaine in the chromatogram obtained with reference
solution (d) (0.5 per cent) ;
disregard limit : 0.5 times the area of the peak due to
lidocaine in the chromatogram obtained with reference
solution (d) (0.05 per cent).
Chlorides (2.4.4) : maximum 35 ppm.

D. N-(2,6-dimethylphenyl)-2-(ethylamino)acetamide,

Dissolve 1.4 g in a mixture of 3 ml of dilute nitric acid R

and 12 ml of water R.
Sulphates (2.4.13) : maximum 0.1 per cent.
Dissolve 0.2 g in 5 ml of ethanol (96 per cent) R and dilute
to 20 ml with distilled water R.
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test C. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
Water (2.5.12) : maximum 1.0 per cent, determined on
1.000 g.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.

E. 2,2-iminobis(N-(2,6-dimethylphenyl)acetamide),

F. 2-(diethylamino)-N-(2,3-dimethylphenyl)acetamide,

ASSAY
To 0.200 g add 50 ml of anhydrous acetic acid R and stir
until dissolution is complete. Titrate with 0.1 M perchloric
acid, determining the end-point potentiometrically (2.2.20).
1 ml of 0.1 M perchloric acid is equivalent to 23.43 mg
of C14H22N2O.

G. N-(2,6-dimethylphenyl)-2-((1-methylethyl)amino)acetamide,

IMPURITIES
Specified impurities : A.
Other detectable impurities (the following substances
H. 2-chloro-N-(2,6-dimethylphenyl)acetamide,
would, if present at a sufficient level, be detected by one
or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified
impurities and/or by the general monograph Substances for
pharmaceutical use (2034). It is therefore not necessary to
identify these impurities for demonstration of compliance.
See also 5.10. Control of impurities in substances for
pharmaceutical use) : B, C, D, E, F, G, H, I, J.
I. 2-(diethylamino)-N-(2,4-dimethylphenyl)acetamide,
(35) A slight carry-over might occasionally be observed ; this can be resolved by rinsing the injector.

450

PHARMEUROPA Vol. 18, No. 3, July 2006

Magnesium carbonate, heavy

Appearance of solution. Solution S is not more intensely

coloured than reference solution B4 (2.2.2, Method II).
Soluble substances : maximum 1.0 per cent.
Mix 2.00 g with 100 ml of water R and boil for 5 min. Filter
whilst hot through a sintered-glass filter (40), allow to cool,
and dilute to 100 ml with water R. Evaporate 50 ml of the
filtrate to dryness and dry at 100-105 C. The residue weighs
J. 2-(diethylamino)-N-(2,5-dimethylphenyl)acetamide.
a maximum of 10 mg.
Substances insoluble in acetic acid : maximum 0.05 per
Reagents
cent.
2-Chloro-N-(2,6-dimethylphenyl)acetamide. C10H12ClNO.
Any residue obtained during the preparation of solution S,
(Mr 197.7). XXXXXXX. [1131-01-7].
washed, dried, and ignited at 600 C, weighs a maximum
of 2.5 mg.
Chlorides (2.4.4) : maximum 700 ppm.
Dilute 1.5 ml of solution S to 15 ml with water R.
Reference: PA/PH/Exp. INC/T (06) 14 ANP
Sulphates (2.4.13) : maximum 0.6 per cent.
Dilute 0.5 ml of solution S to 15 ml with distilled water R.
NOTE ON THE MONOGRAPH
Arsenic (2.4.2, Method A) : maximum 2 ppm, determined
The European Pharmacopoeia Commission has decided
on 10 ml of solution S.
that more attention will be paid to functionality-related
Calcium (2.4.3) : maximum 0.75 per cent.
characteristics (FRCs) in monographs on excipients.
Dilute 2.6 ml of solution S to 150 ml with distilled water R.
While these characteristics are of importance for the
15 ml of the solution complies with the test.
intended use of many excipients, they are essentially a
matter for agreement between the excipient supplier and
Iron (2.4.9) : maximum 400 ppm.
the manufacturer of a medicinal product. For this reason,
Dissolve 0.1 g in 3 ml of dilute hydrochloric acid R and
FRCs are to be dealt with in a specific non-mandatory
dilute to 10 ml with water R. Dilute 2.5 ml of the solution
section of the monograph, and the particular use or uses
to 10 ml with water R.
for which a given FRC is relevant are to be specified.
Heavy metals (2.4.8) : maximum 20 ppm.
FRCs are very diverse in nature, and their treatment in
monographs will have to be adapted to each type.
To 20 ml of solution S add 15 ml of hydrochloric acid R1
XXXX:0043 and shake with 25 ml of methyl isobutyl ketone R for 2 min.
Allow to stand, then separate and evaporate the aqueous
MAGNESIUM CARBONATE, HEAVY lower layer to dryness. Dissolve the residue in 1 ml of acetic
acid R and dilute to 20 ml with water R. 12 ml of the solution
complies with test A. Prepare the reference solution using
Magnesii subcarbonas ponderosus
lead standard solution (1 ppm Pb) R.
DEFINITION
Hydrated basic magnesium carbonate.
Content : 40.0 per cent to 45.0 per cent, calculated as MgO
(Mr 40.30).
CHARACTERS
Appearance : white or almost white powder.
Solubility : practically insoluble in water. It dissolves in
dilute acids with effervescence.
IDENTIFICATION
A. 15 g has an apparent volume (2.9.15) before settling of
not more than 60 ml.
B. It gives the reaction of carbonates (2.3.1).
C. Dissolve about 15 mg in 2 ml of dilute nitric acid R and
neutralise with dilute sodium hydroxide solution R. The
solution gives the reaction of magnesium (2.3.1).
TESTS
Solution S. Dissolve 5.0 g in 100 ml of dilute acetic acid R.
When the effervescence has ceased, boil for 2 min, allow to
cool, and dilute to 100 ml with dilute acetic acid R. Filter, if
necessary, through a previously ignited and tared porcelain
or silica filter crucible of suitable porosity to give a clear
filtrate.

ASSAY
Dissolve 0.150 g in a mixture of 2 ml of dilute hydrochloric
acid R and 20 ml of water R. Carry out the complexometric
titration of magnesium (2.5.11).
1 ml of 0.1 M sodium edetate is equivalent to 4.030 mg
of MgO.
FUNCTIONALITY-RELATED CHARACTERISTICS
This section provides information on characteristics that
are recognised as being relevant control parameters for
one or more functions of the substance when used as an
excipient (see general chapter 5.15)(36). This section is
a non-mandatory part of the monograph and it is not
necessary to verify the characteristics to demonstrate
compliance. Control of these characteristics can however
contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process.
Where control methods are cited, they are recognised as
being suitable for the purpose, but other methods can also
be used. Wherever results for a particular characteristic
are reported, the control method must be indicated.
The following characteristics may be relevant for heavy
magnesium carbonate used as a filler in tablets.
Particle-size distribution (2.9.38) or (2.9.31).
Bulk density and tapped density (2.9.34)(37).

(36) See draft in this issue.

(37) See draft in Pharmeuropa 16.2.

PHARMEUROPA Vol. 18, No. 3, July 2006

451

Magnesium gluconate, hydrated

Reference: PA/PH/Exp. INC/T (06) 8 ANP

NOTE ON THE MONOGRAPH
This monograph is harmonised with the current Ph. Eur.
monograph for Calcium gluconate (0172). It is intended
for material for oral administration only. Higher-purity
material may be required for magnesium gluconate
intended for parenteral use. The Ph. Eur. monograph for
Calcium gluconate for injection (0979) is more stringent
than those of the other gluconates. If similar logic is
followed for magnesium gluconate it would be preferable to
have a separate monograph with additional tests.
Identification : as identification of magnesium by the
reaction described in the Ph. Eur. has proven to be
inapplicable for the gluconate, an alternative method is
proposed.
XXXX:2161

B. To 10 ml of solution S (see Tests) add 3 ml of ammonium

chloride solution R. A slightly hazy precipitate may be
observed. Add 10 ml of disodium hydrogen phosphate
solution R. A white precipitate is formed that does not
dissolve upon the addition of 2 ml of dilute ammonia R1.

TESTS
Solution S. Dissolve 1.0 g in water R and dilute to 50 ml
with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y7 (2.2.2,
Method II).
Sucrose and reducing sugars. Dissolve 0.5 g in a mixture
of 2 ml of hydrochloric acid R1 and 10 ml of water R. Boil
for 5 min, allow to cool, add 10 ml of sodium carbonate
solution R and allow to stand for 10 min. Dilute to 25 ml
with water R and filter. To 5 ml of the filtrate add 2 ml of
cupri-tartaric solution R and boil for 1 min. Allow to stand
for 2 min. No red precipitate is formed.
MAGNESIUM GLUCONATE,
Chlorides (2.4.4) : maximum 500 ppm.
HYDRATED
Dilute 5 ml of solution S to 15 ml with water R.
Magnesii gluconas hydricus
Sulphates (2.4.13) : maximum 500 ppm.
Dissolve 2.0 g in a mixture of 10 ml of acetic acid R and
90 ml of distilled water R.
Heavy metals (2.4.8) : maximum 10 ppm.
Dissolve 2.0 g in 20 ml of water R. 12 ml of the solution
complies with test A. Prepare the reference solution using
lead standard solution (1 ppm Pb) R.
C12H22MgO14,xH2O
Mr 414.3 (anhydrous substance) Water (2.5.32) : maximum 12.0 per cent, determined on
0.080 g.
DEFINITION
Microbial contamination. Total viable aerobic count (2.6.12)
Hydrate magnesium D-gluconate.
not more than 103 micro-organisms per gram, determined
Content : 98.0 per cent to 102.0 per cent (anhydrous
by plate count.
substance).
ASSAY
CHARACTERS
Dissolve 0.350 g in 100 ml of water R and carry out the
Appearance : white or almost white, amorphous, crystalline complexometric titration of magnesium (2.5.11).
or granular powder.
1 ml of 0.1 M sodium edetate is equivalent to 41.43 mg of
Solubility : freely soluble in water, slightly soluble in ethanol C12H22MgO14.
(96 per cent), very slightly soluble in methylene chloride.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 20 mg of the substance to be
examined in 1 ml of water R.
Reference solution. Dissolve 20 mg of calcium
gluconate CRS in 1 ml of water R, heating if necessary in
a water-bath at 60 C.
Plate : TLC silica gel plate R (5-40 m) [or TLC silica gel
plate R (2-10 m)].
Mobile phase : concentrated ammonia R, ethyl acetate R,
water R, ethanol (96 per cent) R (10:10:30:50 V/V/V/V).
Application : 1 l.
Development : over 3/4 of the plate.
Drying : at 100-105 C for 20 min, then allow to cool to
room temperature.
Detection : spray with a solution containing 25 g/l of
ammonium molybdate R and 10 g/l of cerium sulphate R
in dilute sulphuric acid R, and heat at 100-105 C for
about 10 min.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with the reference solution.
452

Reference: PA/PH/Exp. INC/T (06) 1 ANP

NOTE ON THE MONOGRAPH
This monograph is harmonised with the current monograph
for Calcium gluconate (0172).
Identification : as identification of manganese by the
reaction described in the Ph. Eur. has proven to be
inapplicable for the gluconate, an alternative method is
proposed.
XXXX:2162

MANGANESE GLUCONATE,
HYDRATED
Mangani gluconas hydricus

C12H22MnO14,xH2O

Mr 445.2 (anhydrous substance)

PHARMEUROPA Vol. 18, No. 3, July 2006

Marbofloxacin for veterinary use

DEFINITION
Hydrated manganese (II) D-gluconate.
Content : 98.0 per cent to 102.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or pale pink, slightly hygroscopic,
crystalline powder.
Solubility : soluble in water, practically insoluble in ethanol
(96 per cent), insoluble in methylene chloride.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 20 mg of substance to be examined
in 1 ml of water R.
Reference solution. Dissolve 20 mg of calcium
gluconate CRS in 1 ml of water R, heating if necessary in
a water-bath at 60 C.
Plate : TLC silica gel plate R (5-40 m) [or TLC silica gel
plate R (2-10 m)].
Mobile phase : concentrated ammonia R, ethyl acetate R,
water R, ethanol (96 per cent) R (10:10:30:50 V/V/V/V).
Application : 1 l.
Development : over 3/4 of the plate.
Drying : at 100-105 C for 20 min, then allow to cool to
room temperature.
Detection : spray with a solution containing 25 g/l of
ammonium molybdate R and 10 g/l of cerium sulphate R
in dilute sulphuric acid R, and heat at 100-105 C for
about 10 min.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with the reference solution.
B. Dissolve 50 mg in 5 ml of water R. Add 0.5 ml of
ammonium sulphide solution R. A pale pink precipitate
is formed that dissolves upon the addition of 1 ml of
glacial acetic acid R.
TESTS
Solution S. Dissolve 1.0 g in water R and dilute to 50 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 intensity 6 of the range of reference solutions
of the most appropriate colour (2.2.2, Method II).
Sucrose and reducing sugars. Dissolve 0.5 g in a mixture
of 2 ml of hydrochloric acid R1 and 10 ml of water R. Boil
for 5 min, allow to cool, add 10 ml of sodium carbonate
solution R and allow to stand for 10 min. Dilute to 25 ml
with water R and filter. To 5 ml of the filtrate add 2 ml of
cupri-tartaric solution R and boil for 1 min. Allow to stand
for 2 min. No red precipitate is formed.
Chlorides (2.4.4) : maximum 500 ppm.
Dilute 5 ml of solution S to 15 ml with water R.
Sulphates (2.4.13) : maximum 500 ppm.
Dissolve 2.0 g in a mixture of 10 ml of acetic acid R and
90 ml of distilled water R.
Zinc : maximum 50 ppm.
To 10 ml of solution S add 1 ml of sulphuric acid R and
0.1 ml of potassium ferrocyanide solution R. After 30 s, any
opalescence in the solution is not more intense than that
in a mixture of 1.0 ml of zinc standard solution (10 ppm
PHARMEUROPA Vol. 18, No. 3, July 2006

Zn) R, 9 ml of water R, 1 ml of sulphuric acid R and 0.1 ml

of potassium ferrocyanide solution R.
Heavy metals (2.4.8) : maximum 10 ppm.
Dissolve 2.0 g in 20 ml of water R, heating in a water-bath at
60 C. 12 ml of the solution complies with test A. Prepare
the reference solution using lead standard solution (1 ppm
Pb) R.
Water (2.5.32) : maximum 9.0 per cent, determined on
0.080 g.
Microbial contamination. Total viable aerobic count (2.6.12)
not more than 103 micro-organisms per gram, determined
by plate count.
ASSAY
Dissolve 0.400 g in 50 ml of water R. Add 10 mg of ascorbic
acid R, 20 ml of ammonium chloride buffer solution
pH 10.0 R and 0.2 ml of a 2 g/l solution of mordant black
11 R in triethanolamine R. Titrate with 0.1 M sodium
edetate until the colour changes from violet to pure blue.
1 ml of 0.1 M sodium edetate is equivalent to 44.52 mg
of C12H22MnO14.
STORAGE
In a non-metallic, airtight container.

Reference: PA/PH/Exp. P4/T (05) 20 ANP

XXXX:2233

MARBOFLOXACIN FOR VETERINARY

USE
Marbofloxacinum ad usum veterinarium

C17H19FN4O4

Mr 362.4

DEFINITION
9-Fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3dihydro-7H-[1,3,4]oxadiazino[6,5,4-ij]quinoline-6-carboxylic
acid.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : light yellow crystalline powder.
Solubility : slightly soluble in water, sparingly soluble or
slightly soluble in methylene chloride, very slightly soluble
in ethanol (96 per cent).
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : marbofloxacin CRS.
TESTS
Absorbance (2.2.25) : maximum 0.20 by measuring at the
absorption maximum at 450 nm.
Dissolve 0.400 g in borate buffer solution pH 10.4 R and
dilute to 10.0 ml with the same buffer solution.
453

Marbofloxacin for veterinary use

Related substances. Liquid chromatography (2.2.29). Carry

out the test protected from light.
Solvent mixture : methanol R, water R (23:77 V/V).
Test solution. To 100.0 mg of the substance to be examined
add 80 ml of the solvent mixture, sonicate until dissolution
and dilute to 100.0 ml with the solvent mixture.
Reference solution (a). Dilute 5.0 ml of the test solution
to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this
solution to 50.0 ml with the mobile phase.
Reference solution (b). Dissolve 10 mg of marbofloxacin for
peak identification CRS (containing impurities A, B, C, D
and E) in the solvent mixture and dilute to 10.0 ml with the
solvent mixture.
Column :
size : l = 0.15 m, = 4.6 mm ;
stationary phase: end-capped polar-embedded
octadecylsilyl amorphous organosilica polymer R(38)
(3.5 m) ;
temperature : 40 C.
Mobile phase : mix 230 volumes of methanol R and
5 volumes of glacial acetic acid R with 770 volumes of
a 2.70 g/l solution of sodium dihydrogen phosphate R
containing 3.50 g/l of sodium octanesulphonate R,
previously adjusted to pH 2.5 with phosphoric acid R.
Flow rate : 1.2 ml/min.
Detection : spectrophotometer at 315 nm.
Injection: 10 l.
Run time : 2.5 times the retention time of marbofloxacin.
Relative retention with reference to marbofloxacin
(retention time = about 33 min) : impurity C = about 0.9 ;
impurity D = about 1.3 ; impurity E = about 1.5.
System suitability : reference solution (b) :
resolution : minimum 1.5 between the peaks due to
impurity C and marbofloxacin, and minimum 4.0 between
the peaks due to marbofloxacin and impurity D.
Limits :

correction factor: for the calculation of content, multiply

the peak area of impurity E by 1.5 ;
impurities A, B : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent) ;
impurities C, D, E : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent) ;
unspecified impurities : for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.10 per cent) ;
total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
Dissolve 0.5 g in dilute acetic acid R and dilute to 30 ml with
the same solvent. Adding 2 ml of water R instead of 2 ml of
buffer solution pH 3.5 R, the filtrate complies with test E.
Prepare the reference solution using 5 ml of lead standard
solution (2 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying at 100-105 C for 4 h.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g in a platinum crucible.
ASSAY
Dissolve 0.300 g in 80 ml of glacial acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1 ml of 0.1 M perchloric acid is equivalent to 36.24 mg of
C17H19FN4O4.
STORAGE
Protected from light.

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

Figure 2233.-1. Chromatogram for the test for related substances of marbofloxacin for veterinary use : solution of
marbofloxacin spiked with impurities A, B, C, D, E and F
(38) X-Terra RP18 is suitable.

454

PHARMEUROPA Vol. 18, No. 3, July 2006

Measles, mumps and rubella vaccine (live)

IMPURITIES
Specified impurities : A, B, C, D, E.
Other detectable impurities (the following substances
would, if present at a sufficient level, be detected by one
or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified
impurities and/or by the general monograph Substances for
pharmaceutical use (2034). It is therefore not necessary to
identify these impurities for demonstration of compliance.
See also 5.10. Control of impurities in substances for
pharmaceutical use) : F.

A. 6,7-difluoro-8-hydroxy-1-(methylamino)-4-oxo-1,4dihydroquinoline-3-carboxylic acid,

B. 9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,3,4]oxadiazino[6,5,4-ij]quinoline-6-carboxylic acid,

Reference: PA/PH/Exp. 15/T (04) 74 ANP 1R

NOTE ON THE MONOGRAPH
Thermal stability test and Assay : a revision proposal
including additional acceptance criteria was published in
Pharmeuropa 17.2. In light of comments received, a new
revision proposal is shown below. The main changes are :
deletion of details regarding cell cultures and dilution
steps ; these details are an unnecessary restriction since
the validity criteria give a better assurance of correct
design ;
limits are expressed as logarithms and in such a way
that excessive rounding is avoided ;
addition of a sentence referring to the use of neutralising
antisera for the assay ;
since the aim is to confirm the suitable design of the test,
validity criteria apply to the reference preparation only,
and on the combined value obtained from 3 replicates ;
a manufacturers reference preparation may be
used provided it is regularly compared with the
appropriate BRP ;
introduction of control charts in addition to the criteria
0.5 log CCID50, in accordance with good practice ;
deletion of the requirement on the range of virus
concentration found for the replicates ; the requirement
for closeness to the historical value for the reference
preparation, together with the use of a control chart, is
considered to give better control;
introduction of details regarding repetition of the test
and combination of valid results ;
reference to chapter 5.3 for calculations.
XXXX:1057

MEASLES, MUMPS AND RUBELLA

VACCINE (LIVE)
Vaccinum morbillorum, parotitidis et
rubellae vivum

C. R = F : 6,8-difluoro-1-(methylamino)-7-(4-methylpiperazin1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid,
D. R = OH : 6-fluoro-8-hydroxy-1-(methylamino)-7-(4methylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3carboxylic acid,
E. R = O-CH2-CH3 : 8-ethoxy-6-fluoro-1-(methylamino)-7(4-methylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3carboxylic acid,

F. 9-fluoro-3-methyl-10-(4-methyl-4-oxidopiperazin-1-yl)-7oxo-2,3-dihydro-7H-[1,3,4]oxadiazino[6,5,4-ij]quinoline-6carboxylic acid.
PHARMEUROPA Vol. 18, No. 3, July 2006

DEFINITION
Measles, mumps and rubella vaccine (live) is a freeze-dried
preparation of suitable attenuated strains of measles virus,
mumps virus and rubella virus.
The vaccine is reconstituted immediately before use, as
stated on the label, to give a clear liquid that may be coloured
owing to the presence of a pH indicator.
PRODUCTION
The 3 components are prepared as described in the
monographs on Measles vaccine (live) (0213), Mumps
vaccine (live) (0538) and Rubella vaccine (live) (0162) and
comply with the requirements prescribed therein.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
FINAL BULK VACCINE
Virus harvests for each component are pooled and clarified
to remove cells. A suitable stabiliser may be added and the
pooled harvests diluted as appropriate. Suitable quantities
of the pooled harvest for each component are mixed.
Only a final bulk vaccine that complies with the following
requirement may be used in the preparation of the final lot.
Bacterial and fungal contamination. Carry out the test for
sterility (2.6.1), using 10 ml for each medium.
455

Measles, mumps and rubella vaccine (live)

each dilution step using at least 5 cell cultures for each 0.5
log10 dilution step or by a method of equal precision. Use
Titrate 1 vial of the appropriate virus reference preparation
in triplicate to validate each assay. Calculate the individual
virus concentration for each vial of vaccine and for each
replicate of the reference preparation as well as the
Only a final lot that complies with the requirements for
corresponding combined virus concentrations, using the
minimum virus concentration of each component for release, usual statistical methods (for example, 5.3).
with the following requirement for thermal stability and with
The estimated combined estimates of the measles, mumps
each of the requirements given below under Identification
and Tests may be released for use. Provided that the tests for and rubella virus concentrations for the 3 vials of vaccine are
bovine serum albumin and, where applicable, for ovalbumin not less than that stated on the label ; the minimum measles
virus concentration stated on the label is not less than
have been carried out with satisfactory results on the final
1x103 CCID50 3.0 log CCID50 per single human dose ; the
bulk vaccine, they may be omitted on the final lot.
minimum mumps virus concentration stated on the label is
Thermal stability. Maintain samples at least 3 vials of
not less than 5x103 CCID50 3.7 log CCID50 per single human
the final lot of freeze-dried vaccine in the dry state at
dose ; the minimum rubella virus concentration stated on
37 1 C for 7 days. Determine the virus concentration as
the label is not less than 1x103 CCID50 3.0 log CCID50 per
described under Assay in parallel for the heated vaccine and single human dose.
for unheated vaccine stored at 5 3 C the temperature
The assay is not valid if the confidence interval (P = 0.95) of
recommended for storage. For each component, the virus
concentration of the heated vaccine is not more than 1.0 log the logarithm of the virus concentration is greater than 0.3.
lower than that of the unheated vaccine.
The assay is not valid if:
FINAL LOT
For each component, a minimum virus concentration for
release of the product is established such as to ensure, in
light of stability data, that the minimum concentration stated
on the label will be present at the end of the period of validity.

The test is not valid if :

the confidence interval (P = 0.95) of the estimated
virus concentration of the reference preparation for the
3 replicates combined is greater than 0.3 log CCID50 ;
the virus concentration of the reference preparation,
monitored using control charts, differs by more than
0.5 log CCID50 from the value established on a historical
basis in the particular laboratory. The relation with the
appropriate BRP biological reference preparation is
established and monitored at regular intervals when a
manufacturers reference preparation is used.

the confidence interval (P = 0.95) of the estimated

virus concentration of the reference preparation for the
3 replicates combined is greater than 0.3 log CCID50 ;
the virus concentration of the reference preparation,
monitored using control charts, differs by more than
0.5 log CCID50 from the value established on a historical
basis in the particular laboratory. The relation with the
appropriate BRP biological reference preparation is
established and monitored at regular intervals when a
manufacturers reference preparation is used.

The assay is repeated if the confidence interval (P = 0.95) of

the combined virus concentration of the vaccine is greater
than 0.3 log CCID50 ; data generated from valid assays only
IDENTIFICATION
are combined by the usual statistical methods (for example,
When the vaccine reconstituted as stated on the label is
5.3) to calculate the virus concentration of the sample.
mixed with antibodies specific for measles virus, mumps virus The confidence interval (P = 0.95) of the combined virus
and rubella virus, it is no longer able to infect cell cultures
concentration is not greater than 0.3 log CCID50.
susceptible to these viruses. When the vaccine reconstituted
Measles vaccine (live) BRP is suitable for use as a reference
as stated on the label is mixed with quantities of specific
antibodies sufficient to neutralise any two viral components, preparation.
the third viral component infects susceptible cell cultures.
Mumps vaccine (live) BRP is suitable for use as a reference
preparation.
TESTS
Rubella vaccine (live) BRP is suitable for use as a reference
Bacterial and fungal contamination. The reconstituted
vaccine complies with the test for sterility (2.6.1).

preparation.

Bovine serum albumin. Not more than 50 ng per single

human dose, determined by a suitable immunochemical
method (2.7.1).

