EUROPEAN PHARMACOPOEIA 6.

0 Apomorphine hydrochloride
the name and amount of stabiliser, where applicable,
the dilution to be made before use of the product.
01/2008:0136
corrected 6.0
APOMORPHINE HYDROCHLORIDE
Apomorphini hydrochloridum
C
17
H
18
ClNO
2
,1/2H
2
O M
r
312.8
[41372-20-7]
DEFINITION
(6aR)-6-Methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,
g]quinoline-10,11-diol hydrochloride hemihydrate.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance: white or slightly yellowish-brown or
green-tinged greyish, crystalline powder or crystals; on
exposure to air and light, the green tinge becomes more
pronounced.
Solubility: sparingly soluble in water and in alcohol,
practically insoluble in toluene.
IDENTIFICATION
First identification: B, D.
Second identification: A, C, D.
A. Dissolve 10.0 mg in 0.1 M hydrochloric acid and dilute
to 100.0 ml with the same acid. Dilute 10.0 ml of the
solution to 100.0 ml with 0.1 M hydrochloric acid.
Examined between 230 nm and 350 nm (2.2.25), the
solution shows an absorption maximum at