LABELLING

Ovalbumin. If the mumps component is produced in chick

embryos, the vaccine contains not more than 1 g of
ovalbumin per single human dose, determined by a suitable
immunochemical method (2.7.1).

the strains of virus used in the preparation of the vaccine ;

Water (2.5.12). Not more than 3.0 per cent, determined by

the semi-micro determination of water.
ASSAY
The cell lines and/or neutralising antisera are chosen to
ensure that each component is assayed without interference
from the other 2 components.
Titrate the vaccine for infective measles, mumps and rubella
virus at least in triplicate, using at least 3 separate vials
of vaccine and inoculating a suitable number of wells for
456

The label states :

where applicable, that chick embryos have been used for
the preparation of the vaccine ;
the type and origin of the cells used for the preparation
of the vaccine ;
the minimum virus concentration for each component of
the vaccine ;
that contact with disinfectants is to be avoided ;
the time within which the vaccine must be used after
reconstitution ;
that the vaccine must not be given to a pregnant woman
and that a woman must not become pregnant within
2 months after having the vaccine.
PHARMEUROPA Vol. 18, No. 3, July 2006

Measles vaccine (live)

Reference: PA/PH/Exp. 15/T (04) 77 ANP 1R

NOTE ON THE MONOGRAPH
Thermal stability test and Assay : a revision proposal
including additional acceptance criteria was published in
Pharmeuropa 17.2. In light of comments received, a new
revision proposal is shown below. The main changes are :
deletion of details regarding cell cultures and dilution
steps ; these details are an unnecessary restriction since
the validity criteria give a better assurance of correct
design ;
limits are expressed as logarithms and in such a way
that excessive rounding is avoided ;
since the aim is to confirm the suitable design of the test,
validity criteria apply to the reference preparation only,
and on the combined value obtained from 3 replicates ;
a manufacturers reference preparation may be
used provided it is regularly compared with the
appropriate BRP ;
introduction of control charts in addition to the criteria
0.5 log CCID50, in accordance with good practice ;
deletion of the requirement on the range of virus
concentration found for the replicate ; the requirement
for closeness to the historical value for the reference
preparation, together with the use of a control chart, is
considered to give better control ;
introduction of details regarding repetition of the test
and combination of valid results ;
reference to chapter 5.3 for calculations.
Neurovirulence : the present requirements for
neurovirulence testing were reviewed at a joint
EDQM-WHO-IABs scientific workshop on neurovirulence
tests for live virus vaccines in January 2005. On the
basis of the conclusions of the workshop, it is proposed
to revise the present testing scheme to foresee the study
of potential neurovirulence of the vaccine strains during
preclinical development instead of performing the test
for neurovirulence of live virus vaccines (2.6.18) on the
seed lot. This study is based on available data, notably for
wild type virus. Where necessary, a risk analysis study is
considered and, where applicable, a test for neurovirulence
is carried out using an animal model that differentiates
wild type and attenuated virus.
XXXX:0213

MEASLES VACCINE (LIVE)

Vaccinum morbillorum vivum
DEFINITION
Measles vaccine (live) is a freeze-dried preparation of a
suitable attenuated strain of measles virus. The vaccine is
reconstituted immediately before use, as stated on the label,
to give a clear liquid that may be coloured owing to the
presence of a pH indicator.
PRODUCTION
The production of vaccine is based on a virus seed-lot system
and, if the virus is propagated in human diploid cells, a
cell-bank system. The production method shall have been
shown to yield consistently live measles vaccines of adequate
immunogenicity and safety in man. Unless otherwise
justified and authorised, the virus in the final vaccine shall
have undergone no more passages from the master seed
lot than were used to prepare the vaccine shown in clinical
PHARMEUROPA Vol. 18, No. 3, July 2006

studies to be satisfactory with respect to safety and efficacy ;

even with authorised exceptions, the number of passages
beyond the level used for clinical studies shall not exceed 5.
The potential neurovirulence of the vaccine strain is
considered during preclinical development, based on
available data, notably for wild type virus. Where necessary
in light of a risk analysis, a test is carried out on the vaccine
strain using an animal model that differentiates wild type and
attenuated virus ; tests on strains of intermediate attenuation
may also be needed.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9).
SUBSTRATE FOR VIRUS PROPAGATION
The virus is propagated in human diploid cells (5.2.3) or in
cultures of chick-embryo cells derived from a chicken flock
free from specified pathogens (5.2.2).
SEED LOT
The strain of measles virus used shall be identified by
historical records that include information on the origin of
the strain and its subsequent manipulation. To avoid the
unnecessary use of monkeys in the test for neurovirulence,
Virus seed lots are prepared in large quantities and stored at
temperatures below 20 C if freeze-dried, or below 60 C
if not freeze-dried.
Only a seed lot that complies with the following requirements
may be used for virus propagation.
Identification. The master and working seed lots are
identified as measles virus by serum neutralisation in cell
culture, using specific antibodies.
Virus concentration. The virus concentration of the master
and working seed lots is determined to monitor consistency
of production.
Extraneous agents (2.6.16). The working seed lot complies
with the requirements for seed lots.
Neurovirulence (2.6.18). The working seed lot complies with
the test for neurovirulence of live virus vaccines. Macaca
and Cercopithecus monkeys susceptible to measles virus
are suitable for the test.
PROPAGATION AND HARVEST
All processing of the cell bank and subsequent cell cultures
is done under aseptic conditions in an area where no other
cells are handled. Suitable animal (but not human) serum
may be used in the growth medium, but the final medium
for maintaining cell growth during virus multiplication
does not contain animal serum. Serum and trypsin used in
the preparation of cell suspensions and culture media are
shown to be free from extraneous agents. The cell culture
medium may contain a pH indicator such as phenol red and
suitable antibiotics at the lowest effective concentration. It
is preferable to have a substrate free from antibiotics during
production. Not less than 500 ml of the production cell
cultures is set aside as uninfected cell cultures (control cells).
The viral suspensions are harvested at a time appropriate to
the strain of virus being used.
Only a single harvest that complies with the following
requirements may be used in the preparation of the final
bulk vaccine.
Identification. The single harvest contains virus that is
identified as measles virus by serum neutralisation in cell
culture, using specific antibodies.
Virus concentration. The virus concentration in the single
harvest is determined as prescribed under Assay to monitor
consistency of production and to determine the dilution to
be used for the final bulk vaccine.
457

Meloxicam

Extraneous agents (2.6.16). The single harvest complies

with the tests for extraneous agents.
Control cells. If human diploid cells are used for production,
the control cells comply with a test for identification. They
comply with the tests for extraneous agents (2.6.16).
FINAL BULK VACCINE
Virus harvests that comply with the above tests are pooled
and clarified to remove cells. A suitable stabiliser may be
added and the pooled harvests diluted as appropriate.
Only a final bulk vaccine that complies with the following
requirement may be used in the preparation of the final lot.
Bacterial and fungal contamination. The final bulk vaccine
complies with the test for sterility (2.6.1), carried out using
10 ml for each medium.
FINAL LOT
A minimum virus concentration for release of the product
is established such as to ensure, in light of stability data,
that the minimum concentration stated on the label will be
present at the end of the period of validity.
Only a final lot that complies with the requirements for
minimum virus concentration for release, with the following
requirement for thermal stability and with each of the
requirements given below under Identification and Tests may
be released for use. Provided that the test for bovine serum
albumin has been carried out with satisfactory results on the
final bulk vaccine, it may be omitted on the final lot.
Thermal stability. Maintain samples at least 3 vials of
the final lot of freeze-dried vaccine in the dry state at
37 1 C for 7 days. Determine the virus concentration as
described under Assay in parallel for the heated vaccine and
for unheated vaccine stored at 5 3 C the temperature
recommended for storage. The virus concentration of the
heated vaccine is not more than 1.0 log lower than that of
the unheated vaccine.
The test is not valid if :
the confidence interval (P = 0.95) of the estimated
virus concentration of the reference preparation for the
3 replicates combined is greater than 0.3 log CCID50 ;
the virus concentration of the reference preparation,
monitored using control charts, differs by more than
0.5 log CCID50 from the value established on a historical
basis in the particular laboratory. The relation with the
appropriate BRP biological reference preparation is
established and monitored at regular intervals when a
manufacturers reference preparation is used.

reference preparation in triplicate to validate each assay.

Calculate the individual virus concentration for each vial of
vaccine and for each replicate of the reference preparation
as well as the corresponding combined virus concentrations,
using the usual statistical methods (for example, 5.3). The
combined estimates of the virus concentration for the 3 vials
of vaccine is not less than that stated on the label ; the
minimum virus concentration stated on the label is not less
than 1 103 CCID50 3.0 log CCID50 per single human dose.
The assay is not valid if the confidence interval (P = 0.95) of
the logarithm of the virus concentration is greater than 0.3.
The assay is not valid if:
the confidence interval (P = 0.95) of the estimated
virus concentration of the reference preparation for the
3 replicates combined is greater than 0.3 log CCID50 ;
the virus concentration of the reference preparation,
monitored using control charts, differs by more than
0.5 log CCID50 from the value established on a historical
basis in the particular laboratory. The relation with the
appropriate BRP biological reference preparation is
established and monitored at regular intervals when a
manufacturers reference preparation is used.
The assay is repeated if the confidence interval (P = 0.95) of
the combined virus concentration of the vaccine is greater
than 0.3 log CCID50 ; data generated from valid assays
only are combined by the usual statistical methods (for
example, 5.3) to calculate the virus concentration of the
sample. The confidence interval (P = 0.95) of the combined
virus concentration is not greater than 0.3 log CCID50.
Measles vaccine (live) BRP is suitable for use as a reference
preparation.

IDENTIFICATION
When the vaccine reconstituted as stated on the label is
mixed with specific measles antibodies, it is no longer able to
infect susceptible cell cultures.

XXXX:2373

LABELLING
The label states :
the strain of virus used for the preparation of the vaccine ;
the type and origin of the cells used for the preparation
of the vaccine ;
the minimum virus concentration ;
that contact with disinfectants is to be avoided ;
the time within which the vaccine must be used after
reconstitution.

Reference: PA/PH/Exp. P4/T (05) 13 ANP

MELOXICAM
Meloxicamum

TESTS
Bacterial and fungal contamination. The reconstituted
vaccine complies with the test for sterility (2.6.1).
Bovine serum albumin. Not more than 50 ng per single
human dose, determined by a suitable immunochemical
method (2.7.1).
Water (2.5.12). Not more than 3.0 per cent, determined by
the semi-micro determination of water.
ASSAY
Titrate the vaccine for infective virus at least in triplicate,
using at least 3 separate vials of vaccine and inoculating a
suitable number of wells for each dilution step using at least
5 cell cultures for each 0.5 log10 dilution step or by a method
of equal precision. Use Titrate 1 vial of an appropriate virus
458

C14H13N3O4S2

Mr 351.4

DEFINITION
4-Hydroxy-2-methyl-N-(5-methyl-1,3-thiazol-2-yl)-2H-1,2benzothiazine-3-carboxamide 1,1-dioxide.
Content : 99.0 per cent to 101.0 per cent (dried substance).
PHARMEUROPA Vol. 18, No. 3, July 2006

Meloxicam

CHARACTERS
Appearance : pale yellow powder.
Solubility : practically insoluble in water, soluble in
dimethylformamide, very slightly soluble in ethanol (96 per
cent).
mp : about 243 C, with decomposition.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : meloxicam CRS.
TESTS
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 40 mg of the substance to be
examined in a mixture of 5 ml of methanol R and 0.3 ml

of 1 M sodium hydroxide and dilute to 20.0 ml with

methanol R.
Reference solution (a). Dilute 2.0 ml of the test solution to
100.0 ml with methanol R. Dilute 5.0 ml of this solution to
100.0 ml with methanol R.
Reference solution (b). Dissolve 2 mg of the substance
to be examined, 2 mg of meloxicam impurity A CRS,
2 mg of meloxicam impurity B CRS, 2 mg of meloxicam
impurity C CRS and 2 mg of meloxicam impurity D CRS in
a mixture of 5 ml of methanol R and 0.3 ml of 1 M sodium
hydroxide and dilute to 25 ml with methanol R.
Column :
size : l = 0.15 m, = 4.6 mm ;
stationary phase : end-capped octadecylsilyl silica gel
for chromatography R (5 m)(39) ;
temperature : 45 C.

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. impurity B

3. impurity A

5. impurity C

2. meloxicam

4. unknown impurity

6. impurity D

Figure 2373.-1. Chromatogram for the test for related substances of meloxicam : reference solution (b) at 260 nm
The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. meloxicam

3. unknown impurity

2. impurity A

4. impurity C

5. impurity D

Figure 2373.-2. Chromatogram for the test for related substances of meloxicam : reference solution (b) at 350 nm

(39) Inertsil ODS 2 is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

459

2.2.60. Melting point - instrumental method

Mobile phase :
mobile phase A : 1 g/l solution of potassium dihydrogen
phosphate R adjusted to pH 6.0 with 1 M sodium
hydroxide ;
mobile phase B : methanol R ;
Time
(min)
0-2

Mobile phase A
(per cent V/V)
60

Mobile phase B
(per cent V/V)
40

2 - 10

60 30

40 70

10 - 15

30

70

Flow rate : 1.0 ml/min.

Detection : spectrophotometer at 260 nm and 350 nm.
Injection: 10 l.
Relative retention with reference to meloxicam
(retention time = about 7 min) : impurity B = about 0.5 ;
impurity A = about 1.4 ; impurity C = about 1.7 ;
impurity D = about 1.9.
System suitability : reference solution (b) :
resolution : minimum 3.0 between the peaks due to
meloxicam and impurity A at 350 nm ; minimum 3.0
between the peaks due to impurity B and meloxicam at
260 nm.
Limits :
correction factor : for the calculation of content, multiply
the peak area of impurity A by 2 ;
impurity A at 350 nm : not more than the area of the
principal peak in the chromatogram obtained with
reference solution (a) at 350 nm (0.1 per cent) ;
impurity B at 260 nm : not more than the area of the
principal peak in the chromatogram obtained with
reference solution (a) at 350 nm (0.1 per cent) ;
impurities C, D at 350 nm : for each impurity, not more
than 0.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a) at
350 nm (0.05 per cent) ;
unspecified impurities : for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (a), at the wavelength
giving the higher value (0.10 per cent) ;
total: not more than 0.3 per cent ;
disregard limit : 0.3 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
at the same wavelength (0.03 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
2.0 g complies with test C. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.00 g by drying in an oven at 100-105 C for 4 h.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Dissolve 0.250 g in a mixture of 5 ml of anhydrous formic
acid R and 50 ml of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1 ml of 0.1 M perchloric acid is equivalent to 35.14 mg
of C14H13N3O4S2.
STORAGE
Protected from light.
460

IMPURITIES
Specified impurities : A, B, C, D.
Other detectable impurities (the following substances
would, if present at a sufficient level, be detected by one
or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified
impurities and/or by the general monograph Substances for
pharmaceutical use (2034). It is therefore not necessary to
identify these impurities for demonstration of compliance.
See also 5.10. Control of impurities in substances for
pharmaceutical use) : E.

A. ethyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3carboxylate 1,1-dioxide,

B. 5-methyl-1,3-thiazol-2-amine,

C. R = CH3 : N-[(2Z)-3,5-dimethyl-1,3-thiazol-2(3H)-ylidene]4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxamide
1,1-dioxide,
D. R = CH2-CH3 : N-[(2Z)-3-ethyl-5-methyl-1,3-thiazol-2(3H)ylidene]-4-hydroxy-2-methyl-2H-1,2-benzothiazine-3carboxamide 1,1-dioxide.

E. methyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3carboxylate 1,1-dioxide.

Reference: PA/PH/Exp. 13H/T (06) 34 ANP

XXXX:20260

2.2.60. MELTING POINT INSTRUMENTAL METHOD

APPARATUS
There are 2 modes of automatic observation arrangements :
mode A : by light transmission through the capillary tube
loaded with the sample ;
mode B : by light being reflected from the sample in the
capillary tube.
PHARMEUROPA Vol. 18, No. 3, July 2006

2.2.60. Melting point - instrumental method

In both modes, the capillary tube sits in a hollow of a

metal block, which is heated electrically and controlled
by a temperature sensor placed in another hollow of
the metal block. The heating block is capable of being
maintained accurately at a pre-defined temperature by the
heating element, and of being heated at a slow and steady,
pre-defined rate after an initial isothermal period.
In mode A, a beam of light shines through a horizontal
hollow and crosses the capillary tube. A sensor detects the
beam at the end of the cylindrical hole after the capillary
tube.
In mode B, a beam of light illuminates the capillary tube
from the front and the sensor records the image.
Some arrangements allow for the determination of the
melting point on more than one capillary tube.
METHOD
Use glass capillary tubes that are open at one end, about
90 mm long, with an external diameter of 1.3-1.5 mm and an
internal diameter of 0.9-1.3 mm. The wall thickness of the
tube ranges from 0.1 mm to 0.2 mm.
Introduce into the capillary tube a sufficient amount of the
substance, previously treated as described in the monograph,
to form in each tube a column about 4 mm high, and allow
the tubes to stand for the appropriate time at the prescribed
temperature.
Place the capillary tube in the heating block with the closed
end downwards. Set the instrument with initial isothermal
conditions and a rate for subsequent heating as described in
the monograph of the substance to be examined. Start the
temperature program. When the substance starts melting, it
changes its appearance in the capillary tube. As a result, the
temperature of the heating block is recorded automatically
following the signal changes from the photo sensor due to
light transmittance (mode A, Figure 2.2.60.-1), or following
image processing (mode B, Figure 2.2.60.-2).

A. glass capillary

C. image-sensor

E. heating block

B. sample

D. temperature sensor

F. light source

Figure 2.2.60.-2. Mode B : reflectance

CALIBRATION
The temperature scale of the instrument needs to be checked
periodically by measuring the melting point of calibration
standards. Pure benzophenone, vanillin, benzoic acid
and caffeine are used, as these substances melt without
decomposition and have melting points that are precisely
known.
Prepare 3 capillary tubes for each of the 4 calibration
standards.
Melting point of benzophenone : 48.1 C.
Melting point of vanillin : 83.2 C.
Melting point of benzoic acid : 122.4 C.
Melting point of caffeine : 236.3 C.

SYSTEM SUITABILITY
A. glass capillary

C. photo-sensor

E. heating block

B. sample

D. temperature sensor

F. light source

Figure 2.2.60.-1. Mode A : transmittance

PHARMEUROPA Vol. 18, No. 3, July 2006

Carry out a verification, generally before the measurement,

using a certified substance whose melting point is close to
that expected for the substance to be examined. A tolerance
of 0.2 C to 0.6 C is applied depending on the melting
point of the certified substance used.

461

Morphine hydrochloride

Reference: PA/PH/Exp. 11/T (06) 42 ANP

NOTE ON THE MONOGRAPH
Related substances : this monograph has already been
revised to replace the TLC by an LC. Following the public
enquiry, the method has been improved to obtain a better
separation of impurities. As this product of natural origin
has a complex impurity profile, it has not proven possible to
apply the general policy for impurities shown in the general
monograph Substance for pharmaceutical use (2034).
It has been necessary to give an acceptance criterion
equivalent to 0.2 per cent for any other impurities.
XXXX:0097

MORPHINE HYDROCHLORIDE
Morphini hydrochloridum

C17H20ClNO3,3H2O

Mr 375.8

DEFINITION
7,8-Didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol
hydrochloride trihydrate.
Content : 98.0 per cent to 102.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or
colourless, silky needles or cubical masses, efflorescent in a
dry atmosphere.
Solubility : soluble in water, slightly soluble in ethanol
(96 per cent), practically insoluble in toluene.
IDENTIFICATION
First identification : A, E.
Second identification : B, C, D, E.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : morphine hydrochloride CRS.
B. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Solution A. Dissolve 25.0 mg in water R and dilute to
25.0 ml with the same solvent.
Test solution (a). Dilute 10.0 ml of solution A to 100.0 ml
with water R.
Test solution (b). Dilute 10.0 ml of solution A to 100.0 ml
with 0.1 M sodium hydroxide.
Spectral range : 250-350 nm for test solutions (a) and (b).
Absorption maximum : at 285 nm for test solution (a) ; at
298 nm for test solution (b).
Specific absorbance at the absorption maximum : 37 to
43 for test solution (a) ; 64 to 72 for test solution (b).
C. To about 1 mg of powdered substance in a porcelain dish
add 0.5 ml of sulphuric acid-formaldehyde reagent R. A
purple colour develops and becomes violet.
D. It gives the reaction of alkaloids (2.3.1).
E. It gives reaction (a) of chlorides (2.3.1).

TESTS
Solution S. Dissolve 0.500 g in carbon dioxide-free water R
and dilute to 25.0 ml with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y6 or BY6
(2.2.2, Method II).
Acidity or alkalinity. To 10 ml of solution S add 0.05 ml
of methyl red solution R. Not more than 0.2 ml of 0.02 M
sodium hydroxide or 0.02 M hydrochloric acid is required
to change the colour of the indicator.
Specific optical rotation (2.2.7) : 110 to 115 (anhydrous
substance), determined on solution S.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.330 g of the substance to be
examined and 0.100 g of sodium octanesulphonate R in the
mobile phase and dilute to 10.0 ml with the mobile phase.
Reference solution (a). Dilute 1.0 ml of the test solution
to 100.0 ml with the mobile phase. Dilute 1.0 ml of this
solution to 10.0 ml with the mobile phase.
Reference solution (b). Dissolve 25 mg of codeine R in
1.0 ml of the test solution and dilute to 50.0 ml with the
mobile phase.
Column :
size : l = 0.25 m, = 4.6 mm,
stationary phase : octylsilyl silica gel for
chromatography R (5 m)(40).
Mobile phase : to 1.08 g of sodium octanesulphonate R add
250 ml of acetonitrile R and 20 ml of glacial acetic acid R
and dilute to 1000 ml with water R.
Flow rate : 2.0 ml/min.
Detection : spectrophotometer at 283 nm.
Injection : 10 l.
Run time : 4 times the retention time of morphine.
Relative retention with reference to morphine (retention
time = about 4 min) : impurity D = about 0.8 ;
impurity A = about 1.6 ; impurity C = about 2.0 ;
impurity B = about 2.4.
System suitability : reference solution (b) :
resolution : minimum 9 between the peaks due to
morphine and impurity A.
Limits :
correction factor : for the calculation of content, multiply
the peak area of impurity C by 0.25,
impurity B : not more than 4 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.4 per cent),
impurities A, C, D : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent),
any other impurity : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent),
total : not more than 10 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (1.0 per cent),
disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Test solution. Dissolve 0.125 g of the substance to be
examined in a 1 per cent V/V solution of acetic acid R and
dilute to 50.0 ml with the same solution.

(40) Spherisorb Octyl is suitable.

462

PHARMEUROPA Vol. 18, No. 3, July 2006

Morphine hydrochloride

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. impurity D

3. morphine

5. impurity A

2. impurity F

4. impurity E

6. impurity C

7. impurity B

Figure 0097.-1. Chromatogram for the test for related substances of morphine hydrochloride
Reference solution (a). Dilute 1.0 ml of the test solution to
100.0 ml with a 1 per cent V/V solution of acetic acid R.
Dilute 2.0 ml of this solution to 10.0 ml with a 1 per cent V/V
solution of acetic acid R.

Relative retention with reference to morphine (retention

time = about 12.5 min) : impurity C = about 1.6 ;
impurity B = about 1.9.
System suitability : reference solution (b) :
Reference solution (b). Dissolve 5 mg of morphine for
resolution : minimum 1.4 between the peaks due to
system suitability CRS (containing impurities B, C and F) in
impurity F and morphine.
a 1 per cent V/V solution of acetic acid R and dilute to 2 ml Limits :
with the same solution.
correction factors : for the calculation of content,
Column :
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity B = 0.25 ;
size : l = 0.15 m, = 4.6 mm ;
impurity C = 0.4 ;
stationary phase : end-capped octadecylsilyl silica gel

impurity
B : not more than twice the area of the principal
for chromatography R (5 m)(41) ;
peak in the chromatogram obtained with reference
solution (a) (0.4 per cent) ;
temperature : 35 C.

impurity
C : not more than the area of the principal peak
Mobile phase :
in the chromatogram obtained with reference solution (a)
mobile phase A : 1.01 g/l solution of sodium
(0.2 per cent) ;
heptanesulphonate R adjusted to pH 2.6 with a 50 per
any other impurity : for each impurity, not more than the
cent V/V solution of phosphoric acid R ;
area of the principal peak in the chromatogram obtained
mobile phase B : methanol R ;
with reference solution (a) (0.2 per cent) ;
total : not more than 5 times the area of the principal peak
Time
Mobile phase A
Mobile phase B
in the chromatogram obtained with reference solution (a)
(min)
(per cent V/V)
(per cent V/V)
(1.0 per cent) ;
0-2
85
15
disregard limit : 0.25 times the area of the principal peak
2 - 35
85 50
15 50
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
35 - 40
50
50
The thresholds indicated under Related substances
40 - 42
50 85
50 15
(Table 2034.-1) in the general monograph Substances for
42 - 47
85
15
pharmaceutical use (2034) do not apply.
Water (2.5.12) : 12.5 per cent to 15.5 per cent, determined
Flow rate : 1.5 ml/min.
on 0.100 g.
Detection : spectrophotometer at 230 nm.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
Injection : 10 l.
(41) Inertsil ODS-2 or Symmetry C18 are suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

463

Morphine sulphate

ASSAY
Dissolve 0.300 g in a mixture of 5 ml of 0.01 M hydrochloric
acid and 30 ml of ethanol (96 per cent) R. Carry out
a potentiometric titration (2.2.20), using 0.1 M sodium
hydroxide. Read the volume added between the 2 points
of inflexion.
1 ml of 0.1 M sodium hydroxide is equivalent to 32.18 mg
of C17H20ClNO3.
STORAGE
Protected from light.
IMPURITIES
Specified impurities : A, B, C, D.
Other detectable impurities (the following substances
would, if present at a sufficient level, be detected by one
or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified
impurities and/or by the general monograph Substances for
pharmaceutical use (2034). It is therefore not necessary to
identify these impurities for demonstration of compliance.
See also 5.10. Control of impurities in substances for
pharmaceutical use) : A, D, E, F.
A. codeine,

F. morphine N-oxyde.

Reference: PA/PH/Exp. 11/T (06) 41 ANP

NOTE ON THE MONOGRAPH
Related substances : this monograph has already been
revised to replace the TLC by an LC. Following the public
enquiry, the method has been improved to obtain a better
separation of impurities. As for this product of natural
origin has a complex impurity profile, it has not proven
possible to apply the general policy for impurities shown
in the general monograph Substance for pharmaceutical
use (2034). It has been necessary to give an acceptance
criterion equivalent to 0.2 per cent for any other
impurities.
XXXX:1244

MORPHINE SULPHATE
Morphini sulfas
B. 7,7,8,8-tetradehydro-4,5:4,5-diepoxy-17,17-dimethyl2,2-bimorphinanyl-3,3,6,6-tetrol (pseudomorphine
2,2-bismorphine),

C34H40N2O10S,5H2O

C. 6,7,8,14-tetradehydro-4,5-epoxy-6-methoxy-17methylmorphinan-3-ol (oripavine),

Mr 759

DEFINITION
Di(7,8-didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol)
sulphate pentahydrate.
Content : 98.0 per cent to 102.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : soluble in water, very slightly soluble in ethanol
(96 per cent), practically insoluble in toluene.

D. 7,8-didehydro-4,5-epoxy-17-methylmorphinan-3,6,
10-triol (10RS-hydroxymorphine),

E. 7,8-didehydro-4,5-epoxy-17-methyl-6-oxomorphinan-3-ol
(morphinone),
464

IDENTIFICATION
First identification : A, E.
Second identification : B, C, D, E.
A. Infrared absorption spectrophotometry (2.2.24).
Preparation : dissolve 20 mg in 1 ml of water R, add
0.05 ml of 1 M sodium hydroxide and shake. A precipitate
is formed. Filter, wash with 2 quantities, each of 0.5 ml, of
water R and dry the precipitate at 145 C for 1 h. Prepare
discs using the dried precipitate.
Comparison : repeat the operations with morphine
sulphate CRS.
B. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
PHARMEUROPA Vol. 18, No. 3, July 2006

Morphine sulphate

Solution A. Dissolve 25.0 mg in water R and dilute to

25.0 ml with the same solvent.
Test solution (a). Dilute 10.0 ml of solution A to 100.0 ml
with water R.
Test solution (b). Dilute 10.0 ml of solution A to 100.0 ml
with 0.1 M sodium hydroxide.
Spectral range : 250-350 nm for test solutions (a) and (b).
Absorption maximum : at 285 nm for test solution (a) ; at
298 nm for test solution (b).
Specific absorbance at the absorption maximum :
37 to 43 for test solution (a) ; 64 to 72 for test solution (b).
C. To about 1 mg of powdered substance in a porcelain dish
add 0.5 ml of sulphuric acid-formaldehyde reagent R. A
purple colour develops and becomes violet.
D. It gives the reaction of alkaloids (2.3.1).
E. It gives the reactions of sulphates (2.3.1).
TESTS
Solution S. Dissolve 0.500 g in carbon dioxide-free water R
and dilute to 25.0 ml with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y6 or BY6
(2.2.2, Method II).
Acidity or alkalinity. To 10 ml of solution S add 0.05 ml
of methyl red solution R. Not more than 0.2 ml of 0.02 M
sodium hydroxide or 0.02 M hydrochloric acid is required
to change the colour of the indicator.
Specific optical rotation (2.2.7) : 107 to 110 (anhydrous
substance), determined on solution S.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.330 g of the substance to be
examined and 0.100 g of sodium octanesulphonate R in the
mobile phase and dilute to 10.0 ml with the mobile phase.
Reference solution (a). Dilute 1.0 ml of the test solution
to 100.0 ml with the mobile phase. Dilute 1.0 ml of this
solution to 10.0 ml with the mobile phase.
Reference solution (b). Dissolve 25 mg of codeine R in
1.0 ml of the test solution and dilute to 50.0 ml with the
mobile phase.
Column :
size : l = 0.25 m, = 4.6 mm,
stationary phase : octylsilyl silica gel for
chromatography R (5 m)(42).
Mobile phase : to 1.08 g of sodium octanesulphonate R, add
250 ml of acetonitrile R and 20 ml of glacial acetic acid R
and dilute to 1000 ml with water R.
Flow rate : 2.0 ml/min.
Detection : spectrophotometer at 283 nm.
Injection : 10 l.
Run time : 4 times the retention time of morphine.
Relative retention with reference to morphine (retention
time = about 4 min) : impurity D = about 0.8 ;
impurity A = about 1.6 ; impurity C = about 2.0 ;
impurity B = about 2.4.
System suitability : reference solution (b) :
resolution : minimum 9 between the peaks due to
morphine and impurity A.
Limits :
correction factor : for the calculation of content, multiply
the peak area of impurity C by 0.25,

impurity B : not more than 4 times the area of the

principal peak in the chromatogram obtained with
reference solution (a) (0.4 per cent),
impurities A, C, D : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent),
any other impurity : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent),
total : not more than 10 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (1.0 per cent),
disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Test solution. Dissolve 0.125 g of the substance to be
examined in a 1 per cent V/V solution of acetic acid R and
dilute to 50.0 ml with the same solution.
Reference solution (a). Dilute 1.0 ml of the test solution to
100.0 ml with a 1 per cent V/V solution of acetic acid R.
Dilute 2.0 ml of this solution to 10.0 ml with a 1 per cent
V/V solution of acetic acid R.
Reference solution (b). Dissolve 5 mg of morphine for
system suitability CRS (containing impurities B, C and F)
in a 1 per cent V/V solution of acetic acid R and dilute to
2 ml with the same solution.
Column :
size : l = 0.15 m, = 4.6 mm ;
stationary phase : end-capped octadecylsilyl silica gel
for chromatography R (5 m)(43) ;
temperature : 35 C.
Mobile phase :
mobile phase A : 1.01 g/l solution of sodium
heptanesulphonate R adjusted to pH 2.6 with a 50 per
cent V/V solution of phosphoric acid R ;
mobile phase B : methanol R ;
Time
(min)
0-2

Mobile phase A
(per cent V/V)
85

Mobile phase B
(per cent V/V)
15

2 - 35

85 50

15 50

35 - 40

50

50

40 - 42

50 85

50 15

42 - 47

85

15

Flow rate : 1.5 ml/min.

Detection : spectrophotometer at 230 nm.
Injection : 10 l.
Relative retention with reference to morphine (retention
time = about 12.5 min) : impurity C = about 1.6 ;
impurity B = about 1.9.
System suitability : reference solution (b) :
resolution : minimum 1.4 between the peaks due to
impurity F and morphine.
Limits :
correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity B = 0.25 ;
impurity C = 0.4 ;

(42) Spherisorb octyl is suitable.

(43) Inertsil ODS-2 or Symmetry C18 are suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

465

Morphine sulphate

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. impurity D

3. morphine

5. impurity A

2. impurity F

4. impurity E

6. impurity C

7. impurity B

Figure 1244.-1. Chromatogram for the test for related substances of morphine sulphate
impurity B : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.4 per cent) ;
impurity C : not more than the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.2 per cent) ;
any other impurity : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.2 per cent) ;
total: not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(1.0 per cent) ;
disregard limit : 0.25 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
The thresholds indicated under Related substances
(Table 2034.-1) in the general monograph Substances for
pharmaceutical use (2034) do not apply.
Iron (2.4.9) : maximum 5 ppm.
Dissolve the residue from the test for sulphated ash in
water R and dilute to 10.0 ml with the same solvent.
Water (2.5.12) : 10.4 per cent to 13.4 per cent, determined
on 0.100 g.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.

IMPURITIES
Specified impurities : A, B, C, D.
Other detectable impurities (the following substances
would, if present at a sufficient level, be detected by one
or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified
impurities and/or by the general monograph Substances for
pharmaceutical use (2034). It is therefore not necessary to
identify these impurities for demonstration of compliance.
See also 5.10. Control of impurities in substances for
pharmaceutical use) : A, D, E, F.
A. codeine,

B. 7,7,8,8-tetradehydro-4,5:4,5-diepoxy-17,17-dimethyl2,2-bimorphinanyl-3,3,6,6-tetrol (pseudomorphine)
(2,2-bismorphine),

ASSAY
Dissolve 0.500 g in 120 ml of anhydrous acetic acid R.
Titrate with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1 ml of 0.1 M perchloric acid is equivalent to 66.88 mg
of C34H40N2O10S.
STORAGE
Protected from light.
466

C. 6,7,8,14-tetradehydro-4,5-epoxy-6-methoxy-17methylmorphinan-3-ol (oripavine),
PHARMEUROPA Vol. 18, No. 3, July 2006

Mumps vaccine (live)

D. 7,8-didehydro-4,5-epoxy-17-methylmorphinan-3,6,
10-triol (10RS-hydroxymorphine),

basis of the conclusions of the workshop, it is proposed

to revise the present testing scheme to foresee the study
of potential neurovirulence of the vaccine strains during
preclinical development instead of performing the test
for neurovirulence of live virus vaccines (2.6.18) on the
seed lot. This study is based on available data, notably for
wild type virus. Where necessary, a risk analysis study is
considered and, where applicable, a test for neurovirulence
is carried out using an animal model that differentiates
wild type and attenuated virus.
XXXX:0538

MUMPS VACCINE (LIVE)

Vaccinum parotitidis vivum
E. 7,8-didehydro-4,5-epoxy-17-methyl-6-oxomorphinan-3-ol
(morphinone),

DEFINITION
Mumps vaccine (live) is a freeze-dried preparation of a
suitable attenuated strain of mumps virus. The vaccine is
reconstituted immediately before use, as stated on the label,
to give a clear liquid that may be coloured owing to the
presence of a pH indicator.

PRODUCTION
The production of vaccine is based on a virus seed-lot system
and, if the virus is propagated in human diploid cells, a
cell-bank system. The production method shall have been
shown to yield consistently live mumps vaccines of adequate
immunogenicity and safety in man. Unless otherwise
justified and authorised, the virus in the final vaccine shall
F. morphine N-oxyde.
have undergone no more passages from the master seed
lot than were used to prepare the vaccine shown in clinical
studies to be satisfactory with respect to safety and efficacy.
The potential neurovirulence of the vaccine strain is
Reference: PA/PH/Exp. 15/T (04) 76 ANP 1R
considered during preclinical development, based on
available data, notably for wild type virus. Where necessary
NOTE ON THE MONOGRAPH
in light of a risk analysis, a test is carried out on the vaccine
Thermal stability test and Assay : a revision proposal
strain using an animal model that differentiates wild type and
including additional acceptance criteria was published in attenuated virus ; tests on strains of intermediate attenuation
Pharmeuropa 17.2. In light of comments received, a new
may also be needed.
revision proposal is shown below. The main changes are :
The production method is validated to demonstrate that the
deletion of details regarding cell cultures and dilution
product, if tested, would comply with the test for abnormal
steps ; these details are an unnecessary restriction since toxicity for immunosera and vaccines for human use (2.6.9).
the validity criteria give a better assurance of correct
SUBSTRATE FOR VIRUS PROPAGATION
design ;
The virus is propagated in human diploid cells (5.2.3) or in
limits are expressed as logarithms and in such a way
chick-embryo cells or in the amniotic cavity of chick embryos
that excessive rounding is avoided ;
derived from a chicken flock free from specified pathogens
since the aim is to confirm the suitable design of the test, (5.2.2).
validity criteria apply to the reference preparation only ;
and on the combined value obtained from 3 replicates ; SEED LOT
The strain of mumps virus used shall be identified by
a manufacturers reference preparation may be
historical records that include information on the origin of
used provided it is regularly compared with the
the strain and its subsequent manipulation. To avoid the
appropriate BRP ;
unnecessary use of monkeys in the test for neurovirulence,
introduction of control charts in addition to the criteria
Virus seed lots are prepared in large quantities and stored at
0.5 log CCID50, in accordance with good practice ;
temperatures below 20 C if freeze-dried, or below 60 C
deletion of the requirement on the range of virus
if not freeze-dried.
concentration found for the replicate ; the requirement
Only a seed lot that complies with the following requirements
for closeness to the historical value for the reference
preparation, together with the use of a control chart, is may be used for virus propagation.
Identification. The master and working seed lots are
considered to give better control ;
identified as mumps virus by serum neutralisation in cell
introduction of details regarding repetition of the test
culture, using specific antibodies.
and combination of valid results ;
Virus concentration. The virus concentration of the master
reference to chapter 5.3 for calculations.
and working seed lots is determined to ensure consistency
Neurovirulence : the present requirements for
of production.
neurovirulence testing were reviewed at a joint
EDQM-WHO-IABs scientific workshop on neurovirulence
Extraneous agents (2.6.16). The working seed lot complies
tests for live virus vaccines in January 2005. On the
with the requirements for seed lots.
PHARMEUROPA Vol. 18, No. 3, July 2006

467

Mumps vaccine (live)

Neurovirulence (2.6.18). The working seed lot complies with recommended for storage. The virus concentration of the
heated vaccine is not more than 1.0 log lower than that of
the test for neurovirulence of live virus vaccines. Macaca
the unheated vaccine.
and Cercopithecus monkeys are suitable for the test.
The test is not valid if:
PROPAGATION AND HARVEST
All processing of the cell bank and subsequent cell cultures the confidence interval (P = 0.95) of the estimated
is done under aseptic conditions in an area where no other
virus concentration of the reference preparation for the
cells are handled. Suitable animal (but not human) serum
3 replicates combined is greater than 0.3 log CCID50 ;
may be used in the culture media. Serum and trypsin used
the virus concentration of the reference preparation,
in the preparation of cell suspensions and culture media are
monitored using control charts, differs by more than
shown to be free from extraneous agents. The cell culture
0.5 log CCID50 from the value established on a historical
medium may contain a pH indicator such as phenol red and
basis in the particular laboratory. The relation with the
suitable antibiotics at the lowest effective concentration.
appropriate BRP biological reference preparation is
It is preferable to have a substrate free from antibiotics
established and monitored at regular intervals when a
during production. Not less than 500 ml of the production
manufacturers reference preparation is used.
cell cultures is set aside as uninfected cell cultures (control
cells). If the virus is propagated in chick embryos, 2 per
IDENTIFICATION
cent but not less than 20 eggs are set aside as uninfected
When the vaccine reconstituted as stated on the label is
control eggs. The viral suspensions are harvested at a time
mixed with specific mumps antibodies, it is no longer able to
appropriate to the strain of virus being used.
infect susceptible cell cultures.
Only a single harvest that complies with the following
TESTS
requirements may be used in the preparation of the final
bulk vaccine.
Bacterial and fungal contamination. The reconstituted
vaccine complies with the test for sterility (2.6.1).
Identification. The single harvest contains virus that is
identified as mumps virus by serum neutralisation in cell
Bovine serum albumin. Not more than 50 ng per single
culture, using specific antibodies.
human dose, determined by a suitable immunochemical
Virus concentration. The virus concentration in the single method (2.7.1).
harvest is determined as prescribed under Assay to monitor Ovalbumin. If the vaccine is produced in chick embryos,
consistency of production and to determine the dilution to
it contains not more than 1 g of ovalbumin per single
be used for the final bulk vaccine.
human dose, determined by a suitable immunochemical
method (2.7.1).
Extraneous agents (2.6.16). The single harvest complies
with the tests for extraneous agents.
Water (2.5.12). Not more than 3.0 per cent, determined by
the semi-micro determination of water.
Control cells or eggs. If human diploid cells are used
for production, the control cells comply with a test for
identification ; the control cells and the control eggs comply ASSAY
Titrate the vaccine for infective virus at least in triplicate,
with the tests for extraneous agents (2.6.16).
using at least 3 separate vials of vaccine and inoculating a
FINAL BULK VACCINE
suitable number of wells for each dilution step using at least
Single harvests that comply with the above tests are pooled 5 cell cultures for each 0.5 log10 dilution step or by a method
of equal precision. Use Titrate 1 vial of an appropriate virus
and clarified to remove cells. A suitable stabiliser may be
reference preparation in triplicate to validate each assay.
added and the pooled harvests diluted as appropriate.
Calculate the individual virus concentration for each vial of
vaccine and for each replicate of the reference preparation
Only a final bulk vaccine that complies with the following
requirement may be used in the preparation of the final lot. as well as the corresponding combined virus concentrations,
using the usual statistical methods (for example, 5.3). The
Bacterial and fungal contamination. The final bulk vaccine combined estimates of the virus concentration for the 3 vials
complies with the test for sterility (2.6.1), carried out using of vaccine is not less than that stated on the label ; the
10 ml for each medium.
minimum virus concentration stated on the label is not less
than 5 103 CCID50 3.7 log CCID50 per single human dose.
FINAL LOT
The assay is not valid if the confidence interval (P = 0.95) of
A minimum virus concentration for release of the product
the logarithm of the virus concentration is greater than 0.3.
is established such as to ensure, in light of stability data,
that the minimum concentration stated on the label will be The assay is not valid if:
present at the end of the period of validity.
the confidence interval (P = 0.95) of the estimated
virus concentration of the reference preparation for the
Only a final lot that complies with the requirements for
3 replicates combined is greater than 0.3 log CCID50 ;
minimum virus concentration for release, with the following
requirement for thermal stability and with each of the
the virus concentration of the reference preparation,
requirements given below under Identification and Tests
monitored using control charts, differs by more than
may be released for use. Provided that the tests for bovine
0.5 log CCID50 from the value established on a historical
serum albumin and, where applicable, for ovalbumin have
basis in the particular laboratory. The relation with the
been carried out with satisfactory results on the final bulk
appropriate BRP biological reference preparation is
vaccine, they may be omitted on the final lot.
established and monitored at regular intervals when a
manufacturers reference preparation is used.
Thermal stability. Maintain samples at least 3 vials of
the final lot of freeze-dried vaccine in the dry state at
The assay is repeated if the confidence interval (P = 0.95) of
37 1 C for 7 days. Determine the virus concentration as
the combined virus concentration of the vaccine is greater
described under Assay in parallel for the heated vaccine and than 0.3 log CCID50 ; data generated from valid assays only
for unheated vaccine stored at 5 3 C the temperature
are combined by the usual statistical methods (for example,
468

PHARMEUROPA Vol. 18, No. 3, July 2006

Niflumic acid

5.3) to calculate the virus concentration of the sample.

The confidence interval (P = 0.95) of the combined virus
concentration is not greater than 0.3 log CCID50.
Mumps vaccine (live) BRP is suitable for use as a reference
preparation.

Reference solution. Dissolve 25 mg of 3-trifluoromethylaniline R (impurity C) in 20 ml of methanol R and dilute to

100 ml with the same solvent. Dilute 1.0 ml of this solution
to 100 ml with methanol R.
Plate : TLC silica gel F254 plate R(44).
Mobile phase : acetic acid R, ethyl acetate R, toluene R
LABELLING
(5:25:90 V/V/V).
The label states :
Application : 10 l.
the strain of virus used for the preparation of the vaccine ;
Development : over 3/4 of the plate.
that the vaccine has been prepared in chick embryos or
Drying : in air, until the solvents have evaporated.
the type and origin of cells used for the preparation of
Detection : spray with 4-dimethylaminocinnamaldehyde
the vaccine ;
solution
R and heat at 60 C for 10 min.
the minimum virus concentration ;
Limit
:
that contact with disinfectants is to be avoided ;
impurity C : any spot due to impurity C is not more
the time within which the vaccine must be used after
intense than the principal spot in the chromatogram
reconstitution.
obtained with the reference solution (50 ppm).
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 20.0 mg of the substance to be
examined in 10 ml of acetonitrile R and dilute to 20.0 ml
Reference: PA/PH/Exp. 10D/T (06) 5 ANP
with water R.
XXXX:2115 Reference solution (a). Dissolve 10.0 mg of niflumic acid
impurity A CRS, 10.0 mg of niflumic acid impurity B CRS
and 10 mg of niflumic acid impurity E CRS in 45 ml of
NIFLUMIC ACID
acetonitrile R, add 10.0 ml of the test solution and dilute
to 100.0 ml with water R. Dilute 1.0 ml of this solution to
100.0
ml with a mixture of equal volumes of acetonitrile R
Acidum niflumicum
and water R.
Reference solution (b). Dilute 5.0 ml of reference solution (a)
to 10.0 ml with a mixture of equal volumes of acetonitrile R
and water R.
Column :
size : l = 0.125 m, = 4.0 mm ;
stationary phase : octylsilyl silica gel for
chromatography R (5 m)(45) ;
C13H9F3N2O2
Mr 282.2 temperature : 25 C.
Mobile phase : phosphoric acid R, acetonitrile R, water R
DEFINITION
(2.5:500:500 V/V/V).
2-((3-(Trifluoromethyl)phenyl)amino)pyridine-3-carboxylic
Flow rate : 1.0 ml/min.
acid.
Detection : spectrophotometer at 267 nm and 330 nm.
Content : 98.5 per cent to 101.5 per cent (dried substance).
Injection : 10 l.
CHARACTERS
Run time : 4 times the retention time of niflumic acid.
Appearance : pale yellow, crystalline powder.
Relative retention with reference to niflumic acid
(retention time = about 5.5 min) : impurity A = about 0.25 ;
Solubility : practically insoluble in water, freely soluble in
impurity B = about 0.57 ; impurity E = about 0.64.
acetone, soluble in ethanol (96 per cent) and methanol.
System suitability : reference solution (a) :
mp : 202 C to 206 C.
resolution at 330 nm : minimum 1.5 between the peaks
IDENTIFICATION
due to impurities E and B.
Infrared absorption spectrophotometry (2.2.24).
Limits :
Comparison : niflumic acid CRS.
impurity A at 267 nm : not more than the area of the
corresponding peak in the chromatogram obtained with
TESTS
reference solution (a) (0.1 per cent) ;
Appearance of solution. The solution is clear (2.2.1).
impurity B at 330 nm : not more than 4 times the area
Dissolve 0.30 g in methanol R and dilute to 100 ml with the
of the corresponding peak in the chromatogram obtained
same solvent.
with reference solution (a) (0.4 per cent) ;
Impurity C. Thin-layer chromatography (2.2.27).
unspecified impurities at 267 nm and 330 nm : for
each impurity, not more than the area of the peak due
Test solution. Dissolve 0.50 g of the substance to be
to niflumic acid in the chromatogram obtained with
examined in 5 ml of methanol R and dilute to 10.0 ml with
reference solution (a) (0.10 per cent) ;
the same solvent.
(44) Merck 60F254 is suitable.
(45) Lichrospher C8 is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

469

Niflumic acid

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. impurity A

2. impurity B

3. impurity E

4. niflumic acid

Figure 2115.-1. Chromatogram for the test for related substances of niflumic acid : reference solution (b) at 267 nm
The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. impurity B

2. impurity E

3. niflumic acid

Figure 2115.-2. Chromatogram for the test for related substances of niflumic acid : reference solution (b) at 330 nm
sum of impurities other than B at 267 nm and 330 nm :
not more than twice the area of the peak due to niflumic
acid in the chromatogram obtained with reference
solution (a) (0.2 per cent) ;
disregard limit at 267 nm and 330 nm : the area of the
peak due to niflumic acid in the chromatogram obtained
with reference solution (b) (0.05 per cent).
Chlorides (2.4.4) : maximum 200 ppm.
Dissolve 0.5 g in a mixture of 1 ml of nitric acid R and 10 ml
of methanol R, and dilute to 20 ml with water R. To 10 ml of
this solution add 5 ml of water R.
Phosphates (2.4.11) : maximum 100 ppm.
Dilute 1.0 ml of the solution prepared in the test for heavy
metals to 100 ml with water R.
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test C. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.3 per cent, determined
on 1.000 g by drying in a platinum crucible in an oven at
100-105 C.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Dissolve 0.200 g in a mixture of 10 ml of water R and 40 ml
of ethanol (96 per cent) R. Titrate with 0.1 M sodium
hydroxide, determining the end-point potentiometrically
(2.2.20).
1 ml of 0.1 M sodium hydroxide is equivalent to 28.22 mg
of C13H9F3N2O2.
470

IMPURITIES
Specified impurities : A, B, C.
Other detectable impurities (the following substances
would, if present at a sufficient level, be detected by one
or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified
impurities and/or by the general monograph Substances for
pharmaceutical use (2034). It is therefore not necessary to
identify these impurities for demonstration of compliance.
See also 5.10. Control of impurities in substances for
pharmaceutical use) : D, E, F.

A. 2-chloropyridine-3-carboxylic acid,

B. 2-hydroxy-N-(3-(trifluoromethyl)phenyl)pyridine-3carboxamide,

C. 3-(trifluoromethyl)aniline,
PHARMEUROPA Vol. 18, No. 3, July 2006

Nilutamide

IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : nilutamide CRS.
D. 2-hydroxypyridine-3-carboxylic acid,

E. 6-((3-(trifluoromethyl)phenyl)amino)pyridine-3-carboxylic
acid,

F. methyl 2-((3-(trifluoromethyl)phenyl)amino)pyridine-3carboxylate.
Reagents
3-Trifluoromethylaniline. C7H6F3N. (Mr 161.1 ). XXXXXXX.
[98-16-8].
A colourless liquid.
Density : 1.30 g/cm3 (20 C).

Reference: PA/PH/Exp. 10B/T (06) 30 ANP

XXXX:2256

NILUTAMIDE

TESTS
Appearance of solution. The solution is clear (2.2.1) and
not more intensely coloured than reference solution GY6
(2.2.2, Method II).
Dissolve 0.4 g in anhydrous ethanol R and dilute to 20.0 ml
with the same solvent.
Related substances. Liquid chromatography (2.2.29).
Prepare the solutions immediately before use.
Solvent mixture : acetonitrile for chromatography R,
water R (35:65 V/V).
Test solution. Dissolve 0.10 g of the substance to be
examined in the solvent mixture and dilute to 100.0 ml with
the solvent mixture.
Reference solution (a). Dilute 20.0 ml of the test solution
to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this
solution to 100.0 ml with the solvent mixture.
Reference solution (b). Dissolve 5.0 mg of the substance
to be examined and 5.0 mg of nifeline CRS (impurity B) in
the solvent mixture and dilute to 100.0 ml with the solvent
mixture.
Column :
size : l = 0.15 m, = 4.6 mm ;
stationary phase : spherical octadecylsilyl silica gel for
chromatography R (5 m)(46).
Mobile phase :
mobile phase A : 2.0 g/l solution of potassium
dihydrogen phosphate R adjusted to pH 7.5 with 1 M
sodium hydroxide ;
mobile phase B : acetonitrile for chromatography R ;
Time
(min)
0-8

Mobile phase A
(per cent V/V)
55

Mobile phase B
(per cent V/V)
45

8 - 30

55 30

45 70

30 - 31

30 55

70 45

31 - 45

55

45

Flow rate : 1.5 ml/min.

Detection : spectrophotometer at 230 nm.
Injection : 20 l.
Relative retention with reference to nilutamide (retention
time = about 5.3 min) : impurity B = about 0.9.
System suitability : reference solution (b) :
resolution : minimum 3.0 between the peaks due to
impurity B and nilutamide.
C12H10N3O4F3
Mr 317.2
Limits :
DEFINITION
unspecified impurities : for each impurity, not more
5,5-Dimethyl-3-(4-nitro-3-(trifluoromethyl)phenyl)imidazothan 0.5 times the area of the principal peak in the
lidine-2,4-dione.
chromatogram obtained with reference solution (a)
(0.10 per cent) ;
Content : 98.0 per cent to 102.0 per cent (anhydrous
substance).
total : not more than the area of the principal peak in
the chromatogram obtained with reference solution (a)
CHARACTERS
(0.2 per cent) ;
Appearance : white or almost white powder.
disregard limit : 0.25 times the area of the principal peak
Solubility : very slightly soluble in water, freely soluble in
in the chromatogram obtained with reference solution (a)
acetone, soluble in methanol.
(0.05 per cent).

Nilutamidum

(46) Kromasil ODS and Zorbax SB-C18 are suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

471

Nilutamide

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

Figure 2256.-1. Chromatogram for the test for related substances of nilutamide : solution of nilutamide spiked with
impurities A, B, C and D
Heavy metals (2.4.8) : maximum 20 ppm.
Dissolve 0.5 g in a mixture of 10 volumes of water R and
90 volumes of acetone R and dilute to 20 ml with the same
mixture of solvents. 12 ml of the solution complies with
test B. Prepare the reference solution using lead standard
solution (0.5 ppm Pb) obtained by diluting lead standard
solution (100 ppm Pb) R with a mixture of 10 volumes of
water R and 90 volumes of acetone R. Filter the solutions
through a membrane filter (0.45 m).
Water (2.5.12) : maximum 0.5 per cent, determined on
0.500 g.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Liquid chromatography (2.2.29). The solutions are stable
for 24 h at room temperature and in daylight.
Solvent mixture : acetonitrile for chromatography R,
water R (35:65 V/V).
Test solution. Dissolve 50.0 mg of the substance to be
examined in the solvent mixture and dilute to 100.0 ml with
the solvent mixture.
Reference solution (a). Dissolve 50.0 mg of nilutamide CRS
in the solvent mixture and dilute to 100.0 ml with the solvent
mixture.
Reference solution (b). Dissolve 5.0 mg of the substance
to be examined and 5.0 mg of nifeline CRS (impurity B) in
the solvent mixture and dilute to 100.0 ml with the solvent
mixture.
Column :

size : l = 0.15 m, = 4.6 mm ;

stationary phase : spherical octadecylsilyl silica gel for
chromatography R (5 m)(47).
Mobile phase : mix 40 volumes of acetonitrile R and
60 volumes of a 2.0 g/l solution of potassium dihydrogen
phosphate R adjusted to pH 7.5 with 1 M sodium hydroxide.
Flow rate : 1.5 ml/min.
Detection : spectrophotometer at 267 nm.
Injection : 20 l.
Run time : 1.7 times the retention time of nilutamide
(retention time = about 9 min).
System suitability : reference solution (b) :
relative retention with reference to nilutamide (retention
time = about 9 min) : impurity B = about 0.8.
Calculate the percentage content of C12H10N3O4F3 from the
declared content of nilutamide CRS.
STORAGE
Protected from light.
IMPURITIES
Other detectable impurities (the following substances
would, if present at a sufficient level, be detected by one
or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified
impurities and/or by the general monograph Substances for
pharmaceutical use (2034). It is therefore not necessary to
identify these impurities for demonstration of compliance.
See also 5.10. Control of impurities in substances for
pharmaceutical use) : A, B, C, D.

(47) Kromasil ODS and Zorbax SB-C18 are suitable.

472

PHARMEUROPA Vol. 18, No. 3, July 2006

Poliomyelitis vaccine (oral)

XXXX:0215

POLIOMYELITIS VACCINE (ORAL)

Vaccinum poliomyelitidis perorale
A. 5-imino-4,4-dimethyl-1-(4-nitro-3-(trifluoromethyl)phenyl)imidazolidin-2-one,

B. 4-nitro-3-(trifluoromethyl)aniline (nifeline),

C. 5,5-dimethyl-3-(4-nitro-3-(trifluoromethyl)phenyl)-1,3oxazolidine-2,4-dione,

D. N,N-bis(4-nitro-3-(trifluoromethyl)phenyl)urea.

Reference: PA/PH/Exp. 15/T (06) 20 ANP

NOTE ON THE MONOGRAPH
Thermal stability test and Assay : this monograph was
revised following revision by the WHO and was published
in Supplement 5.3 with additional acceptance criteria. The
revised draft below proposes the same changes regarding
the acceptance criteria as those proposed for other live viral
vaccine monographs (Measles, Mumps, Rubella, Varicella
or Oral poliomyelitis vaccine):
deletion of details regarding the method used, only
the cell line remains ; these details are an unnecessary
restriction since the validity criteria give a better
assurance of correct design ;
limits are expressed as logarithms and in such a way
that excessive rounding is avoided ;
since the aim is to confirm the suitable design of the test,
validity criteria apply to the reference preparation only,
and on the combined value obtained from 3 replicates ;
a manufacturers reference preparation may be
used provided it is regularly compared with the
appropriate BRP ;
introduction of control charts in addition to the criteria
0.5 log CCID50, in accordance with good practice ;
deletion of the requirement on the range of virus
concentration found for the replicates, the requirement
for closeness to the historical value for the reference
preparation, together with the use of a control chart, is
considered to give better control ;
introduction of details regarding repetition of the test
and combination of valid results ;
reference to chapter 5.3 for calculations.
PHARMEUROPA Vol. 18, No. 3, July 2006

DEFINITION
Oral poliomyelitis vaccine is a preparation of approved
strains of live attenuated poliovirus type 1, 2 or 3 grown in
in vitro cultures of approved cells, containing any one type
or any combination of the 3 types of Sabin strains, presented
in a form suitable for oral administration.
The vaccine is a clear liquid that may be coloured owing to
the presence of a pH indicator.
PRODUCTION
The vaccine strains and the production method shall have
been shown to yield consistently vaccines that are both
immunogenic and safe in man.
The production of vaccine is based on a virus seed-lot system.
Cell lines are used according to a cell-bank system. If primary
monkey kidney cell cultures are used, production complies
with the requirements indicated below. Unless otherwise
justified and authorised, the virus in the final vaccine shall
not have undergone more than 2 passages from the master
seed lot.
REFERENCE STANDARDS
Poliomyelitis vaccine (oral) types 1, 2, 3 BRP is suitable for
use as a virus reference preparation for the assay.
The International Standards for poliovirus type 2 (Sabin) for
MAPREC (Mutant Analysis by PCR and Restriction Enzyme
Cleavage) assays and poliovirus, type 3 (Sabin) synthetic
DNA for MAPREC assays are suitable for use in the tests
for genetic markers and the molecular tests for consistency
of production.
Reference preparations of each poliovirus type at the Sabin
Original + 2 passage level, namely WHO (SO + 2)/I for
type 1 virus, WHO (SO + 2)/II for type 2 virus and WHO
(SO + 2)/III for type 3 virus are available for comparison of
the in vivo neurovirulence with that of homotypic vaccines.
Requests for the WHO reference preparations for in vivo
neurovirulence tests are to be directed to WHO, Biologicals,
Geneva, Switzerland.
A suitable reference preparation is to be included in each test.
SUBSTRATE FOR VIRUS PROPAGATION
The virus is propagated in human diploid cells (5.2.3), in
continuous cell lines (5.2.3) or in primary monkey kidney
cell cultures (including serially passaged cells from primary
monkey kidney cells).
Primary monkey kidney cell cultures. The following special
requirements for the substrate for virus propagation apply
to primary monkey kidney cell cultures.
Monkeys used for preparation of primary monkey kidney
cell cultures and for testing of virus. If the vaccine is
prepared in primary monkey kidney cell cultures, animals
of a species approved by the competent authority, in good
health, kept in closed or intensively monitored colonies and
not previously employed for experimental purposes shall be
used.
The monkeys shall be kept in well-constructed and
adequately ventilated animal rooms in cages spaced as far
apart as possible. Adequate precautions shall be taken
to prevent cross-infection between cages. Not more than
2 monkeys shall be housed per cage and cage-mates shall not
be interchanged. The monkeys shall be kept in the country of
manufacture of the vaccine in quarantine groups for a period
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Poliomyelitis vaccine (oral)

of not less than 6 weeks before use. A quarantine group is

a colony of selected, healthy monkeys kept in one room,
with separate feeding and cleaning facilities, and having no
contact with other monkeys during the quarantine period. If
at any time during the quarantine period the overall death
rate of a shipment consisting of one or more groups reaches
5 per cent (excluding deaths from accidents or where the
cause was specifically determined not to be an infectious
disease), monkeys from that entire shipment shall continue
in quarantine from that time for a minimum of 6 weeks.
The groups shall be kept continuously in isolation, as in
quarantine, even after completion of the quarantine period,
until the monkeys are used. After the last monkey of a group
has been taken, the room that housed the group shall be
thoroughly cleaned and decontaminated before being used
for a fresh group. If kidneys from near-term monkeys are
used, the mother is quarantined for the term of pregnancy.
Monkeys from which kidneys are to be removed shall be
anaesthetised and thoroughly examined, particularly for
evidence of tuberculosis and cercopithecid herpesvirus 1
(B virus) infection.
If a monkey shows any pathological lesion relevant to the use
of its kidneys in the preparation of a seed lot or vaccine, it
shall not be used, nor shall any of the remaining monkeys of
the quarantine group concerned be used unless it is evident
that their use will not impair the safety of the product.
All the operations described in this section shall be
conducted outside the areas where the vaccine is produced.
The monkeys used shall be shown to be free from antibodies
to simian virus 40 (SV40), simian immunodeficiency virus
and spumaviruses. The blood sample used in testing for
SV40 antibodies must be taken as close as possible to the
time of removal of the kidneys. If Macaca spp. are used for
production, the monkeys shall also be shown to be free from
antibodies to cercopithecid herpesvirus 1 (B virus). Human
herpesvirus has been used as an indicator for freedom from
B virus antibodies on account of the danger of handling
cercopithecid herpesvirus 1 (B virus). Monkeys used for
the production of new seed lots are shown to be free from
antibodies to simian cytomegalovirus (sCMV).
Primary monkey kidney cell cultures for vaccine
production. Kidneys that show no pathological signs are
used for preparing cell cultures. If the monkeys are from a
colony maintained for vaccine production, serially passaged
monkey kidney cell cultures from primary monkey kidney
cells may be used for virus propagation, otherwise the
monkey kidney cells are not propagated in series. Virus for
the preparation of vaccine is grown by aseptic methods in
such cultures. If animal serum is used in the propagation of
the cells, the maintenance medium after virus inoculation
shall contain no added serum.
Each group of cell cultures derived from a single monkey or
from foetuses from no more than 10 near-term monkeys is
prepared and tested as an individual group.
VIRUS SEED LOTS
The strains of poliovirus used shall be identified by
historical records that include information on the origin and
subsequent manipulation of the strains.
Working seed lots are prepared by a single passage from a
master seed lot and at an approved passage level from the
original Sabin virus. Virus seed lots are prepared in large
quantities and stored at a temperature below 60 C.
Only a virus seed lot that complies with the following
requirements may be used for virus propagation.
Identification. Each working seed lot is identified as
poliovirus of the given type, using specific antibodies.
474

Virus concentration. Determined by the method described

below, the virus concentration is the basis for the quantity of
virus used in the neurovirulence test.
Extraneous agents (2.6.16). If the working seed lot is
produced in human diploid cells or in a continuous cell
line, it complies with the requirements for seed lots for
virus vaccines. If the working seed lot is produced in
primary monkey kidney cell cultures, it complies with the
requirements given below under Virus Propagation and
Harvest and Monovalent Pooled Harvest and with the tests
in adult mice, suckling mice and guinea-pigs given in chapter
2.6.16.
In addition to the requirements in chapter 2.6.16, for
vaccines produced in cell lines and when the seed lot was
produced in primary monkey kidney cell cultures, a validated
test for sCMV is performed.
Working seed lots shall be free from detectable DNA
sequences from simian virus 40 (SV40).
Neurovirulence. Each master and working seed lot
complies with the test for neurovirulence of poliomyelitis
vaccine (oral) in monkeys (2.6.19). In addition, at least the
first 4 consecutive batches of monovalent pooled harvest
prepared from these seed lots shall be shown to comply
with the test for neurovirulence of poliomyelitis vaccine
(oral) in monkeys (2.6.19) before the seed lot is deemed
suitable for use. Furthermore, the seed lot shall cease to
be used in vaccine production if the frequency of failure of
the monovalent pooled harvests produced from it is greater
than predicted statistically. This statistical prediction is
calculated after each test on the basis of all the monovalent
pooled harvests tested ; it is equal to the probability of false
rejection on the occasion of a first test (i.e. 1 per cent), the
probability of false rejection on retest being negligible. If the
test is carried out only by the manufacturer, the test slides
are provided to the control authority for assessment.
Genetic markers. Each working seed lot is tested for its
replicating properties at temperatures ranging from 36 C
to 40 C as described under Monovalent pooled harvest. A
profile (i.e. percentage of mutant) of the seed virus using the
MAPREC assay is prepared. Type 3 virus seed lots comply
with the MAPREC assay as described under Monovalent
pooled harvest.
VIRUS PROPAGATION AND HARVEST
All processing of the cell banks and subsequent cell cultures
is done under aseptic conditions in an area where no
other cells are handled. Approved animal (but not human)
serum may be used in the media, but the final medium for
maintaining cell growth during virus multiplication does
not contain animal serum. Serum and trypsin used in
the preparation of cell suspensions and media are shown
to be free from live extraneous agents. The cell-culture
medium may contain a pH indicator such as phenol red and
approved antibiotics at the lowest effective concentration. It
is preferable to have a substrate free from antibiotics during
production. On the day of inoculation with the virus working
seed lot, not less than 5 per cent or 1000 ml, whichever is the
less, of the cell cultures employed for vaccine production are
set aside as uninfected cell cultures (control cells). Special
requirements, given below, apply to control cells when the
vaccine is produced in primary monkey kidney cell cultures.
The virus suspension is harvested not later than 4 days
after virus inoculation. After inoculation of the production
cell culture with the virus working seed lot, inoculated
cells are maintained at a fixed temperature, shown to be
suitable, within the range 33 C to 35 C ; the temperature
is maintained constant to 0.5 C ; control cell cultures are
maintained at 33-35 C for the relevant incubation periods.
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Poliomyelitis vaccine (oral)

Only a single virus harvest that complies with the following

requirements may be used in the preparation of the
monovalent pooled harvest.
Virus concentration. The virus concentration of virus
harvests is determined as prescribed under Assay to monitor
consistency of production and to determine the dilution to
be used for the final bulk vaccine.
Molecular tests for consistency of production. The
MAPREC assay is performed on each virus harvest. The
acceptance/rejection criteria for consistency of production
are determined for each manufacturer and for each working
seed by agreement with the competent authority. These
criteria are periodically reviewed and updated to the
satisfaction of the competent authority. An investigation of
consistency occurs if a virus harvest gives results that are
inconsistent with previous production history.
Control cells. The control cells of the production cell culture
from which the virus harvest is derived comply with a test
for identity and with the requirements for extraneous agents
(2.6.16) or, where primary monkey kidney cell cultures are
used, as shown below.
Primary monkey kidney cell cultures. The following special
requirements apply to virus propagation and harvest in
primary monkey kidney cell cultures.
Cell cultures. On the day of inoculation with virus working
seed lot, each cell culture is examined for degeneration
caused by an infective agent. If, in this examination, evidence
is found of the presence in a cell culture of any extraneous
agent, the entire group of cultures concerned shall be
rejected.
On the day of inoculation with the virus working seed lot,
a sample of at least 30 ml of the pooled fluid removed from
the cell cultures of the kidneys of each single monkey or
from foetuses from not more than 10 near-term monkeys
is divided into 2 equal portions. 1 portion of the pooled
fluid is tested in monkey kidney cell cultures prepared from
the same species, but not the same animal, as that used for
vaccine production. The other portion of the pooled fluid is,
where necessary, tested in monkey kidney cell cultures from
another species so that tests on the pooled fluids are done in
cell cultures from at least 1 species known to be sensitive to
SV40. The pooled fluid is inoculated into bottles of these cell
cultures in such a way that the dilution of the pooled fluid in
the nutrient medium does not exceed 1 in 4. The area of the
cell sheet is at least 3 cm2/ml of pooled fluid. At least 1 bottle
of each kind of cell culture remains uninoculated to serve as
a control. If the monkey species used for vaccine production
is known to be sensitive to SV40, a test in a second species is
not required. Animal serum may be used in the propagation
of the cells, provided that it does not contain SV40 antibody,
but the maintenance medium after inoculation of test
material contains no added serum except as described below.
The cultures are incubated at a temperature of 35-37 C and
are observed for a total period of at least 4 weeks. During
this observation period and after not less than 2 weeks
incubation, at least 1 subculture of fluid is made from
each of these cultures in the same cell culture system. The
subcultures are also observed for at least 2 weeks.
Serum may be added to the original culture at the time of
subculturing, provided that the serum does not contain
SV40 antibody.
Fluorescent-antibody techniques may be useful for detecting
SV40 virus and other viruses in the cells.
A further sample of at least 10 ml of the pooled fluid is
tested for cercopithecid herpesvirus 1 (B virus) and other
viruses in rabbit kidney cell cultures. Serum used in the
PHARMEUROPA Vol. 18, No. 3, July 2006

nutrient medium of these cultures shall have been shown

to be free from inhibitors of B virus. Human herpesvirus
has been used as an indicator for freedom from B virus
inhibitors on account of the danger of handling cercopithecid
herpesvirus 1 (B virus). The sample is inoculated into bottles
of these cell cultures in such a way that the dilution of the
pooled fluid in the nutrient medium does not exceed 1 in 4.
The area of the cell sheet is at least 3 cm2/ml of pooled fluid.
At least 1 bottle of the cell cultures remains uninoculated
to serve as a control.
The cultures are incubated at a temperature of 35-37 C and
observed for at least 2 weeks.
A further sample of 10 ml of the pooled fluid removed from
the cell cultures on the day of inoculation with the seed
lot virus is tested for the presence of extraneous agents by
inoculation into human cell cultures sensitive to measles
virus.
The tests are not valid if more than 20 per cent of the culture
vessels have been discarded for non-specific accidental
reasons by the end of the respective test periods.
If, in these tests, evidence is found of the presence of an
extraneous agent, the single harvest from the whole group
of cell cultures concerned is rejected.
If the presence of cercopithecid herpesvirus 1 (B virus) is
demonstrated, the manufacture of oral poliomyelitis vaccine
shall be discontinued and the competent authority shall
be informed. Manufacturing shall not be resumed until a
thorough investigation has been completed and precautions
have been taken against any reappearance of the infection,
and then only with the approval of the competent authority.
If these tests are not done immediately, the samples of
pooled cell-culture fluid shall be kept at a temperature of
60 C or below, with the exception of the sample for the
test for B virus, which may be held at 4 C, provided that the
test is done not more than 7 days after it has been taken.
Control cell cultures. On the day of inoculation with the
virus working seed lot, 25 per cent (but not more than
2.5 litres) of the cell suspension obtained from the kidneys
of each single monkey or from not more than 10 near-term
monkeys is taken to prepare uninoculated control cell
cultures. These control cell cultures are incubated in the
same conditions as the inoculated cultures for at least
2 weeks and are examined during this period for evidence
of cytopathic changes. The tests are not valid if more than
20 per cent of the control cell cultures have been discarded
for non-specific, accidental reasons. At the end of the
observation period, the control cell cultures are examined
for degeneration caused by an infectious agent. If this
examination or any of the tests required in this section
shows evidence of the presence in a control culture of any
extraneous agent, the poliovirus grown in the corresponding
inoculated cultures from the same group shall be rejected.
Tests for haemadsorbing viruses. At the time of harvest or
within 4 days of inoculation of the production cultures with
the virus working seed lot, a sample of 4 per cent of the
control cell cultures is taken and tested for haemadsorbing
viruses. At the end of the observation period, the remaining
control cell cultures are similarly tested. The tests are carried
out as described in chapter 2.6.16.
Tests for other extraneous agents. At the time of harvest,
or within 7 days of the day of inoculation of the production
cultures with the working seed lot, a sample of at least 20 ml
of the pooled fluid from each group of control cultures is
taken and tested in 2 kinds of monkey kidney cell culture, as
described above.
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Poliomyelitis vaccine (oral)

At the end of the observation period for the original control

cell cultures, similar samples of the pooled fluid are taken
and the tests referred to in this section in the 2 kinds of
monkey kidney cell culture and in the rabbit cell cultures are
repeated, as described above under Cell cultures.
If the presence of cercopithecid herpesvirus 1 (B virus) is
demonstrated, the production cell cultures shall not be used
and the measures concerning vaccine production described
above must be undertaken.
The fluids collected from the control cell cultures at the time
of virus harvest and at the end of the observation period may
be pooled before testing for extraneous agents. A sample of
2 per cent of the pooled fluid is tested in each of the cell
culture systems specified.
Single harvests
Tests for neutralised single harvests in primary monkey
kidney cell cultures. A sample of at least 10 ml of each
single harvest is neutralised by a type-specific poliomyelitis
antiserum prepared in animals other than monkeys. In
preparing antisera for this purpose, the immunising antigens
used shall be prepared in non-simian cells.
Half of the neutralised suspension (corresponding to at
least 5 ml of single harvest) is tested in monkey kidney cell
cultures prepared from the same species, but not the same
animal, as that used for vaccine production. The other half of
the neutralised suspension is tested, if necessary, in monkey
kidney cell cultures from another species so that the tests on
the neutralised suspension are done in cell cultures from at
least 1 species known to be sensitive to SV40.
The neutralised suspensions are inoculated into bottles of
these cell cultures in such a way that the dilution of the
suspension in the nutrient medium does not exceed 1 in 4.
The area of the cell sheet is at least 3 cm2/ml of neutralised
suspension. At least 1 bottle of each type of cell culture
remains uninoculated to serve as a control and is maintained
by nutrient medium containing the same concentration of
the specific antiserum used for neutralisation.
Animal serum may be used in the propagation of the cells,
provided that it does not contain SV40 antibody, but the
maintenance medium, after the inoculation of the test
material, contains no added serum other than the poliovirus
neutralising antiserum, except as described below.
The cultures are incubated at a temperature of 35-37 C
and observed for a total period of at least 4 weeks. During
this observation period and after not less than 2 weeks
incubation, at least 1 subculture of fluid is made from
each of these cultures in the same cell-culture system. The
subcultures are also observed for at least 2 weeks.
Serum may be added to the original cultures at the time
of subculturing, provided that the serum does not contain
SV40 antibody.
Additional tests are made for extraneous agents on a further
sample of the neutralised single harvests by inoculation of
10 ml into human cell cultures sensitive to measles virus.
This test is also validated for the detection of sCMV.
Fluorescent-antibody techniques may be useful for detecting
SV40 virus and other viruses in the cells.
The tests are not valid if more than 20 per cent of the culture
vessels have been discarded for non-specific accidental
reasons by the end of the respective test periods.
If any cytopathic changes occur in any of the cultures, the
causes of these changes are investigated. If the cytopathic
changes are shown to be due to unneutralised poliovirus,
the test is repeated. If there is evidence of the presence of
SV40 or other extraneous agents attributable to the single
harvest, that single harvest is rejected.
476

MONOVALENT POOLED HARVEST

Monovalent pooled harvests are prepared by pooling a
number of satisfactory single harvests of the same virus
type. Monovalent pooled harvests from continuous cell lines
may be purified. Each monovalent pooled harvest is filtered
through a bacteria-retentive filter.
Only a monovalent pooled harvest that complies with the
following requirements may be used in the preparation of
the final bulk vaccine.
Identification. Each monovalent pooled harvest is identified
as poliovirus of the given type, using specific antibodies.
Virus concentration. The virus concentration is determined
by the method described below and serves as the basis for
calculating the dilutions for preparation of the final bulk, for
the quantity of virus used in the neurovirulence test and to
establish and monitor production consistency.
Genetic markers. For Sabin poliovirus type 3, a validated
MAPREC assay is performed. In this analysis the amount
of the mutation at position 472 of the genome (472-C)
is estimated and expressed as a ratio relative to the
International Standard for MAPREC analysis of poliovirus
type 3 (Sabin). A poliovirus type 3 monovalent pooled
harvest found to have significantly more 472-C than the
International Standard for MAPREC analysis of poliovirus
type 3 (Sabin) fails in the MAPREC assay.
The MAPREC analysis of poliovirus type 3 (Sabin) is carried
out using a standard operating procedure approved by the
competent authority. A suitable procedure (Mutant analysis
by PCR and restriction enzyme cleavage (MAPREC) for oral
poliovirus (Sabin) vaccine) is available from WHO, Quality
and Safety of Biologicals (QSB), Geneva. A laboratory must
demonstrate to the competent authority that it is competent
to perform the assay. The manufacturer and the competent
authority shall agree on the procedure and the criteria for
deciding whether a monovalent pooled harvest contains
significantly more 472-C than the International Standard.
Acceptance/rejection criteria for assessment of consistency
of production are determined for each manufacturer and
for each working seed lot by agreement with the competent
authority. These criteria are updated as each new bulk is
prepared and analysed. An investigation of consistency
occurs if a monovalent pooled harvest gives results that are
inconsistent with previous production history.
As the MAPREC assay for type 3 poliovirus (Sabin) is highly
predictive of in vivo neurovirulence, if a filtered monovalent
pooled harvest of type 3 poliovirus (Sabin) fails the MAPREC
assay then this triggers an investigation of the consistency of
the manufacturing process. This investigation also includes
a consideration of the suitability of the working seed lot.
Monovalent pooled harvests passing the MAPREC assay are
subsequently tested for in vivo neurovirulence.
For poliovirus type 3, results from the MAPREC assay and the
monkey neurovirulence test (2.6.19) are used concomitantly
to assess the impact of changes in the production process or
when a new manufacturer starts production.
Pending validation of MAPREC assays for poliovirus types
1 and 2, for these viruses filtered bulk suspension is tested
for the property of reproducing at temperatures of 36 C and
40 C. A ratio of the replication capacities of the virus in the
monovalent pooled harvest is obtained over a temperature
range between 36 C and 40 C in comparison with the seed
lot or a reference preparation for the marker tests and with
appropriate rct/40 and rct/40+ strains of poliovirus of the
same type. The incubation temperatures used in this test are
controlled to within 0.1 C. The monovalent pooled harvest
passes the test if, for both the virus in the harvest and the
appropriate reference material, the titre determined at 36 C
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Poliomyelitis vaccine (oral)

is at least 5.0 log greater than that determined at 40 C. If

growth at 40 C is so low that a valid comparison cannot
be established, a temperature in the region of 39.0-39.5 C
is used, at which temperature the reduction in titre of
the reference material must be in the range 3.0-5.0 log of
its value at 36 C ; the acceptable minimum reduction is
determined for each virus strain at a given temperature. If
the titres obtained for 1 or more of the reference viruses are
not concordant with the expected values, the test must be
repeated.
Neurovirulence (2.6.19). Each monovalent pooled harvest
complies with the test for neurovirulence of poliomyelitis
vaccine (oral). If the monkey neurovirulence test is carried
out only by the manufacturer, the test slides are provided
to the competent authority for assessment. The TgPVR21
transgenic mouse model provides a suitable alternative to
the monkey neurovirulence test for neurovirulence testing
of types 1, 2 or 3 vaccines once a laboratory qualifies as
being competent to perform the test and the experience
gained is to the satisfaction of the competent authority. The
test is carried out using a standard operating procedure
approved by the competent authority. A suitable procedure
(Neurovirulence test of type 1, 2 or 3 live poliomyelitis
vaccines (oral) in transgenic mice susceptible to poliovirus)
is available from WHO, Quality and Safety of Biologicals,
Geneva.
Primary monkey kidney cell cultures. The following special
requirements apply to monovalent pooled harvests derived
from primary monkey kidney cell cultures.
Retroviruses. The monovalent pooled harvest is examined
using a reverse transcriptase assay. No indication of the
presence of retroviruses is found.
Test in rabbits. A sample of the monovalent pooled harvest
is tested for cercopithecid herpesvirus 1 (B virus) and other
viruses by injection of not less than 100 ml into not fewer
than 10 healthy rabbits each weighing 1.5-2.5 kg. Each
rabbit receives not less than 10 ml and not more than 20 ml,
of which 1 ml is given intradermally at multiple sites, and the
remainder subcutaneously. The rabbits are observed for at
least 3 weeks for death or signs of illness.
All rabbits that die after the first 24 h of the test and those
showing signs of illness are examined by autopsy, and
the brain and organs removed for detailed examination to
establish the cause of death.
The test is not valid if more than 20 per cent of the inoculated
rabbits show signs of intercurrent infection during the
observation period. The monovalent pooled harvest passes
the test if none of the rabbits shows evidence of infection
with B virus or with other extraneous agents or lesions of
any kind attributable to the bulk suspension.
If the presence of B virus is demonstrated, the measures
concerning vaccine production described above under Cell
cultures are taken.
Test in guinea-pigs. If the primary monkey kidney cell
cultures are not derived from monkeys kept in a closed
colony, the monovalent pooled harvest shall be shown to
comply with the following test. Administer to not fewer
than 5 guinea-pigs, each weighing 350-450 g, 0.1 ml of
the monovalent pooled harvest by intracerebral injection
and 0.5 ml by intraperitoneal injection. Measure the rectal
temperature of each animal on each working day for 6 weeks.
At the end of the observation period carry out autopsy on
each animal.
In addition, administer to not fewer than 5 guinea-pigs
0.5 ml by intraperitoneal injection and observe as described
above for 2-3 weeks. At the end of the observation period,
carry out a passage from these animals to not fewer than
PHARMEUROPA Vol. 18, No. 3, July 2006

5 guinea-pigs using blood and a suspension of liver or

spleen tissue. Measure the rectal temperature of the latter
guinea-pigs for 2-3 weeks. Examine by autopsy all animals
that, after the first day of the test, die or are euthanised
because they show disease, or show on 3 consecutive days a
body temperature higher than 40.1 C ; carry out histological
examination to detect infection with filoviruses ; in addition,
inject a suspension of liver or spleen tissue or of blood
intraperitoneally into not fewer than 3 guinea-pigs. If any
signs of infection with filoviruses are noted, confirmatory
serological tests are carried out on the blood of the affected
animals. The monovalent pooled harvest complies with the
test if not fewer than 80 per cent of the guinea-pigs survive to
the end of the observation period and remain in good health,
and no animal shows signs of infection with filoviruses.
FINAL BULK VACCINE
The final bulk vaccine is prepared from one or more
satisfactory monovalent pooled harvests and may contain
more than one virus type. Suitable flavouring substances
and stabilisers may be added.
Only a final bulk vaccine that complies with the following
requirement may be used in the preparation of the final lot.
Bacterial and fungal contamination. Carry out the test for
sterility (2.6.1), using 10 ml for each medium.
FINAL LOT
Only a final lot that complies with the following requirement
for thermal stability and is satisfactory with respect to each
of the requirements given below under Identification, Tests
and Assay may be released for use.
IDENTIFICATION
The vaccine is shown to contain poliovirus of each type
stated on the label, using specific antibodies.
TESTS
Bacterial and fungal contamination. The vaccine complies
with the test for sterility (2.6.1).
Thermal stability. Maintain not fewer than 3 vials of the
final lot at 37 1 C for 48 h. Determine the total virus
concentration as described under Assay in parallel for the
heated vaccine and for vaccine maintained at the temperature
recommended for storage. The estimated difference between
the total virus concentration of the unheated and heated
vaccines is not greater than 0.5 log infectious virus units
(CCID50) per single human dose.
The test is not valid if:
the confidence interval (P = 0.95) of the logarithm
of the estimated virus concentration of the reference
preparation for the 3 replicates combined is greater than
0.3 log CCID50 ;
the virus concentration of the reference preparation,
monitored using control charts, differs by more than
0.5 log CCID50 from the assigned value established on a
historical basis in the particular laboratory. The relation
with the appropriate BRP biological reference preparation
is established and monitored at regular intervals when a
manufacturers reference preparation is used.
the range of virus concentrations found for the replicates
of any sample is greater than 0.8 log CCID50.
ASSAY
Titrate the vaccine for infectious virus, using not fewer than
3 separate vials of vaccine, following the method described
below. Use Titrate 1 vial of an appropriate virus reference
preparation in triplicate to validate each assay. If the vaccine
contains more than one poliovirus type, titrate each type
477

Rabies vaccine for human use prepared in cell cultures

separately, using appropriate type-specific antiserum (or

preferably a monoclonal antibody) to neutralise each of the
other types present.
Calculate the individual virus concentration for each vial of
vaccine and for each replicate of the reference preparation
as well as the corresponding combined virus concentrations
using the usual statistical methods (for example, 5.3).
For a trivalent vaccine, the combined estimated mean virus
titres per single human dose must be :
not less than 1 106.0 6.0 log infectious virus units
(CCID50) for type 1 ;
not less than 1 105.0 5.0 log infectious virus units
(CCID50) for type 2 ;
and not less than 1 105.5 5.5 log infectious virus units
(CCID50) for type 3.

Reference: PA/PH/Exp. 15/T (05) 58 ANP

NOTE ON THE MONOGRAPH
The monograph presented below has been revised according
to the 3Rs (Reduction, Replacement, Refinement) approach,
in order to introduce the possibility of replacing the lethal
end-point by a more human end-point. Equivalence of
results obtained by both methods is shown on a suitable
number of vaccines batches.
XXXX:0216

RABIES VACCINE FOR HUMAN USE

PREPARED IN CELL CULTURES
Vaccinum rabiei ex cellulis
ad usum humanum

DEFINITION
Rabies vaccine for human use prepared in cell cultures is a
For a monovalent or divalent vaccine, the minimum virus
freeze-dried preparation of a suitable strain of fixed rabies
titres are decided by the competent authority.
virus grown in cell cultures and inactivated by a validated
Method. Inoculate groups of 8 to 12 flat-bottomed a suitable method.
number of wells in a microtitre plate with 0.1 ml a suitable
The vaccine is reconstituted immediately before use as stated
volume of each of the selected dilutions of virus followed
on the label to give a clear liquid that may be coloured owing
by a suitable cell suspension of the Hep-2 (Cincinnati) line.
to the presence of a pH indicator.
Incubate the plates at a suitable temperature. Examine the
PRODUCTION
cultures on days 7-9.
GENERAL PROVISIONS
The assay is not valid if :
The production of the vaccine is based on a virus seed-lot
system and, if a cell line is used for virus propagation, a
the confidence interval (P = 0.95) of the logarithm
cell-bank system. The production method shall have been
of the estimated virus concentration of the reference
preparation for the 3 replicates combined is greater than shown to yield consistently vaccines that comply with the
requirements for immunogenicity, safety and stability. Unless
0.3 log CCID50 ;
otherwise justified and authorised, the virus in the final
vaccine must not have undergone more passages from the
the virus concentration of the reference preparation,
master seed lot than were used to prepare the vaccine shown
monitored using control charts, differs by more than
in clinical studies to be satisfactory with respect to safety
0.5 log CCID50 from the assigned value established on a
historical basis in the particular laboratory. The relation and efficacy ; even with authorised exceptions, the number
with the appropriate BRP biological reference preparation of passages beyond the level used for clinical studies must
is established and monitored at regular intervals when a not exceed 5.
manufacturers reference preparation is used.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
the range of virus concentrations found for the replicates toxicity for immunosera and vaccines for human use (2.6.9).
for any sample is greater than 0.8 log CCID50.
SUBSTRATE FOR VIRUS PROPAGATION
The assay is repeated if the confidence interval (P = 0.95) of The virus is propagated in a human diploid cell line (5.2.3), in
a continuous cell line approved by the competent authority,
the combined virus concentration of the vaccine is greater
than 0.3 log CCID50 ; data generated from valid assays only or in cultures of chick-embryo cells derived from a flock free
are combined by the usual statistical methods (for example, from specified pathogens (5.2.2).
5.3) to calculate the virus concentration of the sample.
SEED LOTS
The confidence interval (P = 0.95) of the combined virus
The strain of rabies virus used shall be identified by historical
concentration is not greater than 0.3 log CCID50.
records that include information on the origin of the strain
and its subsequent manipulation.
Poliomyelitis vaccine (oral) BRP is suitable for use as a
Working seed lots are prepared by not more than 5 passages
reference preparation.
from the master seed lot.
Only a working seed lot that complies with the following
tests may be used for virus propagation.
LABELLING
Identification. Each working seed lot is identified as rabies
The label states :
virus using specific antibodies.
the types of poliovirus contained in the vaccine ;
Virus concentration. The virus concentration of each
working seed lot is determined by a cell culture method using
the minimum amount of virus of each type contained in
immunofluorescence, to ensure consistency of production.
1 single human dose ;
Extraneous agents (2.6.16). The working seed lot complies
the cell substrate used for the preparation of the vaccine ; with the requirements for virus seed lots. If the virus has
been passaged in mouse brain, specific tests for murine
viruses are carried out.
that the vaccine is not to be injected.
478

PHARMEUROPA Vol. 18, No. 3, July 2006

Rabies vaccine for human use prepared in cell cultures

VIRUS PROPAGATION AND HARVEST

All processing of the cell bank and subsequent cell cultures
is done under aseptic conditions in an area where no
other cells are handled. Approved animal (but not human)
serum may be used in the media, but the final medium for
maintaining cell growth during virus multiplication does
not contain animal serum ; the media may contain human
albumin. Serum and trypsin used in the preparation of cell
suspensions and media are shown to be free from extraneous
agents. The cell culture media may contain a pH indicator
such as phenol red and approved antibiotics at the lowest
effective concentration. Not less than 500 ml of the cell
cultures employed for vaccine production are set aside as
uninfected cell cultures (control cells). The virus suspension
is harvested on one or more occasions during incubation.
Multiple harvests from the same production cell culture may
be pooled and considered as a single harvest.
Only a single harvest that complies with the following
requirements may be used in the preparation of the
inactivated viral harvest.
Identification. The single harvest contains virus that is
identified as rabies virus using specific antibodies.

Glycoprotein content. Determine the glycoprotein

content by a suitable immunochemical method (2.7.1),
for example, single-radial immunodiffusion, enzyme-linked
immunosorbent assay or an antibody-binding test. The
content is within the limits approved for the particular
product.
Sterility (2.6.1). The final bulk vaccine complies with the
test for sterility, carried out using 10 ml for each medium.
FINAL LOT
The final bulk vaccine is distributed aseptically into sterile
containers and freeze-dried to a moisture content shown to
be favourable to the stability of the vaccine. The containers
are then closed so as to avoid contamination and the
introduction of moisture.
Only a final lot that complies with each of the requirements
given below under Identification, Tests and Assay may be
released for use. Provided that the test for residual infectious
virus has been carried out with satisfactory results on the
inactivated viral suspension and the test for bovine serum
albumin has been carried out with satisfactory results on the
final bulk vaccine, these tests may be omitted on the final lot.

IDENTIFICATION
The vaccine is shown to contain rabies virus antigen by
a suitable immunochemical method (2.7.1) using specific
antibodies, preferably monoclonal ; alternatively, the assay
Control cells. The control cells of the production cell culture serves also to identify the vaccine.
from which the single harvest is derived comply with a test
TESTS
for identification and with the requirements for extraneous
Residual infectious virus. Inoculate a quantity equivalent to
agents (2.6.16).
not less than 25 human doses of vaccine into cell cultures of
PURIFICATION AND INACTIVATION
the same type as those used for production of the vaccine.
A passage may be made after 7 days. Maintain the cultures
The virus harvest may be concentrated and/or purified
for a total of 21 days and then examine the cell cultures for
by suitable methods ; the virus harvest is inactivated by a
validated method at a fixed, well-defined stage of the process, rabies virus using an immunofluorescence test. No rabies
virus is detected.
which may be before, during or after any concentration
or purification. The method shall have been shown to be
Bovine serum albumin : maximum 50 ng per single human
capable of inactivating rabies virus without destruction of
dose, determined by a suitable immunochemical method
the immunogenic activity. If betapropiolactone is used, the
(2.7.1).
concentration shall at no time exceed 1:3500.
Sterility (2.6.1). It complies with the test.
Only an inactivated viral suspension that complies with the Bacterial endotoxins (2.6.14) : less than 25 IU per single
following requirements may be used in the preparation of
human dose.
the final bulk vaccine.
Pyrogens (2.6.8). Unless otherwise justified and authorised,
Residual infectious virus. Carry out an amplification test for it complies with the test. Unless otherwise justified and
residual infectious rabies virus immediately after inactivation authorised, inject into each rabbit a single human dose of
or using a sample frozen immediately after inactivation and the vaccine diluted to 10 times its volume.
stored at 70 C. Inoculate a quantity of inactivated viral
Water (2.5.12) : maximum 3.0 per cent.
suspension equivalent to not less than 25 human doses of
vaccine into cell cultures of the same type as those used for ASSAY
production of the vaccine. A passage may be made after
The potency of rabies vaccine is determined by comparing
7 days. Maintain the cultures for a total of 21 days and
the dose necessary to protect mice against the effects of
then examine the cell cultures for rabies virus using an
a lethal dose of rabies virus, administered intracerebrally,
immunofluorescence test. No rabies virus is detected.
with the quantity of a reference preparation of rabies
Residual host-cell DNA. If a continuous cell line is used for vaccine necessary to provide the same protection. For
virus propagation, the content of residual host-cell DNA,
this comparison a reference preparation of rabies vaccine,
determined using a suitable method as described in Products calibrated in International Units, and a suitable preparation
of recombinant DNA technology (0784), is not greater than of rabies virus for use as the challenge preparation are
100 pg per single human dose.
necessary.
The International Unit is the activity contained in a stated
FINAL BULK VACCINE
quantity of the International Standard. The equivalence in
The final bulk vaccine is prepared from one or more
International Units of the International Standard is stated
inactivated viral suspensions. An approved stabiliser may
be added to maintain the activity of the product during and by the World Health Organisation.
The test described below uses a parallel-line model with
after freeze-drying.
at least 3 points for the vaccine to be examined and the
Only a final bulk vaccine that complies with the following
reference preparation. Once the analyst has experience with
requirements may be used in the preparation of the final lot. the method for a given vaccine, it is possible to carry out
Virus concentration. Titrate for infective virus in cell
cultures ; the titre is used to monitor consistency of
production.

PHARMEUROPA Vol. 18, No. 3, July 2006

479

Rabies vaccine for human use prepared in cell cultures

a simplified test using a single dilution of the vaccine to

be examined. Such a test enables the analyst to determine
that the vaccine has a potency significantly higher than
the required minimum, but does not give full information
on the validity of each individual potency determination.
The use of a single dilution allows a considerable reduction
in the number of animals required for the test and must
be considered by each laboratory in accordance with the
provisions of the European Convention for the Protection
of Vertebrate Animals used for Experimental and other
Scientific Purposes.
Selection and distribution of the test animals. Use healthy
female mice that are about 4 weeks old, each weighing
11-15 g, and from the same stock. Distribute the mice into 6
groups of a size suitable to meet the requirements for validity
of the test and, for titration of the challenge suspension, 4
groups of 5.
Preparation of the challenge suspension. Inoculate mice
intracerebrally with the Challenge Virus Standard (CVS)
strain of rabies virus and when the mice show signs of rabies,
but before they die, euthanise them, then remove the brains
and prepare a homogenate of the brain tissue in a suitable
diluent. Separate gross particulate matter by centrifugation
and use the supernatant liquid as the challenge suspension.
Distribute the suspension in small volumes in ampoules,
seal and store at a temperature below 60 C. Thaw one
ampoule of the suspension and make serial dilutions in a
suitable diluent. Allocate each dilution to a group of 5 mice
and inject intracerebrally into each mouse 0.03 ml of the
dilution allocated to its group. Observe the mice for 14 days.
Calculate the LD50 of the undiluted suspension using the
number in each group that, between the 5th and 14th days,
die or develop signs of rabies.
Determination of potency of the vaccine. Prepare 3 fivefold
serial dilutions of the vaccine to be examined and 3 fivefold
serial dilutions of the reference preparation. Prepare the
dilutions such that the most concentrated suspensions may
be expected to protect more than 50 per cent of the animals
to which they are administered and the least concentrated
suspensions may be expected to protect less than 50 per
cent of the animals to which they are administered. Allocate
the 6 dilutions, 1 to each of the 6 groups of mice, and inject
intraperitoneally into each mouse 0.5 ml of the dilution
allocated to its group. After 7 days, prepare 3 identical
dilutions of the vaccine to be examined and of the reference
preparation and repeat the injections. 7 days after the
second injection, prepare a suspension of the challenge
virus such that, on the basis of the preliminary titration,
0.03 ml contains about 50 LD50. Inject intracerebrally into
each vaccinated mouse 0.03 ml of this suspension. Prepare
3 suitable serial dilutions of the challenge suspension.
Allocate the challenge suspension and the 3 dilutions,
1 to each of the 4 groups of 5 control mice, and inject
intracerebrally into each mouse 0.03 ml of the suspension
or dilution allocated to its group. Observe the animals in
each group for 14 days and record the number in each group
that die or show signs of rabies in the period 5-14 days after
challenge.
The test is not valid unless :
for both the vaccine to be examined and the reference
preparation the 50 per cent protective dose lies between
the largest and smallest doses given to the mice ;

480

the titration of the challenge suspension shows that

0.03 ml of the suspension contain not less than 10 LD50 ;
the confidence limits (P = 0.95) are not less than 25 per
cent and not more than 400 per cent of the estimated
potency ;
the statistical analysis shows a significant slope and no
significant deviations from linearity or parallelism of the
dose-response curves.
The vaccine complies with the test if the estimated potency
is not less than 2.5 IU per human dose.
Application of alternative end-points. Once a laboratory
has established the above assay for routine use, the lethal
end-point should be replaced by an observation of clinical
signs and application of an end-point earlier than death to
reduce animal suffering. The progress of rabies infection in
mice following intracerebral injection can be represented by
5 stages defined by typical clinical signs :
Stage 1 : ruffled fur, hunched back ;
Stage 2 : slow movements, loss of alertness (circular
movements may also occur) ;
Stage 3 : shaky movements, trembling, convulsions ;
Stage 4 : signs of paralysis ;
Stage 5 : moribund state.
Mice are observed twice daily from day 4 after challenge.
Clinical signs are recorded using a chart such as that shown
in Table 0216.-1. Experience has shown that using stage 3 as
an end-point yields assay results equivalent to those found
when a lethal end-point is used. This should be verified by
each laboratory by scoring a suitable number of assays using
both the clinical signs and the lethal end-point.
Further details of the use of clinical signs are given in the
report of Workshop 48 of the European Centre for the
Validation of Alternative Methods (ECVAM).
Table 0216.-1. Example of a chart used to record clinical
signs in the rabies vaccine potency test
Days after challenge
4

Clinical signs

10

11

Ruffled fur
Hunched back
Slow movements
Circular movements
Trembling
Shaky movements
Convulsions
Paresis
Paralysis
Prostration
Agony, coma

LABELLING
The label states the biological origin of the cells used for the
preparation of the vaccine.

PHARMEUROPA Vol. 18, No. 3, July 2006

Rabies vaccine (inactivated) for veterinary use

Reference: PA/PH/Exp. 15V/T (05) 53 ANP

NOTE ON THE MONOGRAPH
The monograph presented below has been revised according
to the 3Rs (Reduction, Replacement, Refinement) approach,
in order to introduce the possibility of replacing the lethal
end-point by a more humane end-point. Equivalence of
results obtained by both methods is shown on a suitable
number of vaccine batches.
XXXX:0451

RABIES VACCINE (INACTIVATED) FOR

VETERINARY USE
Vaccinum rabiei inactivatum
ad usum veterinarium
DEFINITION
Rabies vaccine (inactivated) for veterinary use is a liquid or
freeze-dried preparation of fixed rabies virus inactivated by a
suitable method.
PRODUCTION
The vaccine is prepared from virus grown either in suitable
cell lines or in primary cell cultures from healthy animals
(5.2.4). The virus suspension is harvested on one or more
occasions within 28 days of inoculation. Multiple harvests
from a single production cell culture may be pooled and
considered as a single harvest. The rabies virus is inactivated
by a suitable method.
Inactivation. The test for residual live rabies virus is carried
out by inoculation of the inactivated virus into the same
type of cell culture as that used in the production of the
vaccine or a cell culture shown to be at least as sensitive ; the
quantity of inactivated virus used in the test is equivalent to
not less than 25 doses of the vaccine. After incubation for
4 days, a subculture is made using trypsinised cells ; after
incubation for a further 4 days, the cultures are examined for
residual live rabies virus by an immunofluorescence test. No
live virus is detected.
Antigen content. The content of rabies virus glycoprotein is
determined by a suitable immunochemical method (2.7.1).
The content is within the limits approved for the particular
preparation.
The vaccine may contain one or more adjuvants.
CHOICE OF VACCINE COMPOSITION
The vaccine is shown to be satisfactory with respect to
immunogenicity for each species for which it is recommended.
The suitability of the vaccine with respect to immunogenicity
for carnivores (cats and dogs) is demonstrated by direct
challenge. For other species, if a challenge test has been
carried out for the vaccine in cats or dogs, an indirect test
is carried out by determining the antibody level following
vaccination of not fewer than 20 animals according to the
recommended schedule ; the vaccine is satisfactory if, after
the period claimed for protection, the mean rabies virus
antibody level in the serum of the animals is not less than
0.5 IU/ml, and not more than 10 per cent of the animals have
an antibody level less than 0.1 IU/ml. The test described
below may be used to demonstrate immunogenicity in cats
and dogs.
Immunogenicity. Use not fewer than 35 susceptible animals
of the minimum age recommended for vaccination. Take
a blood sample from each animal and test each sample
individually for antibodies against rabies virus to determine
susceptibility. Administer one dose of vaccine by the
recommended route to each of not fewer than 25 animals.
PHARMEUROPA Vol. 18, No. 3, July 2006

Keep not fewer than 10 animals as controls. Observe all

the animals for a period equal to the claimed duration
of immunity. No animal shows signs of rabies. On the
last day of the period of claimed duration of immunity or
later, challenge all animals by intramuscular injection of
virulent rabies virus of a strain approved by the competent
authority. Observe the animals for 90 days. Animals that die
from causes not attributable to rabies are eliminated. The
test is not valid if the number of such deaths reduces the
number of vaccinated animals in the test to fewer than 25.
The test is not valid unless at least 8 of the 10 control
animals (or a statistically equivalent number if more than
10 control animals are challenged) show signs of rabies and
the presence of rabies virus in their brain is demonstrated
by the fluorescent-antibody test or another suitable method.
The vaccine complies with the test if not more than 2 of the
25 vaccinated animals (or a statistically equivalent number
if more than 25 vaccinated animals are challenged) show
signs of rabies.
BATCH TESTING
The test described under Potency is not necessarily carried
out for routine testing of batches of vaccine. It is carried out,
for a given vaccine, on one or more occasions, as decided by
or with the agreement of the competent authority. Where
the test is not carried out, a suitable validated alternative
method is used, the criteria for acceptance being set with
reference to a batch of vaccine that has given satisfactory
results in the test described above for immunogenicity or
in the test described under Potency. The following test
may be used after a suitable correlation with the test for
immunogenicity described above or the test described under
Potency has been established.
Batch potency test. Use 5 mice each weighing 18-20 g.
Vaccinate each mouse subcutaneously or intramuscularly
using 1/5 of the recommended dose volume. Take
blood samples 14 days after the injection and test the
sera individually for rabies antibody using the rapid
fluorescent focus inhibition test described for Human rabies
immunoglobulin (0723). The amount of antibody is not less
than that produced by a vaccine that has been found to be
satisfactory with respect to immunogenicity as described
above or in the test described under Potency.
Antigen content. The quantity of rabies virus glycoprotein
per dose, determined by a suitable immunochemical method
(2.7.1), is not significantly lower than that of a batch that has
been found to be satisfactory with respect to immunogenicity
as described above or in the test described under Potency.
IDENTIFICATION
When injected into animals, the vaccine stimulates the
production of specific neutralising antibodies.
TESTS
Safety. If the vaccine is intended for use in more than one
species including one belonging to the order of Carnivora,
carry out the test in dogs. Otherwise, use one of the
species for which the vaccine is intended. Administer by
a recommended route a double dose of vaccine to each
of 2 animals that have no antibodies against rabies virus.
Observe the animals for 14 days. No abnormal local or
systemic reaction occurs.
Inactivation. Carry out the test using a pool of the contents
of 5 containers.
For vaccines that do not contain an adjuvant, carry out
a suitable amplification test for residual infectious rabies
virus, using the same type of cell culture as that used in the
production of the vaccine or a cell culture shown to be at
least as sensitive. No live virus is detected.
481

Rabies vaccine (inactivated) for veterinary use

For vaccines that contain an adjuvant, inject intracerebrally

into each of not fewer than 10 mice, each weighing 11-15 g,
0.03 ml of a pool of at least 5 times the smallest stated dose.
To avoid interference from any antimicrobial preservative
or the adjuvant, the vaccine may be diluted up to 10 times
before injection. In this case, or if the vaccine strain is
pathogenic only for unweaned mice, carry out the test on
mice that are 1-4 days old. Observe the animals for 21 days.
If more than 2 animals die during the first 48 h, repeat the
test. From the 3rd to the 21st day following the injection, the
animals show no signs of rabies, and immunofluorescence
tests carried out on the brains of the animals show no
indication of the presence of rabies virus.

Determination of potency of the vaccine. Prepare at least

3 serial dilutions of the vaccine to be examined and 3 similar
dilutions of the reference preparation. Prepare the dilutions
such that those containing the largest quantity of vaccine
may be expected to protect more than 50 per cent of the
animals into which they are injected and those containing
the smallest quantities of vaccine may be expected to protect
less than 50 per cent of the animals into which they are
injected. Allocate each dilution to a different group of mice
and inject intraperitoneally into each mouse 0.5 ml of the
dilution allocated to its group. 14 days after the injection,
prepare a suspension of the challenge virus such that, on the
basis of the preliminary titration, it contains about 50 ID50
in each 0.03 ml. Inject intracerebrally into each vaccinated
Sterility. The vaccine complies with the test for sterility
mouse 0.03 ml of this suspension. Prepare 3 suitable serial
prescribed in the monograph on Vaccines for veterinary
dilutions of the challenge suspension. Allocate the challenge
use (0062).
suspension and the 3 dilutions, 1 to each of 4 groups of
10 unvaccinated mice, and inject intracerebrally into each
POTENCY
mouse 0.03 ml of the suspension or dilution allocated to its
group. Observe the animals in each group for 14 days. The
The potency of the vaccine is determined by comparing
test is not valid if more than 2 mice of any group die within
the dose necessary to protect mice against the clinical effects the first 4 days after challenge. Record the numbers in each
of the dose of rabies virus defined below, administered
group that show signs of rabies in the period 5-14 days after
intracerebrally, with the quantity of a reference preparation, challenge.
calibrated in International Units, necessary to provide the
same protection.
The test is not valid unless :

The International Unit is the activity of a stated quantity of for both the vaccine to be examined and the reference
the International Standard. The equivalence in International
preparation the 50 per cent protective dose lies between
Units of the International Standard is stated by the World
the smallest and the largest dose given to the mice ;
Health Organisation.
the titration of the challenge suspension shows that
Rabies vaccine (inactivated) for veterinary use BRP is
0.03 ml of the suspension contains at least 10 ID50 ;
calibrated in International Units against the International
the confidence limits (P = 0.95) are not less than 25 per
Standard.
cent and not more than 400 per cent of the estimated
The test described below uses a parallel-line model with
potency ;
at least 3 points for the vaccine to be examined and the
the statistical analysis shows a significant slope and no
reference preparation. Once the analyst has experience
significant deviations from linearity or parallelism of the
with the method for a given vaccine, it is possible to carry
dose-response lines.
out a simplified test using one dilution of the vaccine to be
examined. Such a test enables the analyst to determine
The vaccine complies with the test if the estimated potency
that the vaccine has a potency significantly higher than
is not less than 1 IU in the smallest prescribed dose.
the required minimum, but will not give full information
on the validity of each individual potency determination. It Application of alternative end-points. Once a laboratory
has established the above assay for routine use, the lethal
allows a considerable reduction in the number of animals
end-point should be replaced by an observation of clinical
required for the test and should be considered by each
laboratory in accordance with the provisions of the European signs and application of an end-point earlier than death to
reduce animal suffering. The progress of rabies infection in
Convention for the Protection of Vertebrate Animals used
mice following intracerebral injection can be represented by
for Experimental and Other Scientific Purposes.
5 stages defined by typical clinical signs :
Selection and distribution of the test animals. Use in the
test healthy female mice that are about 4 weeks old and from Stage 1 : ruffled fur, hunched back ;
the same stock. Distribute the mice into at least 10 groups
Stage 2 : slow movements, loss of alertness (circular
of not fewer than 10 mice.
movements may also occur) ;
Preparation of the challenge suspension. Inoculate a
Stage 3 : shaky movements, trembling, convulsions ;
group of mice intracerebrally with the Challenge Virus
Standard (CVS) strain of rabies virus and when the mice
Stage 4 : signs of paralysis ;
show signs of rabies, but before they die, euthanise them,
Stage 5 : moribund state.
then remove the brains and prepare a homogenate of the
brain tissue in a suitable diluent. Separate gross particulate Mice are observed twice daily from day 4 after challenge.
matter by centrifugation and use the supernatant liquid
Clinical signs are recorded using a chart such as that shown
as the challenge suspension. Distribute the suspension in
in Table 0451.-1. Experience has shown that using stage 3 as
small volumes into ampoules, seal and store at a temperature an end-point yields assay results equivalent to those found
below 60 C. Thaw one ampoule of the suspension and
when a lethal end-point is used. This should be verified by
make serial dilutions in a suitable diluent. Allocate each
each laboratory by scoring a suitable number of assays using
dilution to a group of mice and inject intracerebrally into
both the clinical signs and the lethal end-point.
each mouse 0.03 ml of the dilution allocated to its group.
Observe the animals for 14 days and record the number in
Further details of the use of clinical signs are given in the
each group that, between the 5th and 14th day, develop signs report of Workshop 48 of the European Centre for the
of rabies. Calculate the ID50 of the undiluted suspension.
Validation of Alternative Methods (ECVAM).
482

PHARMEUROPA Vol. 18, No. 3, July 2006

Roselle

Table 0451.-1. Example of a chart used to record clinical

signs in the rabies vaccine potency test
Days after challenge
4

Clinical signs

10

11

Ruffled fur
Hunched back
Slow movements
Circular movements
Trembling
Shaky movements
Convulsions
Paresis
Paralysis
Prostration
Agony, coma

LABELLING
The label states :
the type of cell culture used to prepare the vaccine and
the species of origin ;
the minimum number of International Units per dose ;
the minimum period for which the vaccine provides
protection.

the parenchyma containing numerous crystal clusters of

calcium oxalate and, sporadically, mucilage-filled cavities,
sometimes associated with polygonal epidermal cells
and anisocytic stomata (2.8.3) ; numerous fragments
of vascular bundles with spiral and reticulate vessels ;
sclerenchymatous fibres with a wide lumen ; rarely,
rectangular, pitted parenchymatous cells ; fragments
of unicellular, smooth, bent covering trichomes and
occasional glandular trichomes ; rounded pollen grains
with a spiny exine.
C. Thin-layer chromatography (2.2.27).
Test solution. To 1.0 g of the powdered drug (355) add
10 ml of ethanol (60 per cent V/V) R. Shake for 15 min
and filter.
Reference solution. Dissolve 2.5 mg of quinaldine red R
and 2.5 mg of sulphan blue R in 10 ml of ethanol (96 per
cent) R methanol R.
Plate : TLC silica gel plate R (5-40 m) [or TLC silica gel
plate R (2-10 m)].
Mobile phase : acetic acid R anhydrous formic acid R,
water R, butanol R (15:30:60 V/V/V) (10:12:40 V/V/V).
Application : 20 l, as bands 10 l as bands of 10 mm [or
2 l as bands of 8 mm].
Development : over a path of 10 cm [or 6 cm].
Drying : in air.
Detection : examine in daylight on the same day.
Results : see below the sequence of the zones present in
the chromatograms obtained with the reference solution
and the test solution. Furthermore, other faint zones
may be present in the chromatogram obtained with the
test solution.
Top of the plate

Reference: PA/PH/Exp. 13B/T (06) 43 ANP

NOTE ON THE MONOGRAPH
In order to validate the separation and spacing between
the zones, a 2nd reference substance has been added to the
reference solution used in the TLC identification test.
XXXX:1623

ROSELLE
Hibisci sabdariffae flos

_______

_______

Quinaldine red : an orange-red

zone
A pale violet zone
Sulphan blue : a blue zone
_______

A violet-blue zone An intense

violet zone
_______
A violet-blue zone An intense
violet-blue zone
A violet-blue zone

DEFINITION
Reference solution
Test solution
Whole or cut dried calyces and epicalyces of Hibiscus
sabdariffa L. collected during fruiting.
TESTS
Content : minimum 13.5 per cent of acids, expressed as citric
Foreign matter (2.8.2) : maximum 2 per cent of fragments of
acid (C6H8O7 ; Mr 192.1) (dried drug).
fruits (red funicles and parts of the 5-caverned capsule with
CHARACTERS
yellowish-grey pericarp, whose thin walls consist of several
layers of differently directed fibres ; flattened, reniform seeds
Acidic taste.
with a dotted surface).
IDENTIFICATION
Loss on drying (2.2.32) : maximum 11.0 per cent, determined
A. The calyx is joined in the lower half to form an urceolate on 1.000 g of the powdered drug (355) by drying in an oven
structure, the upper half dividing to form 5 long
at 100-105 C for 2 h.
acuminate recurved tips. The tips have a prominent,
Total ash (2.4.16) : maximum 10.0 per cent.
slightly protruding midrib and a large, thick nectary
gland about 1 mm in diameter. The epicalyx consists of
Colouring power intensity. Reduce 100 g to a coarse
8-12 small, obovate leaflets, which are adnate to the base powder (1400) and homogenise. Reduce about 10 g of this
of the calyx. The calyx and epicalyx are fleshy, dry, easily mixture to a powder (355). To 1.0 g of the powdered drug
fragmented and bright red or deep purple, somewhat
(355) add 25 ml of boiling water R in a 100 ml flask and heat
lighter at the base of the inner side.
for 15 min on a water-bath with frequent shaking. Filter the
hot mixture into a 50 ml graduated flask ; rinse successively
B. Reduce to a powder (355). The powder is red or
purplish-red. Examine under a microscope using chloral the 100 ml flask and the filter with 3 quantities, each of 5 ml,
of warm water R. After cooling, dilute to 50 ml with water R.
hydrate solution R. The powder shows the following
Dilute 5 ml of this solution to 50 ml with water R. Measure
diagnostic characters : predominantly red fragments of
PHARMEUROPA Vol. 18, No. 3, July 2006

483

Rubella vaccine (live)

the absorbance (2.2.25) at 520 nm using water R as the

compensation liquid. The absorbance is not less than 0.350
for the whole drug and not less than 0.250 for the cut drug.
ASSAY
Shake 1.000 g of the powdered drug (355) with 100 ml of
carbon dioxide-free water R for 15 min. Filter. To 50.0 ml
of the filtrate add 100 ml of carbon dioxide-free water R.
Titrate with 0.1 M sodium hydroxide to pH 7.0, determining
the end-point potentiometrically (2.2.20).
1 ml of 0.1 M sodium hydroxide is equivalent to 6.4 mg
of citric acid.

XXXX:0162

RUBELLA VACCINE (LIVE)

Vaccinum rubellae vivum
DEFINITION
Rubella vaccine (live) is a freeze-dried preparation of a
suitable attenuated strain of rubella virus. The vaccine is
reconstituted immediately before use, as stated on the label,
to give a clear liquid that may be coloured owing to the
presence of a pH indicator.

PRODUCTION
The production of vaccine is based on a virus seed-lot
system and a cell-bank system. The production method shall
have been shown to yield consistently live rubella vaccines
of adequate immunogenicity and safety in man. Unless
otherwise justified and authorised, the virus in the final
vaccine shall have undergone no more passages from the
master seed lot than were used to prepare the vaccine shown
Reference: PA/PH/Exp. 15/T (04) 75 ANP 1R
in clinical studies to be satisfactory with respect to safety
and efficacy.
NOTE ON THE MONOGRAPH
The potential neurovirulence of the vaccine strain is
considered during preclinical development, based on
Thermal stability test and Assay : a revision proposal
including additional acceptance criteria was published in available data, notably for wild type virus. Where necessary
in light of a risk analysis, a test is carried out on the vaccine
Pharmeuropa 17.2. In light of comments received, a new
revision proposal is shown below. The main changes are : strain using an animal model that differentiates wild type and
attenuated virus ; tests on strains of intermediate attenuation
deletion of details regarding cell cultures and dilution
may also be needed.
steps ; these details are an unnecessary restriction since
The production method is validated to demonstrate that the
the validity criteria give a better assurance of correct
product, if tested, would comply with the test for abnormal
design ;
toxicity for immunosera and vaccines for human use (2.6.9).
limits are expressed as logarithms and in such a way
SUBSTRATE FOR VIRUS PROPAGATION
that excessive rounding is avoided ;
The virus is propagated in human diploid cells (5.2.3).
since the aim is to confirm the suitable design of the test, SEED LOT
validity criteria apply to the reference preparation only, The strain of rubella virus used shall be identified by
and on the combined value obtained from 3 replicates ; historical records that include information on the origin of
the strain and its subsequent manipulation. To avoid the
a manufacturers reference preparation may be
unnecessary
use of monkeys in the test for neurovirulence,
used provided it is regularly compared with the
Virus seed lots are prepared in large quantities and stored at
appropriate BRP ;
temperatures below 20 C if freeze-dried, or below 60 C
introduction of control charts in addition to the criteria if not freeze-dried.
0.5 log CCID50, in accordance with good practice ;
Only a seed lot that complies with the following requirements
may be used for virus propagation.
deletion of the requirement on the range of virus
concentration found for the replicate ; the requirement
Identification. The master and working seed lots are
for closeness to the historical value for the reference
identified as rubella virus by serum neutralisation in cell
preparation, together with the use of a control chart, is culture, using specific antibodies.
considered to give better control ;
Virus concentration. The virus concentration of the master
and working seed lots is determined to ensure consistency
introduction of details regarding repetition of the test
of production.
and combination of valid results ;
Extraneous agents (2.6.16). The working seed lot complies
reference to chapter 5.3 for calculations.
with the requirements for seed lots.
Neurovirulence : the present requirements for
Neurovirulence (2.6.18). The working seed lot complies with
neurovirulence testing were reviewed at a joint
the test for neurovirulence of live virus vaccines. Macaca
EDQM-WHO-IABs scientific workshop on neurovirulence
and Cercopithecus monkeys are suitable for the test.
tests for live virus vaccines in January 2005. On the
PROPAGATION AND HARVEST
basis of the conclusions of the workshop, it is proposed
All processing of the cell bank and subsequent cell cultures
to revise the present testing scheme to foresee the study
is done under aseptic conditions in an area where no other
of potential neurovirulence of the vaccine strains during
cells are handled. Suitable animal (but not human) serum
preclinical development instead of performing the test
may be used in the growth medium, but the final medium
for neurovirulence of live virus vaccines (2.6.18) on the
seed lot. This study is based on available data, notably for for maintaining cell growth during virus multiplication
does not contain animal serum. Serum and trypsin used in
wild type virus. Where necessary, a risk analysis study is
considered and, where applicable, a test for neurovirulence the preparation of cell suspensions and culture media are
shown to be free from extraneous agents. The cell culture
is carried out using an animal model that differentiates
medium may contain a pH indicator such as phenol red and
wild type and attenuated virus.
484

PHARMEUROPA Vol. 18, No. 3, July 2006

Rubella vaccine (live)

suitable antibiotics at the lowest effective concentration.

It is preferable to have a substrate free from antibiotics
during production. Not less than 500 ml of the production
cell cultures is set aside as uninfected cell cultures (control
cells). The temperature of incubation is controlled during
the growth of the virus. The virus suspension is harvested,
on one or more occasions, within 28 days of inoculation.
Multiple harvests from the same production cell culture may
be pooled and considered as a single harvest.
Only a single harvest that complies with the following
requirements may be used in the preparation of the final
bulk vaccine.
Identification. The single harvest contains virus that is
identified as rubella virus by serum neutralisation in cell
culture, using specific antibodies.
Virus concentration. The virus concentration in the single
harvest is determined as prescribed under Assay to monitor
consistency of production and to determine the dilution to
be used for the final bulk vaccine.
Extraneous agents (2.6.16). The single harvest complies
with the tests for extraneous agents.
Control cells. The control cells comply with a test for
identification and with the tests for extraneous agents
(2.6.16).
FINAL BULK VACCINE
Single harvests that comply with the above tests are pooled
and clarified to remove cells. A suitable stabiliser may be
added and the pooled harvests diluted as appropriate.
Only a final bulk vaccine that complies with the following
requirement may be used in the preparation of the final lot.
Bacterial and fungal contamination. The final bulk vaccine
complies with the test for sterility (2.6.1), carried out using
10 ml for each medium.
FINAL LOT
A minimum virus concentration for release of the product
is established such as to ensure, in light of stability data,
that the minimum concentration stated on the label will be
present at the end of the period of validity.
Only a final lot that complies with the requirements for
minimum virus concentration for release, with the following
requirement for thermal stability and with each of the
requirements given below under Identification and Tests may
be released for use. Provided that the test for bovine serum
albumin has been carried out with satisfactory results on the
final bulk vaccine, it may be omitted on the final lot.
Thermal stability. Maintain samples at least 3 vials of
the final lot of freeze-dried vaccine in the dry state at
37 1 C for 7 days. Determine the virus concentration as
described under Assay in parallel for the heated vaccine and
for unheated vaccine stored at 5 3 C the temperature
recommended for storage. The virus concentration of the
heated vaccine is not more than 1.0 log lower than that of
the unheated vaccine.
The test is not valid if :
the confidence interval (P = 0.95) of the estimated
virus concentration of the reference preparation for the
3 replicates combined is greater than 0.3 log CCID50 ;
the virus concentration of the reference preparation,
monitored using control charts, differs by more than
0.5 log CCID50 from the value established on a historical
basis in the particular laboratory. The relation with the
appropriate BRP biological reference preparation is
established and monitored at regular intervals when a
manufacturers reference preparation is used.
PHARMEUROPA Vol. 18, No. 3, July 2006

IDENTIFICATION
When the vaccine reconstituted as stated on the label is
mixed with specific rubella antibodies, it is no longer able to
infect susceptible cell cultures.
TESTS
Bacterial and fungal contamination. The reconstituted
vaccine complies with the test for sterility (2.6.1).
Bovine serum albumin. Not more than 50 ng per single
human dose, determined by a suitable immunochemical
method (2.7.1).
Water (2.5.12). Not more than 3.0 per cent, determined by
the semi-micro determination of water.
ASSAY
Titrate the vaccine for infective virus at least in triplicate,
using at least 3 separate vials of vaccine and inoculating a
suitable number of wells for each dilution step using at least
5 cell cultures for each 0.5 log10 dilution step or by a method
of equal precision. Use Titrate 1 vial of an appropriate virus
reference preparation in triplicate to validate each assay.
Calculate the individual virus concentration for each vial of
vaccine and for each replicate of the reference preparation
as well as the corresponding combined virus concentrations,
using the usual statistical methods (for example, 5.3). The
combined estimates of the virus concentration for the 3 vials
of vaccine is not less than that stated on the label ; the
minimum virus concentration stated on the label is not less
than 1x103CCID50 3.0 log CCID50 per single human dose. The
assay is not valid if the confidence interval (P = 0.95) of the
logarithm of the virus concentration is greater than 0.3.
The assay is not valid if:
the confidence interval (P = 0.95) of the estimated
virus concentration of the reference preparation for the
3 replicates combined is greater than 0.3 log CCID50 ;
the virus concentration of the reference preparation,
monitored using control charts, differs by more than
0.5 log CCID50 from the value established on a historical
basis in the particular laboratory. The relation with the
appropriate BRP biological reference preparation is
established and monitored at regular intervals when a
manufacturers reference preparation is used.
The assay is repeated if the confidence interval (P = 0.95) of
the combined virus concentration of the vaccine is greater
than 0.3 log CCID50 ; data generated from valid assays only
are combined by the usual statistical methods (for example,
5.3) to calculate the virus concentration of the sample.
The confidence interval (P = 0.95) of the combined virus
concentration is not greater than 0.3 log CCID50.
Rubella vaccine (live) BRP is suitable for use as a reference
preparation.
LABELLING
The label states :
the strain of virus used for the preparation of the vaccine ;
the type and origin of the cells used for the preparation
of the vaccine ;
the minimum virus concentration ;
that contact with disinfectants is to be avoided ;
the time within which the vaccine must be used after
reconstitution ;
that the vaccine must not be given to a pregnant woman
and that a woman must not become pregnant within
2 months after having the vaccine.
485

Selamectin for veterinary use

Reference: PA/PH/Exp. 7/T (05) 67 ANP

TESTS
Related substances. Liquid chromatography (2.2.29).
XXXX:2268
Solvent mixture : water R, acetonitrile R (800:1200 V/V).
solution. Dissolve 25 mg of the substance to be
SELAMECTIN FOR VETERINARY USE Test
examined in the solvent mixture and dilute to 50.0 ml with
the solvent mixture.
Selamectinum ad usum veterinarium
Reference solution (a). Dilute 1.0 ml of the test solution to
100.0 ml with the solvent mixture.
Reference solution (b). Dissolve 5 mg of selamectin for
system suitability CRS (containing impurities A, B, C and
D) in the solvent mixture and dilute to 100.0 ml with the
solvent mixture.
Column :
size : l = 0.15 m, = 3.9 mm ;
stationary phase : end-capped octadecylsilyl silica gel
for chromatography R (4 m)(48) ;
temperature : 30 C.
Mobile phase :
mobile phase A : water R ;
mobile phase B : acetonitrile R ;
C43H63NO11
Mr 770
DEFINITION
(2aE,4E,5S,6S,6S,7S,8E,11R,13R,15S,17aR,20aR,
20bS)-6-Cyclohexyl-7-[(2,6-dideoxy-3-O-methyl-L-arabino-hexopyranosyl)oxy]-3,4,5,6,6,7,10,
11,14,15,20a,20b-dodecahydro-20b-hydroxy-5,6,
8,19-tetramethylspiro[11,15-methano-2H,13H,17H-furo[4,3,2-pq][2,6]benzodioxacyclooctadecin-13,2[2H]pyran]-17,20(17aH)-dione 20-oxime.
Semi-synthetic product derived from a fermentation product.
Content : 96.0 per cent to 102.0 per cent (anhydrous and
solvent-free substance).
CHARACTERS
Appearance : white or almost white powder.
Solubility : practically insoluble in water, freely soluble
in isopropyl alcohol, soluble in acetone and in methylene
chloride, sparingly soluble in methanol.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : selamectin CRS.

Time
(min)
0 - 28

Mobile phase A
(per cent V/V)
40

Mobile phase B
(per cent V/V)
60

28 - 45

40 20

60 80

45 - 46

20 40

80 60

46 - 48

40

60

Flow rate : 2.0 ml/min.

Detection : spectrophotometer at 243 nm.
Injection : 20 l.
Identification of impurities : use the chromatogram
supplied with selamectin for system suitability CRS and
the chromatogram obtained with reference solution (b) to
identify the peaks due to impurities A, B, C and D.
Relative retention with reference to selamectin
(retention time = about 22 min) : impurity A = about 0.2 ;
impurity B = about 0.4 ; impurity C = about 0.5 ;
impurity D = about 1.7.
System suitability : reference solution (b) :
resolution : minimum 10 between the peaks due to
impurities B and C.

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. impurity A

2. impurity B

3. impurity C

4. selamectin

5. impurity D

Figure 2268.-1. Chromatogram for the test for related substances of selamectin for veterinary use
(48) Nova-Pak C18 is suitable.

486

PHARMEUROPA Vol. 18, No. 3, July 2006

Selamectin for veterinary use

Limits :

IMPURITIES

correction factor : for the calculation of content, multiply

the peak area of impurity D by 1.5 ;

Specified impurities : A, B, C, D.

impurities A, B : for each impurity, not more than twice

the area of the principal peak in the chromatogram
obtained with reference solution (a) (2.0 per cent) ;
impurities C, D : for each impurity, not more than 1.5 times
the area of the principal peak in the chromatogram
obtained with reference solution (a) (1.5 per cent) ;
any other impurity : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (1.0 per cent) ;
total: not more than 4 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(4.0 per cent) ;
disregard limit : 0.2 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.2 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test C. Prepare the reference solution
using 2 ml of lead standard solution (10 ppm Pb) R.

A. (5Z,23S,25R)-25-cyclohexyl-4-O-de(2,6-dideoxy-3O-methyl--L-arabino-hexopyranosyl)-5-demethoxy25-de(1-methylpropyl)-22,23-dihydro-23-hydroxy-5hydroxyiminoavermectin A1a,

Water (2.5.12, Method A) : maximum 7.0 per cent, determined

on 0.20 g.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Liquid chromatography (2.2.29).
Test solution. Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 250.0 ml with
the mobile phase.
Reference solution. Dissolve 50.0 mg of selamectin CRS
in the mobile phase and dilute to 250.0 ml with the mobile
phase.
Column :
size : l = 0.15 m, = 3.9 mm ;
stationary phase: end-capped octadecylsilyl silica gel
for chromatography R (4 m)(49) ;

B. (5Z,25R)-25-cyclohexyl-4-O-de(2,6-dideoxy-3-O-methyl-L-arabino-hexopyranosyl)-5-demethoxy-25-de(1methylpropyl)-5-hydroxyiminoavermectin A1a,

temperature : 30 C.
Mobile phase : water R, acetonitrile R (400:1600 V/V).
Flow rate : 1.0 ml/min.
Detection : spectrophotometer at 243 nm.
Injection: 20 l.
Run time : twice the retention time of selamectin.
Retention time : selamectin = about 9 min.
Calculate the percentage content of C43H63NO11 from the
declared content of selamectin CRS.
STORAGE
In an airtight container.

C. (5Z,13S,25S)-25-cyclohexyl-25-demethyl-5-deoxy-13hydroxy-5-hydroxyiminomilbemycin 1,

(49) Nova-Pak C18 is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

487

Sodium phenylbutyrate

Impurity C. Gas chromatography (2.2.28).

Silylation solution. To 2 ml of N,O-bis(trimethylsilyl)trifluoroacetamide R add 0.04 ml of chlorotrimethylsilane R
and mix.
Test solution. Dissolve 50.0 mg of the substance to be
examined in 3 ml of water R and add 0.5 ml of hydrochloric
acid R. Extract with 2 quantities, each of 5 ml, of methylene
chloride R. Evaporate the combined methylene chloride
extracts to dryness in a vial with a screw cap and add 0.5 ml
of the silylation solution. Seal the vial and heat at 70 5 C
for 20 min.
Reference solution (a). Dissolve 5.0 mg of sodium
phenylbutyrate impurity C CRS in methylene chloride R
and dilute to 10.0 ml with the same solvent.
D. (5Z,25S)-25-cyclohexyl-5-demethoxy-25-de(1-methylpropyl)-22,23-dihydro-5-hydroxyiminoavermectin A1a.
Reference solution (b). Dilute 1.0 ml of reference solution (a)
to 10.0 ml with methylene chloride R. Place 1.0 ml of this
solution in a vial with a screw cap, evaporate to dryness and
add 0.5 ml of the silylation solution. Seal the vial and heat at
70 5 C for 20 min.
Reference: PA/PH/Exp. 10D/T (04) 24 ANP 1R
Reference solution (c). Dissolve 10 mg of the substance to
NOTE ON THE MONOGRAPH
be examined in 25 ml of water R. To 3 ml of the solution add
0.1 ml of hydrochloric acid R. Extract with 2 quantities, each
This monograph has already been published in
of 5 ml, of methylene chloride R. Combine the methylene
Pharmeuropa 17.1. Further changes are proposed.
chloride extracts and add 2 ml of reference solution (a).
Definition : the lower limit for the content has been
Evaporate to dryness in a vial with a screw cap and add
tightened, based on batch data.
0.5 ml of the silylation solution. Seal the vial and heat at
Related substances : the daily dose exceeds 2 g, therefore
70 5 C for 20 min.
the limit for unspecified impurities is 0.05 per cent and
Column :
the disregard level is 0.02 per cent ; it is also proposed
material : fused silica ;
to increase the level for impurity A from 0.05 per cent to
0.1 per cent, as it is a degradation product.
size : l = 25 m, = 0.25 mm ;
Heavy metals : method G has been replaced by method B.
stationary phase : poly(dimethyl)(diphenyl)siloxane R
XXXX:2183
(film thickness 1.0 m)(50).
Carrier gas : helium for chromatography R.
SODIUM PHENYLBUTYRATE
Flow rate : 0.9 ml/min.
Split ratio : 1:100.
Natrii phenylbutyras
Temperature :

Column

C10H11NaO2

Mr 186.2

DEFINITION
Sodium 4-phenylbutanoate.
Content : 98.0 99.0 per cent to 101.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or yellowish-white powder.
Solubility : freely soluble in water and methanol, practically
insoluble in methylene chloride.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : sodium phenylbutyrate CRS.
B. Dissolve 0.15 g in 2 ml of water R. The solution gives
reaction (a) of sodium (2.3.1).
TESTS
pH (2.2.3) : 6.5 to 7.5.
Dissolve 0.20 g in carbon dioxide-free water R and dilute to
10 ml with the same solvent.

Time
(min)
0-5

Temperature
(C)
50

5 - 27

50 270

27 - 32

270

Injection port

270

Detector

270

Detection : flame ionisation.

Injection : 1 l.
Relative retention with reference to phenylbutyrate
(retention time = about 20 min) : impurity C = about 0.98.
System suitability : reference solution (c) :
resolution : minimum 3.0 between the peaks due to
impurity C and phenylbutyrate.
Limit :
impurity C : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (b) (0.1 per cent).
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.20 g of the substance to be
examined in 10 ml of methanol R and dilute to 50.0 ml with
water R.

(50) HP-5 is suitable.

488

PHARMEUROPA Vol. 18, No. 3, July 2006

Sodium phenylbutyrate

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. impurity A

2. impurity B

3. phenylbutyrate

Figure 2183.-1. Chromatogram for the test for related substances of sodium phenylbutyrate
Reference solution (a). Dissolve 4.0 mg of -tetralone R
(impurity B) in 10 ml of methanol R and dilute to 200.0 ml
with the same solvent.
Reference solution (b). Dissolve 0.20 g of the substance to
be examined in 10 ml of methanol R, add 1 ml of reference
solution (a) and dilute to 50.0 ml with water R.
Reference solution (b) (c). Dilute 1.0 ml of reference
solution (a) to 50.0 ml with water R.
Reference solution (c) (d). Dissolve 5.0 mg of
3-benzoylpropionic acid R (impurity A) in 2.5 ml of
methanol R and dilute to 50.0 ml with the same solvent.
Dilute 1.0 ml of this solution to 50.0 ml with water R.
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase : base-deactivated end-capped
octadecylsilyl silica gel for chromatography R (5 m)(51).
Mobile phase : glacial acetic acid R, methanol R, water R
(1:49:50 V/V/V).
Flow rate : 1.3 ml/min.
Detection : spectrophotometer at 245 nm.
Injection: 20 l of the test solution and reference
solutions (b), (c) and (d).
Run time : twice the retention time of phenylbutyrate.
Relative retention with reference to phenylbutyrate
(retention time = about 17 min) : impurity A = about 0.3 ;
impurity B = about 0.7.
System suitability : reference solution (a) (b) :
resolution : minimum 6 between the peaks due to
impurity B and phenylbutyrate.
Limits :
impurity B : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (b) (c) (0.01 per cent) ;
impurity A : not more than twice the area of the
corresponding peak in the chromatogram obtained with
reference solution (c) (d) (0.05 0.1 per cent) ;
unspecified impurities : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (c) (d) (0.1 0.05 per cent) ;

sum of impurities other than A and B : not more than

twice the area of the principal peak in the chromatogram
obtained with reference solution (c) (d) (0.1 per cent) ;
disregard limit of impurities other than A and
B : 0.4 times the area of the principal peak in the
chromatogram obtained with reference solution (c) (d)
(0.05 0.02 per cent).
Heavy metals (2.4.8) : maximum 10 ppm.
0.5 g complies with test G. Prepare the reference solution
using 0.5 ml of lead standard solution (10 ppm Pb) R.
Dissolve 2.0 g in a mixture of 25 volumes of water R and
75 volumes of anhydrous ethanol R and dilute to 20 ml
with the same mixture of solvents. 12 ml of the solution
complies with test B. Prepare the reference solution using
lead standard solution (1 ppm Pb), obtained by diluting
lead standard solution (100 ppm Pb) R with a mixture
of 25 volumes of water R and 75 volumes of anhydrous
ethanol R.
Water (2.5.12) : maximum 0.5 per cent, determined on 1.000
2.000 g.
ASSAY
Dissolve 0.150 g in 50 ml of anhydrous acetic acid R. The
solution obtained is not clear ; during the titration, the
opalescence disappears. Titrate with 0.1 M perchloric acid,
determining the end-point potentiometrically (2.2.20).
1 ml of 0.1 M perchloric acid is equivalent to 18.62 mg
of C10H11NaO2.
IMPURITIES
Specified impurities : A, B, C.

A. 4-oxo-4-phenylbutanoic acid (3-benzoylpropionic acid),

B. 3,4-dihydronaphtalen-1(2H)-one (-tetralone),

(51) Kromasil C18, endcapped, base-deactivated, is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

489

Sucrose mono- and distearate

C. 4-cyclohexylbutanoic acid.
Reagents
3-Benzoylpropionic acid. C10H10O3. (Mr 178.2). XXXXXXX.
[2051-95-8]. 4-Oxo-4-phenylbutanoic acid.
mp : about 118 C.
-Tetralone. C10H10O. (Mr 146.2). XXXXXXX. [529-34-0].
1-Oxotetraline. 3,4-Dihydro-1(2H)-naphthalen-1(2H)-one.
bp : about 115 C.
mp : about 5 C.

Reference: PA/PH/Exp. 13H/T (04) 26 ANP

XXXX:2317

SUCROSE MONO- AND DISTEARATE

Sacchari mono- et distearas
DEFINITION
Mixture of sucrose mono- and diesters, mainly sucrose monoand distearate, obtained by transesterification of stearic acid
methyl esters of vegetable origin with sucrose (0204). It
contains variable quantities of mono- and diesters.
Content :
monoesters : 20.0 per cent to 45.0 per cent ;
diesters : 30.0 per cent to 40.0 per cent ;
sum of triesters and polyesters : maximum 15.0 per cent.

Test solution. Dissolve 0.200 g of the substance to be

examined in the solvent mixture and dilute to 4.0 ml with the
solvent mixture.
Reference solution. Dissolve 50.0 mg of sucrose CRS in
the solvent mixture and dilute to 10.0 ml with the solvent
mixture. Dilute 1.0 ml of this solution to 10.0 ml with the
solvent mixture.
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase : spherical aminopropylsilyl silica gel
for chromatography R (4 m)(52).
Mobile phase :
mobile phase A : 0.01 g/l solution of ammonium
acetate R in acetonitrile for chromatography R ;
mobile phase B : 0.01 g/l solution of ammonium
acetate R in a mixture of 10 volumes of water for
chromatography R and 90 volumes of tetrahydrofuran
for chromatography R ;
Time
(min)
0-1

Mobile phase A
(per cent V/V)
100

Mobile phase B
(per cent V/V)
0

Flow rate
(litres/min)
1.0

1-9

100 0

0 100

1.0

9 - 16

100

1.0

16 - 16.01

100

1.0 2.5

16.01 - 32

100

2.5

32 - 33

0 100

100 0

2.5

33 - 36

100

2.5 1.0

Detection : evaporative light-scattering detector(53) ; the

following settings have been found to be suitable ; if the
detector has different setting parameters, adjust the detector
settings so as to comply with the system suitability criterion :
carrier gas : nitrogen R ;
CHARACTERS
flow rate : 1.0 litres/min ;
Appearance : white or almost white, unctuous powder.
evaporator temperature : 45 C ;
Solubility : very slightly soluble in water, sparingly soluble
nebuliser temperature : 40 C.
in ethanol (96 per cent).
Injection : 20 l.
IDENTIFICATION
Retention time : about 26 min.
A. Composition of fatty acids (see Tests).
System suitability : reference solution :
B. It complies with the limits of the assay.
signal-to-noise ratio : minimum 10.
Limit : maximum 4.0 per cent.
TESTS
Water (2.5.12) : maximum 4.0 per cent, determined on 0.2 g.
Acid value (2.5.1) : maximum 6.0, determined on 3.00 g.
Use a freshly neutralised mixture of 1 volume of water R and Total ash (2.4.16) : maximum 1.5 per cent.
2 volumes of 2-propanol R as solvent and heat gently.
ASSAY
Composition of fatty acids (2.4.22, Method C). Use the
Size-exclusion chromatography (2.2.30) : use the
mixture of calibrating substances in Table 2.4.22.-1.
normalisation procedure.
Composition of the fatty-acid fraction of the substance :
Test solution. Dissolve 60.0 mg of the substance to be
lauric acid : maximum 3.0 per cent ;
examined in tetrahydrofuran R and dilute to 4.0 ml with the
same solvent.
myristic acid : maximum 3.0 per cent ;
Column :
palmitic acid : 25.0 per cent to 40.0 per cent ;
size : l = 0.6 m, = 7 mm ;
stearic acid : 55.0 per cent to 75.0 per cent ;
stationary phase : styrene-divinylbenzene copolymer R
sum of the contents of palmitic acid and stearic acid :
(5 m) with a pore size of 10 nm(54).
minimum 90.0 per cent.
Mobile phase : tetrahydrofuran R.
Free sucrose. Liquid chromatography (2.2.29) : use the
Flow rate : 1.2 ml/min.
normalisation procedure.
Detection : differential refractometer.
Solvent mixture : water for chromatography R,
tetrahydrofuran for chromatography R (12.5:87.5 V/V).
Injection : 20 l.

(52) Nova-Pak in a Waters High Performance Carbohydrate column is suitable.

(53) Polymer Laboratories PL-ELS 1000 is suitable.
(54) Polymer Laboratories gel column reference 1110-8520 is suitable.

490

PHARMEUROPA Vol. 18, No. 3, July 2006

Sucrose monopalmitate

Relative retention with reference to monoesters (retention

time = about 10 min) : diesters = about 0.90 ; triesters and
polyesters = about 0.92.
Calculations :
free fatty acids : calculate the percentage content (D) of
free fatty acids, using the following expression :

IA

= acid value,

monoesters : calculate the percentage content of

monoesters using the following expression :

monoesters : minimum 55.0 per cent ;

diesters : maximum 40.0 per cent ;
sum of triesters and polyesters: maximum 20.0 per cent.
CHARACTERS
Appearance : white or almost white, unctuous powder.
Solubility : very slightly soluble in water, sparingly soluble
in ethanol (96 per cent).
IDENTIFICATION
A. Composition of fatty acids (see Tests).
B. It complies with the limits of the assay.

TESTS
Acid value (2.5.1) : maximum 6.0, determined on 3.00 g.
Use a freshly neutralised mixture of 1 volume of water R and
2 volumes of 2-propanol R as solvent and heat gently.
Composition of fatty acids (2.4.22, Method C). Use the
A
= percentage content of monoesters determined
mixture of calibrating substances in Table 2.4.22.-1.
by the normalisation procedure ;
Composition of the fatty-acid fraction of the substance :
S
= percentage content of sucrose determined in
lauric acid : maximum 3.0 per cent ;
the test for free sucrose ;
myristic acid : maximum 3.0 per cent ;
diesters : calculate the percentage content of diesters
palmitic acid : 70.0 per cent to 85.0 per cent ;
using the following expression :
stearic acid : 10.0 per cent to 25.0 per cent ;
sum of the contents of palmitic acid and stearic acid :
minimum 90.0 per cent.
Free sucrose. Liquid chromatography (2.2.29) : use the
normalisation procedure.
B
= percentage content of diesters determined by
Solvent mixture : water for chromatography R,
the normalisation procedure ;
tetrahydrofuran for chromatography R (12.5:87.5 V/V).
sum of triesters and polyesters : calculate the sum of the
percentage contents of triesters and polyesters using the Test solution. Dissolve 0.200 g of the substance to be
examined in the solvent mixture and dilute to 4.0 ml with the
following expression :
solvent mixture.
Reference solution. Dissolve 50.0 mg of sucrose CRS in
the solvent mixture and dilute to 10.0 ml with the solvent
mixture. Dilute 1.0 ml of this solution to 10.0 ml with the
solvent mixture.
C
= sum of the percentage contents of triesters and Column :
polyesters determined by the normalisation
size : l = 0.25 m, = 4.6 mm ;
procedure.
stationary phase : spherical aminopropylsilyl silica gel
for chromatography R (4 m)(55).
STORAGE
Mobile phase :
Protected from humidity.
mobile phase A : 0.01 g/l solution of ammonium
acetate R in acetonitrile for chromatography R ;
mobile phase B : 0.01 g/l solution of ammonium
acetate R in a mixture of 10 volumes of water for
Reference: PA/PH/Exp. 13H/T (03) 85 ANP
chromatography R and 90 volumes of tetrahydrofuran
for chromatography R ;
XXXX:2319

SUCROSE MONOPALMITATE
Sacchari monopalmitas
DEFINITION
Mixture of sucrose monoesters, mainly sucrose
monopalmitate, obtained by transesterification of palmitic
acid methyl esters of vegetable origin with sucrose (0204). It
contains variable quantities of mono- and diesters.
Content :

Time
(min)
0-1

Mobile phase A
(per cent V/V)
100

Mobile phase B
(per cent V/V)
0

Flow rate
(litres/min)
1.0

1-9

100 0

0 100

1.0

9 - 16

100

1.0

16 - 16.01

100

1.0 2.5

16.01 - 32

100

2.5

32 - 33

0 100

100 0

2.5

33 - 36

100

2.5 1.0

(55) Nova-Pak in a Waters High Performance Carbohydrate column is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

491

Sucrose monostearate

diesters : calculate the percentage content of diesters

Detection : evaporative light-scattering detector(56) ; the
using the following expression :
following settings have been found to be suitable ; if the
detector has different setting parameters, adjust the detector
settings so as to comply with the system suitability criterion :
carrier gas : nitrogen R ;
flow rate : 1.0 litres/min ;
evaporator temperature : 45 C ;
nebuliser temperature : 40 C.
Injection: 20 l.
Retention time : about 26 min.

= percentage content of diesters determined by

the normalisation procedure ;
sum of triesters and polyesters: calculate the sum of the
percentage contents of triesters and polyesters using the
following expression :

System suitability : reference solution :

signal-to-noise ratio : minimum 10.
Limit : maximum 4.0 per cent.
Water (2.5.12) : maximum 4.0 per cent, determined on 0.2 g.
Total ash (2.4.16) : maximum 1.5 per cent.
ASSAY
Size-exclusion chromatography (2.2.30) : use the
normalisation procedure.
Test solution. Dissolve 60.0 mg of the substance to be
examined in tetrahydrofuran R and dilute to 4.0 ml with the
same solvent.

= sum of the percentage contents of triesters and

polyesters determined by the normalisation
procedure.

STORAGE
Protected from humidity.

Reference: PA/PH/Exp. 13H/T (03) 84 ANP

XXXX:2318

Column :
size : l = 0.6 m, = 7 mm ;
stationary phase: styrene-divinylbenzene copolymer R
(5 m) with a pore size of 10 nm(57).
Mobile phase : tetrahydrofuran R.
Flow rate : 1.2 ml/min.
Detection : differential refractometer.
Injection: 20 l.
Relative retention with reference to monoesters (retention
time = about 10 min) : diesters = about 0.90 ; triesters and
polyesters = about 0.92.
Calculations :
free fatty acids : calculate the percentage content (D) of
free fatty acids, using the following expression :

IA

= acid value ;

monoesters : calculate the percentage content of

monoesters using the following expression :

A
S

= percentage content of monoesters determined

by the normalisation procedure ;
= percentage content of sucrose determined in
the test for free sucrose ;

SUCROSE MONOSTEARATE
Sacchari monostearas
DEFINITION
Mixture of sucrose monoesters, mainly sucrose monostearate,
obtained by transesterification of stearic acid methyl esters
of vegetable origin with sucrose (0204). It contains variable
quantities of mono- and diesters.
Content :
monoesters : minimum 50.0 per cent ;
diesters : maximum 40.0 per cent ;
sum of triesters and polyesters: maximum 25.0 per cent.
CHARACTERS
Appearance : white or almost white, unctuous powder.
Solubility : very slightly soluble in water, sparingly soluble
in ethanol (96 per cent).
IDENTIFICATION
A. Composition of fatty acids (see Tests).
B. It complies with the limits of the assay.
TESTS
Acid value (2.5.1) : maximum 6.0, determined on 3.00 g.
Use a freshly neutralised mixture of 1 volume of water R and
2 volumes of 2-propanol R as solvent and heat gently.
Composition of fatty acids (2.4.22, Method C). Use the
mixture of calibrating substances in Table 2.4.22.-1.
Composition of the fatty-acid fraction of the substance :
lauric acid : maximum 3.0 per cent ;
myristic acid : maximum 3.0 per cent ;
palmitic acid : 25.0 per cent to 40.0 per cent ;

(56) Polymer Laboratories PL-ELS 1000 is suitable.

(57) Polymer Laboratories gel column reference 1110-8520 is suitable.

492

PHARMEUROPA Vol. 18, No. 3, July 2006

Sucrose monostearate

stearic acid : 55.0 per cent to 75.0 per cent ;

sum of the contents of palmitic acid and stearic acid :
minimum 90.0 per cent.
Free sucrose. Liquid chromatography (2.2.29) : use the
normalisation procedure.
Solvent mixture : water for chromatography R,
tetrahydrofuran for chromatography R (12.5:87.5 V/V).
Test solution. Dissolve 0.200 g of the substance to be
examined in the solvent mixture and dilute to 4.0 ml with the
solvent mixture.
Reference solution. Dissolve 50.0 mg of sucrose CRS in
the solvent mixture and dilute to 10.0 ml with the solvent
mixture. Dilute 1.0 ml of this solution to 10.0 ml with the
solvent mixture.
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase: spherical aminopropylsilyl silica gel
for chromatography R (4 m)(58).
Mobile phase :
mobile phase A : 0.01 g/l solution of ammonium
acetate R in acetonitrile for chromatography R ;
mobile phase B : 0.01 g/l solution of ammonium
acetate R in a mixture of 10 volumes of water for
chromatography R and 90 volumes of tetrahydrofuran
for chromatography R ;
Time
(min)
0-1

Mobile phase A Mobile phase B

(per cent V/V) (per cent V/V)
0
100

Flow rate
(litres/min)
1.0

1-9

100 0

0 100

1.0

9 - 16

100

1.0

16 - 16.01

100

1.0 2.5

16.01 - 32

100

2.5

32 - 33

0 100

100 0

2.5

33 - 36

100

2.5 1.0

Test solution. Dissolve 60.0 mg of the substance to be

examined in tetrahydrofuran R and dilute to 4.0 ml with the
same solvent.
Column :
size : l = 0.6 m, = 7 mm ;
stationary phase : styrene-divinylbenzene copolymer R
(5 m) with a pore size of 10 nm(60).
Mobile phase : tetrahydrofuran R.
Flow rate : 1.2 ml/min.
Detection : differential refractometer.
Injection : 20 l.
Relative retention with reference to monoesters (retention
time = about 10 min) : diesters = about 0.90 ; triesters and
polyesters = about 0.92.
Calculations :
free fatty acids : calculate the percentage content (D) of
free fatty acids, using the following expression :

IA

= acid value ;

monoesters : calculate the percentage content of

monoesters using the following expression :

A
S

= percentage content of monoesters determined

by the normalisation procedure ;
= percentage content of sucrose determined in
the test for free sucrose ;

diesters : calculate the percentage content of diesters

using the following expression :

Detection : evaporative light-scattering detector(59) ; the

following settings have been found to be suitable ; if the
detector has different setting parameters, adjust the detector
settings so as to comply with the system suitability criterion :
carrier gas : nitrogen R ;
flow rate : 1.0 litres/min ;
B
= percentage content of diesters determined by
the normalisation procedure ;
evaporator temperature : 45 C ;
sum of triesters and polyesters: calculate the sum of the
nebuliser temperature : 40 C.
percentage contents of triesters and polyesters using the
Injection: 20 l.
following expression :
Retention time : about 26 min.
System suitability : reference solution :
signal-to-noise ratio : minimum 10.
Limit : maximum 4.0 per cent.
Water (2.5.12) : maximum 4.0 per cent, determined on 0.2 g.
C
= sum of the percentage contents of triesters and
polyesters determined by the normalisation
Total ash (2.4.16) : maximum 1.5 per cent.
procedure.
ASSAY
STORAGE
Size-exclusion chromatography (2.2.30) : use the
normalisation procedure.
Protected from humidity.
(58) Nova-Pak in a Waters High Performance Carbohydrate column is suitable.
(59) Polymer Laboratories PL-ELS 1000 is suitable.
(60) Polymer Laboratories gel column reference 1110-8520 is suitable.

PHARMEUROPA Vol. 18, No. 3, July 2006

493

Tetracaine hydrochloride

Reference: PA/PH/Exp. 10A/T (05) 68 ANP

NOTE ON THE MONOGRAPH
Characters : revision of the melting point range.
Related substances : TLC replaced by an LC.
XXXX:0057

TETRACAINE HYDROCHLORIDE
Tetracaini hydrochloridum

C15H25ClN2O2

Mr 300.8

DEFINITION
2-(Dimethylamino)ethyl 4-(butylamino)benzoate
hydrochloride.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, slightly hygroscopic,
crystalline powder.
Solubility : freely soluble in water, soluble in ethanol (96 per
cent).
It melts at about 148 C or it may occur in either of 2 other
crystalline forms which melt respectively at about 134 C
and 139 C. Mixtures of these forms melt within the range
134 C to 147 C 152 C.

mixture of 4 volumes of glacial acetic acid R, 16 volumes of

hexane R and 80 volumes of dibutyl ether R. Remove the
plate and dry it in a current of warm air for a few minutes.
Allow the plate to cool before use.
Test solution. Dissolve 1.0 g of the substance to be examined
in water R and dilute to 10 ml with the same solvent.
Reference solution. Dissolve 50 mg of 4-aminobenzoic
acid R in water R and dilute to 100 ml with the same solvent.
Dilute 1 ml of the solution to 10 ml with water R.
Apply to the plate 5 l of each solution. Develop over a
path of 10 cm using a mixture of 4 volumes of glacial acetic
acid R, 16 volumes of hexane R and 80 volumes of dibutyl
ether R. Dry the plate at 100 C to 105 C for 10 min and
examine in ultraviolet light at 254 nm. Any spot in the
chromatogram obtained with the test solution, apart from
the principal spot, is not more intense than the spot in the
chromatogram obtained with the reference solution (0.05 per
cent). The principal spot in the chromatogram obtained with
the test solution remains at the starting point.
Liquid chromatography (2.2.29).
Solvent mixture : acetonitrile R, water R (20:80 V/V).
Test solution. Dissolve 50 mg of the substance to be
examined in the solvent mixture and dilute to 50 ml with the
solvent mixture.
Reference solution (a). Dilute 1.0 ml of the test solution
to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this
solution to 10.0 ml with the solvent mixture.
Reference solution (b). Dissolve the contents of a vial
of tetracaine for system suitability CRS (containing
impurities A, B and C) in 2 ml of the solvent mixture.
Column :
size : l = 0.15 m, = 4.6 mm ;
stationary phase : octadecylsilyl silica gel for
chromatography R (5 m)(61) ;
temperature : 30 C.
Mobile phase :
mobile phase A : dissolve 1.36 g of potassium dihydrogen
phosphate R in water R, add 0.5 ml of phosphoric acid R
and dilute to 1000 ml with water R ;
mobile phase B : acetonitrile R ;

IDENTIFICATION
First identification : A, B, D.
Second identification : B, C, D.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : tetracaine hydrochloride CRS.
B. To 10 ml of solution S (see Tests) add 1 ml of ammonium
Time
Mobile phase A
Mobile phase B
thiocyanate solution R. A white, crystalline precipitate is
(min)
formed which, after recrystallisation from water R and
(per cent V/V)
(per cent V/V)
drying at 80 C for 2 h, melts (2.2.14) at about 131 C.
0-3
80
20
C. To about 5 mg add 0.5 ml of fuming nitric acid R.
80 40
20 60
3 - 18
Evaporate to dryness on a water-bath, allow to cool and
40
60
18 - 23
dissolve the residue in 5 ml of acetone R. Add 1 ml of
0.1 M alcoholic potassium hydroxide. A violet colour
Flow rate : 1.5 ml/min.
develops.
Detection : spectrophotometer at 300 nm.
D. Solution S gives reaction (a) of chlorides (2.3.1).
Injection : 10 l.
TESTS
Identification of impurities : use the chromatogram
supplied with tetracaine for system suitability CRS and
Solution S. Dissolve 5.0 g in carbon dioxide-free water R
the chromatogram obtained with reference solution (b) to
and dilute to 50 ml with the same solvent.
identify the peaks due to impurities A, B and C.
Appearance of solution. The solution is clear (2.2.1) and
Relative retention with reference to tetracaine
colourless (2.2.2, Method II).
(retention time = about 8 min) : impurity A = about 0.3 ;
Dilute 2 ml of solution S to 10 ml with water R.
impurity B = about 1.7 ; impurity C = about 2.1.
pH (2.2.3) : 4.5 to 6.5.
System suitability : reference solution (b) :
Dilute 1 ml of solution S to 10 ml with carbon dioxide-free resolution : minimum 5 between the peaks due to
water R.
tetracaine and impurity B ;
Related substances. Examine by thin-layer chromatography the chromatogram obtained is similar to the
(2.2.27), using a TLC silica gel GF254 plate R. Carry out
chromatogram supplied with tetracaine for system
a preliminary development over a path of 12 cm using a
suitability CRS.
(61) Zorbax AQ, Inertsil ODS 3 and Symmetry C18 are suitable.

494

PHARMEUROPA Vol. 18, No. 3, July 2006

Varicella vaccine (live)

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. impurity A

2. tetracaine

3. impurity B

4. impurity C

Figure 0057.-1. Chromatogram for the test for related substances of tetracaine hydrochloride
Limits :
correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity B = 0.6 ;
impurity C = 0.7 ;
impurity A : not more than 0.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.05 per cent) ;
impurities B, C : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent) ;
unspecified impurities : for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.10 per cent) ;
total: not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
disregard limit : 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Heavy metals (2.4.8) : maximum 10 ppm.
12 ml of solution S complies with test A. Prepare the
reference solution using lead standard solution (1 ppm
Pb) R.
Loss on drying (2.2.32) : maximum 1.0 per cent, determined
on 1.000 g by drying in an oven at 100-105 C.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Dissolve 0.250 g in 50 ml of ethanol (96 per cent) R
and add 5.0 ml of 0.01 M hydrochloric acid. Carry out
a potentiometric titration (2.2.20), using 0.1 M sodium
hydroxide. Read the volume added between the 2 points
of inflexion.
1 ml of 0.1 M sodium hydroxide is equivalent to 30.08 mg
of C15H25ClN2O2.
PHARMEUROPA Vol. 18, No. 3, July 2006

STORAGE
Protected from light.
IMPURITIES
Specified impurities : A, B, C.
A. 4-aminobenzoic acid,

B. 4-(butylamino)benzoic acid,

C. methyl 4-(butylamino)benzoate.

Reference: PA/PH/Exp. 15/T (04) 73 ANP 1R

NOTE ON THE MONOGRAPH
Assay : a revision proposal including additional acceptance
criteria was published in Pharmeuropa 17.2. In light of
comments received, a new revision proposal is shown
below. The main changes are :
deletion of details regarding cell cultures and dilution
steps ; these details are an unnecessary restriction since
the validity criteria give a better assurance of correct
design ;
limits are expressed as logarithms and in such a way
that excessive rounding is avoided ;
495

Varicella vaccine (live)

since the aim is to confirm the suitable design of the test, VIRUS SEED LOT
validity criteria apply to the reference preparation only, The strain of varicella virus shall be identified as being
and on the combined value obtained from 3 replicates ; suitable by historical records which shall include information
on the origin of the strain and its subsequent manipulation.
since the BRP is not yet available, the requirement
regarding the link between the manufacturers reference The virus shall at no time have been passaged in continuous
cell lines. Seed lots are prepared in the same kind of cells
preparation and the BRP is not included in this draft ;
as those used for the production of the final vaccine.
introduction of control charts in addition to the criteria To avoid the unnecessary use of monkeys in the test
0.5 log PFU, in accordance with good practice ;
for neurovirulence, Virus seed lots are prepared in large
quantities and stored at temperatures below 20 C, if
deletion of the requirement on the range of virus
concentration found for the replicates ; the requirement freeze-dried, or below 60 C, if not freeze-dried.
for closeness to the historical value for the reference
Only a virus seed lot that complies with the following
preparation, together with the use of a control chart, is requirements may be used for virus propagation.
considered to give better control ;
Identification. The master and working seed lots are
identified as varicella virus by serum neutralisation in cell
introduction of details regarding repetition of the test
culture, using specific antibodies.
and combination of valid results ;
Virus concentration. The virus concentration of the master
reference to chapter 5.3 for calculations.
and working seed lots is determined as prescribed under
Neurovirulence : the present requirements for
Assay to monitor consistency of production.
neurovirulence testing were reviewed at a joint
Extraneous agents (2.6.16). The working seed lot complies
EDQM-WHO-IABs scientific workshop on neurovirulence
with the requirements for seed lots for live virus vaccines ; a
tests for live virus vaccines in January 2005. On the
sample of 50 ml is taken for the test in cell cultures.
basis of the conclusions of the workshop, it is proposed
to revise the present testing scheme to foresee the study
Neurovirulence (2.6.18). The working seed lot complies
of potential neurovirulence of the vaccine strains during
with the test for neurovirulence of live virus vaccines.
preclinical development instead of performing the test
VIRUS PROPAGATION AND HARVEST
for neurovirulence of live virus vaccines (2.6.18) on the
seed lot. This study is based on available data, notably for All processing of the cell bank and subsequent cell cultures
is done under aseptic conditions in an area where no other
wild type virus. Where necessary, a risk analysis study is
considered and, where applicable, a test for neurovirulence cells are handled. Approved animal (but not human) serum
may be used in the media. Serum and trypsin used in the
is carried out using an animal model that differentiates
preparation of cell suspensions and media are shown to be
wild type and attenuated virus.
XXXX:0648 free from extraneous agents. The cell culture medium may
contain a pH indicator such as phenol red and approved
antibiotics at the lowest effective concentration. It is
preferable to have a substrate free from antibiotics during
VARICELLA VACCINE (LIVE)
production. 5 per cent, but not less than 50 ml, of the cell
cultures employed for vaccine production is set aside as
Vaccinum varicellae vivum
uninfected cell cultures (control cells). The infected cells
constituting a single harvest are washed, released from the
support surface and pooled. The cell suspension is disrupted
DEFINITION
by sonication.
Varicella vaccine (live) is a freeze-dried preparation of a
Only a virus harvest that complies with the following
suitable attenuated strain of Herpesvirus varicellae. The
vaccine is reconstituted immediately before use, as stated on requirements may be used in the preparation of the final
bulk vaccine.
the label, to give a clear liquid that may be coloured owing
Identification. The virus harvest contains virus that is
to the presence of a pH indicator.
identified as varicella virus by serum neutralisation in cell
culture, using specific antibodies.
PRODUCTION
The production of vaccine is based on a virus seed-lot system Virus concentration. The concentration of infective virus
in virus harvests is determined as prescribed under Assay
and a cell-bank system. The production method shall have
to monitor consistency of production and to determine the
been shown to yield consistently live varicella vaccines of
dilution to be used for the final bulk vaccine.
adequate immunogenicity and safety in man. The virus in
the final vaccine shall not have been passaged in cell cultures Extraneous agents (2.6.16). Use 50 ml for the test in cell
cultures.
beyond the 38th passage from the original isolated virus.
Control cells. The control cells of the production cell culture
The potential neurovirulence of the vaccine strain is
from which the single harvest is derived comply with a
considered during preclinical development, based on
available data, notably for wild type virus. Where necessary test for identity and with the requirements for extraneous
in light of a risk analysis, a test is carried out on the vaccine agents (2.6.16).
strain using an animal model that differentiates wild type and FINAL BULK VACCINE
attenuated virus ; tests on strains of intermediate attenuation Virus harvests that comply with the above tests are pooled
may also be needed.
and clarified to remove cells. A suitable stabiliser may be
The production method is validated to demonstrate that the added and the pooled harvests diluted as appropriate.
product, if tested, would comply with the test for abnormal
Only a final bulk vaccine that complies with the following
toxicity for immunosera and vaccines for human use (2.6.9). requirements may be used in the preparation of the final lot.
SUBSTRATE FOR VIRUS PROPAGATION
Bacterial and fungal contamination. Carry out the test for
sterility (2.6.1) using 10 ml for each medium.
The virus is propagated in human diploid cells (5.2.3).
496

PHARMEUROPA Vol. 18, No. 3, July 2006

Willow bark

FINAL LOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers and freeze-dried to a moisture
content shown to be favourable to the stability of the
vaccine. The containers are then closed so as to prevent
contamination and the introduction of moisture.
Only a final lot that is satisfactory with respect to each of the
requirements given below under Identification, Tests and
Assay may be released for use. Provided that the test for
bovine serum albumin has been carried out with satisfactory
results on the final bulk vaccine, it may be omitted on the
final lot.

Reference: PA/PH/Exp. 13B/T (06) 12 ANP

NOTE ON THE MONOGRAPH
It is proposed to revise the monograph in order to harmonise
the assay method with the monograph for Willow bark dry
extract (2312) published in Pharmeuropa 18.1.
In the Characters section it has been decided generally to
delete the cross reference to identification tests A and B
(macroscopic and microscopic description), since the
Identification section is mandatory while the Characters
section is only informative.
XXXX:1583

IDENTIFICATION
When the vaccine reconstituted as stated on the label is
mixed with specific Herpesvirus varicellae antibodies, it is
no longer able to infect susceptible cell cultures.

WILLOW BARK

TESTS
Bacterial and fungal contamination. The reconstituted
vaccine complies with the test for sterility (2.6.1).
Bovine serum albumin. Not more than 0.5 g per human
dose, determined by a suitable immunochemical method
(2.7.1).
Water (2.5.12). Not more than 3.0 per cent, determined by
the semi-micro determination of water.

DEFINITION
Whole or fragmented dried bark of young branches or whole
dried pieces of current-year twigs of various species of genus
Salix including S. purpurea L., S. daphnoides Vill. and
S. fragilis L.
Content : minimum 1.5 per cent of total salicylic derivatives,
expressed as salicin (C13H18O7 ; Mr 286.3) (dried drug).

ASSAY
Titrate the vaccine for infective virus, using at least
3 separate vials of vaccine using at least 10 cell cultures
for each fourfold dilution or by a technique of equal
precision. Use Titrate 1 vial of an appropriate virus reference
preparation in triplicate to validate each assay. Calculate
the individual virus concentration for each vial of vaccine
and for each replicate of the reference preparation as well as
the corresponding combined virus concentrations, using the
usual statistical methods (for example, 5.3). The combined
estimates of the virus concentration for the 3 vials of vaccine
is not less than that stated on the label.
The assay is not valid if :
the confidence interval (P = 0.95) of the estimated
virus concentration of the reference preparation for the
3 replicates combined is greater than 0.3 log PFU ;
the virus concentration of the reference preparation,
monitored using control charts, differs by more than
0.5 log PFU from the value established on a historical
basis in the particular laboratory.
The assay is repeated if the confidence interval (P = 0.95) of
the combined virus concentration of the vaccine is greater
than 0.3 log PFU ; data generated from valid assays
only are combined by the usual statistical methods (for
example, 5.3) to calculate the virus concentration of the
sample. The confidence interval (P = 0.95) of the combined
virus concentration is not greater than 0.3 log PFU.
LABELLING
The label states :
the strain of virus used for the preparation of the vaccine ;
the type and origin of the cells used for the preparation
of the vaccine ;
that contact with disinfectants is to be avoided ;
the minimum virus concentration ;
that the vaccine is not to be administered to pregnant
women ;
the time within which the vaccine must be used after
reconstitution.
PHARMEUROPA Vol. 18, No. 3, July 2006

Salicis cortex

IDENTIFICATION
A. The bark is 1-2 mm thick and occurs in flexible, elongated,
quilled or curved pieces. The outer surface is smooth
or slightly wrinkled longitudinally and greenish-yellow
to brownish-grey. The inner surface is smooth or
finely striated longitudinally and white, pale yellow or
reddish-brown, depending on the species. The fracture is
short in the outer part and coarsely fibrous in the inner
region. The diameter of current-year twigs is not more
than 10 mm. The wood is white or pale yellow.
B. Reduce to a powder (355). The powder is pale yellow,
greenish-yellow or light brown. Examine under a
microscope using chloral hydrate solution R. The powder
shows the following diagnostic characters : bundles of
narrow fibres, up to about 600 m long, with very thick
walls and surrounded by a crystal sheath containing
prism crystals of calcium oxalate ; parenchyma of the
cortex with thick, pitted and deeply beaded walls, and
containing large cluster crystals of calcium oxalate ;
uniseriate medullary rays ; thickened and suberised cork
cells. Groups of brownish collenchyma from the bud
may be present. Twigs show, additionally, fragments of
lignified fibres and vessels from the xylem.
C. Thin-layer chromatography (2.2.27).
Test solution (a). To 1.0 g of the powdered drug (500)
(355) add 20 10 ml of methanol R. Heat in a water-bath
at about 50 C, with frequent shaking, for 10 min. Cool
and filter.
Test solution (b). To 5.0 ml of test solution (a) add 1.0 ml
of a 50 g/l solution of anhydrous sodium carbonate R
and heat in a water-bath at about 60 C for 10 min. Cool
and filter if necessary.
Reference solution. Dissolve 2 mg of salicin R and 2 mg
of chlorogenic acid R in 1.0 ml of methanol R.
Plate : TLC silica gel plate R (5-40 m) [or TLC silica gel
plate R (2-10 m)].
Mobile phase : water R, methanol R, ethyl acetate R
(8:15:77 V/V/V).
Application : 10 l [or 2 l] as bands.
Development : over a path of 15 cm [or 6 cm].
Drying : in a current of warm air.
497

Willow bark

Detection : spray with a mixture of 5 volumes of sulphuric

acid R and 95 volumes of methanol R. Heat at 100-105 C
for 5 min and examine in daylight.
The chromatogram obtained with the reference solution
shows in the middle third a reddish-violet zone due
to salicin. In the chromatogram obtained with test
solution (a), the zone due to salicin appears with only
slight to moderate intensity. In the chromatogram
obtained with test solution (b) the zone due to salicin is
clearly more intense and there are, above the zone due to
salicin, one (salicortin or 2-O-acetylsalicortin, or possibly
two tremulacin) faint reddish-violet zones Other blue,
yellow or brown zones can occur in both chromatograms.
Results : see below the sequence of zones present in the
chromatograms obtained with the reference solution
and test solutions (a) and (b). Furthermore, other zones
may be present in the chromatograms obtained with test
solutions (a) and (b).
Top of the plate
_______

Salicin : a
reddish-violet zone
_______

_______
Several reddishviolet zones may be
present
A weak reddish-violet
zone (salicin)

A reddish-violet zone
(salicin)
_______

Chlorogenic acid : a
brown zone
Reference solution

Test solution (a)

Test solution (b)

TESTS
Foreign matter (2.8.2) : maximum 3 per cent of twigs with a
diameter greater than 10 mm, and maximum 2 per cent of
other foreign matter.
Loss on drying (2.2.32) : maximum 11 per cent, determined
on 1.000 g of the powdered drug (355) by drying in an oven
at 100-105 C for 2 h.
Total ash (2.4.16) : maximum 10 per cent.
ASSAY
Examine by liquid chromatography (2.2.29), using
resorcinol R as the internal standard.
Internal standard solution. Dissolve 50 mg of resorcinol R
in 10 ml of methanol R.
Test solution. To 0.5 g of the powdered drug (355) add
50 ml of methanol R and heat under a reflux condenser for
30 min. Cool and filter. Take up the residue with 50 ml of
methanol R. Proceed as above. Combine the filtrates and
evaporate under reduced pressure. Take up the residue
with 5.0 ml of methanol R, add 5.0 ml of 0.1 M sodium
hydroxide and heat in a water-bath at about 60 C under
a reflux condenser, with frequent shaking for about 1 h.
After cooling, add 0.5 ml of 1 M hydrochloric acid. Dilute
the solution to 20.0 ml with a mixture of 50 volumes of
methanol R and 50 volumes of water R. Add 1.0 ml of the
internal standard solution to 10.0 ml of this solution. Filter
through a membrane filter.
Reference solution (a). Dissolve 18.5 mg of salicin R
in 10.0 ml of a mixture of 20 volumes of water R and
80 volumes of methanol R and add 1.0 ml of the internal
standard solution.
Reference solution (b). Dissolve 1.0 mg of picein R in 1.0 ml
of reference solution (a).

The chromatographic procedure may be carried out using :

a stainless steel column, 0.10 m long and 3 mm or 4 mm
in internal diameter, packed with octadecylsilyl silica gel
for chromatography R (3 m),
as mobile phase at a flow rate of 1.0 ml/min a mixture
of 1.8 volumes of tetrahydrofuran R and 98.2 volumes
of water R, containing 0.5 per cent V/V of phosphoric
acid R,
as detector a spectrophotometer set at 270 nm,
a loop injector.
Inject 10 l of reference solution (b). The assay is not valid
unless the resolution between the peaks corresponding to
salicin and picein and between the peaks corresponding to
picein and resorcinol is at least 1.5.
Inject five times 10 l of reference solution (a).
Inject three times 10 l of the test solution. Continue the
chromatography for four times the retention time of the
peak corresponding to salicin.
Liquid chromatography (2.2.29).
Test solution. To 1.000 g of the powdered drug (355) add
40 ml of methanol R and 40.0 ml of a 4.2 g/l solution of
sodium hydroxide R. Heat in a water-bath at about 60 C
under a reflux condenser, with frequent shaking, for about
1 h. After cooling, add 4.0 ml of a 103.0 g/l solution of
hydrochloric acid R. Filter the suspension into a 100 ml
volumetric flask, wash and dilute to 100.0 ml with a mixture
of 50 volumes of methanol R and 50 volumes of water R.
Filter through a membrane filter (pore size : 0.45 m).
Reference solution. Dissolve 5.0 mg of picein R in 25.0 ml
of a mixture of 20 volumes of water R and 80 volumes of
methanol R (solution A). Dissolve 15.0 mg of salicin CRS in
25 ml of a mixture of 20 volumes of water R and 80 volumes
of methanol R ; add 5.0 ml of solution A and dilute to 50.0 ml
with a mixture of 20 volumes of water R and 80 volumes of
methanol R.
Column:
size : l = 0.10 m, = 4.6 mm ;
stationary phase : octadecylsilyl silica gel for
chromatography R (3 m)(62).
Mobile phase:
mobile phase A : tetrahydrofuran R, 0.5 per cent V/V
solution of phosphoric acid R (1.8:98.2 V/V) ;
mobile phase B : tetrahydrofuran R ;
Time
(min)
0 - 15

Mobile phase A
(per cent V/V)
100

Mobile phase B
(per cent V/V)
0

15 - 17

100 90

0 10

17 - 23

90

10

23 - 25

90 100

10 0

25 - 40

100

Flow rate : 1.0 ml/min.

Detection : spectrophotometer at 270 nm.
Injection : 10 l.
System suitability : reference solution:
retention time : salicin = about 6.4 min ; picein = about
7.7 min ;
resolution : minimum 1.5 between the peaks due to
salicin and picein.

(62) Waters Spherisorb 3 m ODS2 is suitable.

498

PHARMEUROPA Vol. 18, No. 3, July 2006

Yellow fever vaccine (live)

The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.

1. salicin

2. picein

Figure 1583.-1. Chromatogram for the assay of willow bark : reference solution
Calculate the percentage content of total salicylic derivatives, since there is no appropriate BRP available,
the requirement regarding the link between the
expressed as salicin, using the following expression :
manufacturers reference preparation and the BRP is
not included in this draft ;
introduction of control charts in addition to the criteria
0.5 log PFU, in accordance with good practice ;
introduction of details regarding repetition of the test
and combination of valid results ;
A1 = area of the peak due to salicin in the
chromatogram obtained with the test solution ;
reference to chapter 5.3 for calculations.
XXXX:0537
A2 = area of the peak due to salicin in the chromatogram
obtained with the reference solution ;
YELLOW FEVER VACCINE (LIVE)
m1 = mass of the drug to be examined, in milligrams ;
m2 = mass of salicin CRS in the reference solution, in
Vaccinum febris flavae vivum
milligrams ;
p
DEFINITION
= declared content of salicin CRS.
Yellow fever vaccine (live) is a freeze-dried preparation of
the 17D strain of yellow fever virus grown in fertilised hen
eggs. The vaccine is reconstituted immediately before use, as
stated on the label, to give a clear liquid.
PRODUCTION
The production of vaccine is based on a virus seed-lot
system. The production method shall have been shown to
NOTE ON THE MONOGRAPH
yield consistently yellow fever vaccine (live) of acceptable
immunogenicity and safety for man.
Thermal stability test and Assay : the revised draft below
proposes the same changes regarding the acceptance
The production method is validated to demonstrate that
criteria as those proposed for other live viral vaccine
the product, if tested, would comply with the test for
monographs (Measles, Mumps, Rubella, Varicella or Oral
abnormal toxicity for immunosera and vaccines for human
poliomyelitis vaccine) :
use (2.6.9) modified as follows for the test in guinea-pigs :
inject 10 human doses into each guinea-pig and observe for
limits are expressed as logarithms and in such a way
21 days.
that excessive rounding is avoided ;
Reference preparation. In the test for neurotropism,
since the aim is to confirm the suitable design of the test,
a suitable batch of vaccine known to have satisfactory
validity criteria apply to the reference preparation only,
properties in man is used as the reference preparation.
and on the combined value obtained from 3 replicates ;
Reference: PA/PH Exp. 15/T (06) 21 ANP

PHARMEUROPA Vol. 18, No. 3, July 2006

499

Yellow fever vaccine (live)

SUBSTRATE FOR VIRUS PROPAGATION

Virus for the preparation of master and working seed lots
and of all vaccine batches is grown in the tissues of chick
embryos from a flock free from specified pathogens (SPF)
(5.2.2).
SEED LOTS
The 17D strain shall be identified by historical records
that include information on the origin of the strain and its
subsequent manipulation. Virus seed lots are prepared in
large quantities and stored at a temperature below 60 C.
Master and working seed lots shall not contain any human
protein or added serum.
Unless otherwise justified and authorised, the virus in the
final vaccine shall be between passage levels 204 and 239
from the original isolate of strain 17D. A working seed lot
shall be only 1 passage from a master seed lot. A working
seed lot shall be used without intervening passage as the
inoculum for infecting the tissues used in the production of
a vaccine lot, so that no vaccine virus is more than 1 passage
from a seed lot that has passed all the safety tests.
Only a virus seed lot that complies with the following
requirements may be used for virus propagation.
Identification. The master and working seed lots are
identified as containing yellow fever virus by serum
neutralisation in cell culture, using specific antibodies.
Extraneous agents (2.6.16). Each master seed lot complies
with the following tests :
test in guinea-pigs (as described in chapter 2.6.16 under
Virus seed lot) ;
bacterial and fungal sterility (as described in chapter
2.6.16 under Virus seed lot and virus harvests) ;
mycoplasmas (as described in chapter 2.6.16 under Virus
seed lot and virus harvests).
Avian leucosis viruses (2.6.24). Each master seed lot
complies with the test for avian leucosis viruses.
Extraneous agents (2.6.16). Each working seed lot complies
with the following tests :
test in adult mice (intraperitoneal inoculation only) (as
described in chapter 2.6.16 under Virus seed lot) ;
test in guinea-pigs (as described in chapter 2.6.16 under
Virus seed lot) ;
bacterial and fungal sterility (as described in chapter
2.6.16 under Virus seed lot and virus harvests) ;
mycoplasmas (as described in chapter 2.6.16 under Virus
seed lot and virus harvests) ;
mycobacteria (as described in chapter 2.6.16 under Virus
seed lot and virus harvests) ;
test in cell culture for other extraneous agents (as
described in chapter 2.6.16 under Virus seed lot and
virus harvests) ;
avian viruses (as described in chapter 2.6.16 under Virus
seed lot and virus harvests).
Avian leucosis viruses (2.6.24). Each working seed lot
complies with the test for avian leucosis viruses.
Tests in monkeys. Each master and working seed lot
complies with the following tests in monkeys for viraemia
(viscerotropism), immunogenicity and neurotropism.
The monkeys shall be Macaca sp. susceptible to yellow fever
virus and shall have been shown to be non-immune to yellow

500

fever at the time of injecting the seed virus. They shall be

healthy and shall not have received previously intracerebral
or intraspinal inoculation. Furthermore, they shall not have
been inoculated by other routes with neurotropic viruses or
with antigens related to yellow fever virus. Not fewer than
10 monkeys are used for each test.
Use a test dose of 0.25 ml containing the equivalent
of not less than 5000 mouse LD50 and not more than
50 000 mouse LD50, determined by a titration for infectious
virus and using the established equivalence between virus
concentration and mouse LD50 (see under Assay). Inject
the test dose into 1 frontal lobe of each monkey under
anaesthesia and observe the monkeys for not less than
30 days.
Viraemia (Viscerotropism). Viscerotropism is indicated
by the amount of virus present in serum. Take blood
from each of the test monkeys on the 2nd, 4th and 6th days
after inoculation and prepare serum from each sample.
Prepare 1:10, 1:100 and 1:1000 dilutions from each serum
and inoculate each dilution into a group of at least 6 cell
culture vessels used for the determination of the virus
concentration. The seed lot complies with the test if
none of the sera contains more than the equivalent of
500 mouse LD50 in 0.03 ml and at most 1 serum contains
more than the equivalent of 100 mouse LD50 in 0.03 ml.
Immunogenicity. Take blood from each monkey 30 days
after the injection of the test dose and prepare serum
from each sample. The seed lot complies with the test if
at least 90 per cent of the test monkeys are shown to be
immune, as determined by examining their sera in the test
for neutralisation of yellow fever virus described below.
It has been shown that a low dilution of serum (for
example, 1:10) may contain non-specific inhibitors that
influence this test ; such serum shall be treated to remove
inhibitors. Mix dilutions of at least 1:10, 1:40 and 1:160
of serum from each monkey with an equal volume of
17D vaccine virus at a dilution that will yield an optimum
number of plaques with the titration method used. Incubate
the serum-virus mixtures in a water-bath at 37 C for 1 h
and then cool in iced water ; add 0.2 ml of each serum-virus
mixture to each of 4 cell-culture plates and proceed as
for the determination of virus concentration. Inoculate
similarly 10 plates with the same amount of virus, plus an
equal volume of a 1:10 dilution of monkey serum known to
contain no neutralising antibodies to yellow fever virus. At
the end of the observation period, compare the mean number
of plaques in the plates receiving virus plus non-immune
serum with the mean number of plaques in the plates
receiving virus plus dilutions of each monkey serum. Not
more than 10 per cent of the test monkeys have serum that
fails to reduce the number of plaques by 50 per cent at the
1:10 dilution.
Neurotropism. Neurotropism is assessed from clinical
evidence of encephalitis, from incidence of clinical
manifestations and by evaluation of histological lesions, in
comparison with 10 monkeys injected with the reference
preparation. The seed lot is not acceptable if either the onset
and duration of the febrile reaction or the clinical signs of
encephalitis and pathological findings are such as to indicate
a change in the properties of the virus.

PHARMEUROPA Vol. 18, No. 3, July 2006

Yellow fever vaccine (live)

Clinical evaluation
The monkeys are examined daily for 30 days by personnel
familiar with clinical signs of encephalitis in primates (if
necessary, the monkeys are removed from their cage and
examined for signs of motor weakness or spasticity). The
seed lot is not acceptable if in the monkeys injected with
it the incidence of severe signs of encephalitis, such as
paralysis or inability to stand when stimulated, or mortality is
greater than for the reference vaccine. These and other signs
of encephalitis, such as paresis, incoordination, lethargy,
tremors or spasticity are assigned numerical values for the
severity of symptoms by a grading method. Each day each
monkey in the test is given a score based on the following
scale :
grade 1 : rough coat, not eating ;
grade 2 : high-pitched voice, inactive, slow moving ;
grade 3 : shaky, tremors, unco-ordinated, limb weakness ;
grade 4 : inability to stand, limb paralysis or death (a dead
monkey receives a daily score of 4 from the day of death
until day 30).
A clinical score for a particular monkey is the average of its
daily scores ; the clinical score for the seed lot is the mean of
the individual monkey scores. The seed lot is not acceptable
if the mean of the clinical severity scores for the group of
monkeys inoculated with it is significantly greater (P = 0.95)
than the mean for the group of monkeys injected with the
reference preparation. In addition, special consideration is
given to any animal showing unusually severe signs when
deciding on the acceptability of the seed lot.
Histological evaluation
5 levels of the brain are examined including :
block I : the corpus striatum at the level of the optic
chiasma ;
block II : the thalamus at the level of the mamillary bodies ;
block III : the mesencephalon at the level of the superior
colliculi ;
block IV : the pons and cerebellum at the level of the
superior olives ;
block V : the medulla oblongata and cerebellum at the
level of the mid-inferior olivary nuclei.
Cervical and lumbar enlargements of the spinal cord are each
divided equally into 6 blocks ; 15 m sections are cut from
the tissue blocks embedded in paraffin wax and stained with
gallocyanin. Numerical scores are given to each hemisection
of the cord and to structures in each hemisection of the
brain as listed below. Lesions are scored as follows :
grade 1 - minimal : 1 to 3 small focal inflammatory
infiltrates ; degeneration or loss of a few neurons ;
grade 2 - moderate : 4 or more focal inflammatory
infiltrates ; degeneration or loss of neurons affecting not
more than one third of cells ;
grade 3 - severe : moderate focal or diffuse inflammatory
infiltration ; degeneration or loss of up to two thirds of
the neurons ;
grade 4 - overwhelming : variable but often severe
inflammatory reaction ; degeneration or loss of more than
90 per cent of neurons.
It has been found that inoculation of yellow fever vaccine
into the monkey brain causes histological lesions in different
anatomical formations of the central nervous system with
varying frequency and severity (I. S. Levenbook et al.,
Journal of Biological Standardization, 1987, 15, 305-313).
Based on these 2 indicators, the anatomical structures
can be divided into target, spared and discriminator areas.
PHARMEUROPA Vol. 18, No. 3, July 2006

Target areas are those which show more severe specific

lesions in a majority of monkeys irrespective of the degree
of neurovirulence of the seed lot. Spared areas are those
which show only minimal specific lesions and in a minority
of monkeys. Discriminator areas are those where there
is a significant increase in the frequency of more severe
specific lesions with seed lots having a higher degree of
neurovirulence. Discriminator and target areas for Macaca
cynomolgus and Macaca rhesus monkeys are shown in the
table below.
Type of monkey
Macaca cynomolgus

Discriminator areas

Target areas

Globus pallidus

Substantia nigra

Putamen

Macaca rhesus

Anterior/median
thalamic nucleus
Lateral thalamic
nucleus
Caudate nucleus
Globus pallidus
Putamen

Substantia nigra
Cervical
enlargement
Lumbar enlargement

Anterior/median
thalamic nucleus
Lateral thalamic
nucleus
Cervical enlargement
Lumbar enlargement

Scores for discriminator and target areas are used for the
final evaluation of the seed lot. The individual monkey score
is calculated from the sum of individual target area scores in
each hemisection divided by the number of areas examined.
A separate score is calculated similarly for the discriminator
areas.
Mean scores for the test group are calculated in 2 ways : (1) by
dividing the sum of the individual monkey discriminator
scores by the number of monkeys and (2) by dividing the
sum of the individual monkey target and discriminator
scores by the number of monkeys. These 2 mean scores are
taken into account when deciding on the acceptability of the
seed lot. The seed lot is not acceptable if either of the mean
lesion scores is significantly greater (P = 0.95) than for the
reference preparation.
PROPAGATION AND HARVEST
All processing of the fertilised eggs is done under aseptic
conditions in an area where no other infectious agents or
cells are handled at the same time. At least 2 per cent but
not less than 20 and not more than 80 eggs are maintained
as uninfected control eggs. After inoculation and incubation
at a controlled temperature, only living and typical chick
embryos are harvested. At the time of harvest, the control
eggs are treated in the same way as the inoculated eggs to
obtain a control embryonic pulp. The age of the embryo
at the time of virus harvest is reckoned from the initial
introduction of the egg into the incubator and shall be not
more than 12 days. After homogenisation and clarification
by centrifugation, the extract of embryonic pulp is tested as
described below and kept at 70 C or colder until further
processing. Virus harvests that comply with the prescribed
tests may be pooled. No human protein is added to the virus
suspension at any stage during production. If stabilisers are
added, they shall have been shown to have no antigenic or
sensitising properties for man.
Only a single harvest that complies with the following
requirements may be used in the preparation of the final
bulk vaccine.
501

Yellow fever vaccine (live)

Identification. The single harvest contains virus that is

identified as yellow fever virus by serum neutralisation in
cell culture, using specific antibodies.
Sterility (2.6.1). The harvest complies with the test for
sterility, carried out using 10 ml for each medium.
Mycoplasmas (2.6.7).The single harvest complies with the
test for mycoplasma, carried out using 10 ml.
Mycobacteria (2.6.2). A 5 ml single harvest sample is tested
for the presence of Mycobacterium spp. by culture methods
known to be sensitive for the detection of these organisms.
Embryonic pulp of control eggs. The extract of the control
eggs shows no evidence of the presence of any extraneous
agents in the tests described below.
Test in cell culture for other extraneous agents. Inoculate
a 5 ml sample of embryonic pulp of the control eggs into
continuous simian kidney and human cell cultures as well
as into primary chick-embryo-fibroblast cells. The cells are
incubated at 36 1 C and observed for a period of 14 days.
The embryonic pulp of the control eggs passes the test
if there is no evidence of the presence of any extraneous
agents. The test is not valid unless at least 80 per cent of the
cell cultures remain viable.
Avian viruses. Using 0.1 ml per egg, inoculate the embryonic
pulp of control eggs : by the allantoic route into a group of
10 fertilised SPF eggs (5.2.2) (9 to 11 days old) ; into the
yolk sac of a group of 10 fertilised SPF eggs (5.2.2), 5 to
7 days old. Incubate for 7 days. The embryonic pulp lot
of the control eggs complies if the allantoic and yolk sac
fluids show no signs of haemagglutinating agents and if the
embryos and chorio-allantoic membranes examined to detect
any macroscopic pathology are typical. The test is not valid
unless at least 80 per cent of the inoculated eggs survive
during the 7 day observation period.
Virus concentration. In order to calculate the dilution for
formulation of the final bulk, each single harvest is titrated
as described under Assay.
FINAL BULK VACCINE
Single harvests that comply with the tests prescribed above
are pooled and clarified again. A test for protein nitrogen
content is carried out. A suitable stabiliser may be added
and the pooled harvests diluted as appropriate.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final lot.
Sterility (2.6.1). The final bulk vaccine complies with the
test for sterility, carried out using 10 ml for each medium.
Protein nitrogen content : maximum 0.25 mg per
human dose before the addition of any stabiliser.
FINAL LOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers and freeze-dried to a moisture
content shown to be favourable to the stability of the
vaccine. The containers are then closed so as to prevent
contamination and the introduction of moisture.
Only a final lot that is satisfactory with respect to thermal
stability and each of the requirements given below under
Identification, Tests and Assay may be released for use.
Provided that the test for ovalbumin has been performed
with satisfactory results on the final bulk vaccine, it may be
omitted on the final lot.
Thermal stability. Maintain samples of the final lot of
freeze-dried vaccine in the dry state at 37 C for 14 days.
Determine the virus concentration as described under
Assay in parallel for the heated vaccine and for unheated
vaccine. The difference in the virus concentration between
unheated and heated vaccine does not exceed 1.0 log10 and
502

the virus concentration of the heated vaccine is not less

than the number of plaque-forming units (PFU) equivalent
to 1 103 mouse LD50 per human dose.
The test is not valid if:
the confidence interval (P = 0.95) of the estimated
virus concentration of the reference preparation for the
3 replicates combined is greater than 0.3 log PFU ;
the virus concentration of the reference preparation,
monitored using control charts, differs by more than
0.5 log PFU from the value established on a historical
basis in the particular laboratory.
IDENTIFICATION
When the vaccine reconstituted as stated on the label is
mixed with specific yellow fever virus antibodies, there is a
significant reduction in its ability to infect susceptible cell
cultures.
TESTS
Ovalbumin : maximum 5 g of ovalbumin per human dose,
determined by a suitable immunochemical method (2.7.1).
Water (2.5.12) : maximum 3.0 per cent.
Sterility (2.6.1). The reconstituted vaccine complies with the
test for sterility.
Bacterial endotoxins (2.6.14) : less than 5 IU per single
human dose.
ASSAY
Titrate for infective virus in cell cultures using at least
3 separate vials of vaccine. Use Titrate 1 vial of an
appropriate virus reference preparation in triplicate to
validate each assay.
Calculate the individual virus concentration for each vial of
vaccine and for each replicate of the reference preparation
as well as the corresponding combined virus concentrations
using the usual statistical methods (for example, 5.3).
The combined virus concentration for the 3 vials of vaccine
is not less than the equivalent in PFU of 1 x 103 3.0 log
mouse LD50 per human dose. The relationship between
mouse LD50 and PFU is established by each laboratory and
approved by the competent authority.
The method shown below, or another suitable technique,
may be used to determine the mouse LD50.
Suggested method for determination of the mouse LD50
Mouse LD50. The statistically calculated quantity of virus
suspension that is expected to produce fatal specific
encephalitis in 50 per cent of mice of a highly susceptible
strain, 4 to 6 weeks of age, after intracerebral inoculation.
Appropriate serial dilutions of the reconstituted vaccine are
made in diluent for yellow fever virus (a 7.5 g/l solution of
bovine albumin R in phosphate buffered saline pH 7.4 R, or
any other diluent that has been shown to be equivalent for
maintaining the infectivity of the virus).
Mice of a highly susceptible strain, 4 to 6 weeks of age, are
injected intracerebrally under anaesthesia with 0.03 ml of
the vaccine dilution. Groups of not fewer than 6 mice are
used for each dilution ; the series of dilutions is chosen so
as to cover the range 0-100 per cent mortality of the mice.
Injection of the mice is performed immediately after the
dilutions have been made. The mice are observed for 21 days
and all deaths are recorded.
Only survivors and deaths caused by typical yellow fever
infections are counted in the computations. Mice paralysed
on the 21st day of observation are counted as survivors.
The assay is not valid if:
PHARMEUROPA Vol. 18, No. 3, July 2006

Zinc gluconate, hydrated

the confidence interval (P = 0.95) of the estimated

virus concentration of the reference preparation for the
3 replicates combined is greater than 0.3 log PFU ;
the virus concentration of the reference preparation,
monitored using control charts, differs by more than
0.5 log PFU from the value established on a historical
basis in the particular laboratory.
The assay is repeated if the confidence interval (P = 0.95) of
the combined virus concentration of the vaccine is greater
than 0.3 log PFU ; data generated from valid assays only
are combined by the usual statistical methods (for example,
5.3) to calculate the virus concentration of the sample.
The confidence interval (P = 0.95) of the combined virus
concentration is not greater than 0.3 log PFU.
LABELLING
The label states :
the strain of virus used in preparation of the vaccine ;
that the vaccine has been prepared in chick embryos ;
the minimum virus concentration ;
that contact with disinfectants is to be avoided ;
the period of time within which the vaccine is to be used
after reconstitution.

Mobile phase : concentrated ammonia R, ethyl acetate R,

water R, ethanol (96 per cent) R (10:10:30:50 V/V/V/V).
Application : 1 l.
Development : over 3/4 of the plate.
Drying : at 100-105 C for 20 min, then allow to cool to
room temperature.
Detection : spray with a solution containing 25 g/l of
ammonium molybdate R and 10 g/l of cerium sulphate R
in dilute sulphuric acid R, and heat at 100-105 C for
about 10 min.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with the reference solution.
B. Dissolve 0.1 g in 5 ml of water R. Add 0.5 ml of potassium
ferrocyanide solution R. A white precipitate is formed
that does not dissolve upon the addition of 5 ml of
hydrochloric acid R.

TESTS
Solution S. Dissolve 1.0 g in water R and dilute to 50 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).
Sucrose and reducing sugars. Dissolve 0.5 g in a mixture
Reference: PA/PH/Exp. INC/T (06) 2 ANP
of 2 ml of hydrochloric acid R1 and 10 ml of water R. Boil
for 5 min, allow to cool, add 10 ml of sodium carbonate
NOTE ON THE MONOGRAPH
solution R and allow to stand for 10 min. Dilute to 25 ml
This monograph was elaborated taking into account the
with water R and filter. To 5 ml of the filtrate add 2 ml of
other monographs on gluconates.
cupri-tartaric solution R and boil for 1 min. Allow to stand
XXXX:2164 for 2 min. No red precipitate is formed.
Chlorides (2.4.4) : maximum 500 ppm.
ZINC GLUCONATE, HYDRATED
Dilute 5 ml of solution S to 15 ml with water R.
Sulphates (2.4.13) : maximum 500 ppm.
Zinci gluconas hydricus
Dissolve 2.0 g in a mixture of 10 ml of acetic acid R and
90 ml of distilled water R.
Cadmium : maximum 5.0 ppm.
Atomic absorption spectrometry (2.2.23, Method II).
Test solution. Dissolve 5.00 g in 20 ml of deionised distilled
water R with the aid of ultrasound and dilute to 25.0 ml
C12H22ZnO14,xH2O
Mr 455.7 (anhydrous substance) with the same solvent.
Reference solutions. Prepare the reference solutions using
DEFINITION
cadmium standard solution (0.1 per cent Cd) R, diluted as
necessary with deionised distilled water R.
Hydrated zinc D-gluconate.
Source : cadmium hollow-cathode lamp.
Content : 98.0 per cent to 102.0 per cent (anhydrous
substance).
Wavelength : 228.8 nm.
Atomisation device : air-acetylene flame.
CHARACTERS
Appearance : white or almost white, hygroscopic, crystalline Heavy metals (2.4.8) : maximum 10 ppm.
powder.
Dissolve 2.0 g in 20 ml of water R, heating in a water-bath
Solubility : soluble in water, practically insoluble in ethanol at 60 C. 12 ml of the solution complies with test A.
Prepare the reference solution using lead standard solution
(96 per cent), insoluble in methylene chloride.
(1 ppm Pb) R.
IDENTIFICATION
Water (2.5.32) : maximum 12.0 per cent, determined on
A. Thin-layer chromatography (2.2.27).
0.080 g.
Test solution. Dissolve 20 mg of the substance to be
Microbial contamination. Total viable aerobic count (2.6.12)
examined in 1 ml of water R.
not more than 103 micro-organisms per gram, determined
Reference solution. Dissolve 20 mg of calcium
by plate count.
gluconate CRS in 1 ml of water R, heating if necessary in
ASSAY
a water-bath at 60 C.
Plate : TLC silica gel plate R (5-40 m) [or TLC silica gel Dissolve 0.400 g in 5 ml of dilute acetic acid R. Carry out
the complexometric titration of zinc (2.5.11).
plate R (2-10 m)].
PHARMEUROPA Vol. 18, No. 3, July 2006

503

Zinc gluconate, hydrated

1 ml of 0.1 M sodium edetate is equivalent to 45.57 mg of

C12H22ZnO14.
STORAGE
In a non-metallic, airtight container.

504

PHARMEUROPA Vol. 18, No. 3, July 2006

Arnica flower

Illustrations of Powdered Drugs

in Herbal Monographs
It has been decided to introduce progressively illustrations of powdered drugs into herbal monographs in order to
complement the corresponding identification section (usually Identification B). These drawings will be published
in Pharmeuropa as they become available.

Reference: PA/PH/Exp. 13A/T (06) 42 ANP

NOTE ON THE MONOGRAPH
This monograph is currently published in the 5th Edition.
XXXX:1391

ARNICA FLOWER
Arnicae flos

A. Epidermis of the ligulate corolla E. Epidermis of the corolla with

with covering trichome in surface striated cuticle and biseriate secretory
view (Aa) and in side view (Ab)
trichome in surface view (Ea) and in
side view (Eb)
B. Secretory trichome with
multicellular head

F. Covering trichome of the ovary in

surface view (Fa) and in side view (Fb)

C. Bristles of the calyx

G. Secretory trichome of the ovary

D. Pollen grain

Figure 1391.-1. Illustration of powdered herbal drug of

arnica flower (see identification B)

PHARMEUROPA Vol. 18, No. 3, July 2006

A. Epidermis of the bracts of the D. Epidermis of bracts of the involucre

involucre with covering trichomes with stomata and biseriate secretory
and stomata
trichome
B. Multicellular stalk of covering
trichome

E. Secretory trichome

C. Pollen grain

Figure 1391.-2. Illustration of powdered herbal drug of

arnica flower (see identification B)

505

Butchers broom

Reference: PA/PH/Exp. 13A/T (06) 44 ANP

Reference: PA/PH/Exp. 13A/T (06) 43 ANP

NOTE ON THE MONOGRAPH

NOTE ON THE MONOGRAPH
This monograph is currently published in the 5th Edition.
This monograph is currently published in the 5th Edition.
XXXX:1847
XXXX:1831

BUTCHERS BROOM

GOLDENSEAL RHIZOME

Rusci rhizoma

Hydrastis rhizoma

A. Thick-walled parenchymatous
cells

E. Vessels accompanied by calcium

oxalate fibres (F)

B. Endodermis fragment

F. Calcium oxalate fibres

C. Sclereid cells

G. Dermal tissue of the root

D. Parenchyma, some cells of

which contain raphides of calcium
oxalate

H. Raphides

A. Parenchyma fragment

D. Pitted fibres

B. Vessels

E. Starch granules

C. Fragment of cork from the

rhizome and roots in surface view
(Ca) and in side view (Cb)

Figure 1831.-1. Illustration of powdered herbal drug of

goldenseal rhizome (see identification B)

Figure 1847.-1. Illustration of powdered herbal drug of

butchers broom (see identification B)

506

PHARMEUROPA Vol. 18, No. 3, July 2006

Hop strobile

Reference: PA/PH/Exp. 13A/T (06) 40 ANP

Reference: PA/PH/Exp. 13A/T (06) 41 ANP

NOTE ON THE MONOGRAPH

NOTE ON THE MONOGRAPH
th
This monograph is currently published in the 5 Edition.
This monograph is currently published in the 5th Edition.
XXXX:0909
XXXX:1222

HAMAMELIS LEAF

HOP STROBILE

Hamamelidis folium

Lupuli flos

A. Epidermis of bracts and

bracteoles

E. Multicellular glandular trichome

A. Adaxial epidermis and palisade

parenchyma

F. Spongy mesophyll

B. Covering trichome

F. Glandular trichome in surface

view (Fa) and in side view (Fb)

B. Abaxial epidermis

G. Sclereid

C. Lignified fibres with sheath of

prismatic calcium oxalate crystals

H. Vessels accompanied by fibres

and by sheaths made up of calcium
oxalate tubes

C. Mesophyll containing calcium

oxalate cluster crystals and vessels
D. Anomocytic stoma

G. Sclerenchymatous cells of the

testa

D. Star-shaped covering trichomes

and free covering trichome

J. Isolated prisms of calcium

oxalate

Figure 1222.-1. Illustration of powdered herbal drug of

hop strobile (see identification B)

E. Palisade parenchyma

Figure 0909.-1. Illustration of powdered herbal drug of

hamamelis leaf (see identification B)

PHARMEUROPA Vol. 18, No. 3, July 2006

507

508

PHARMEUROPA Vol. 18, No. 3, July 2006

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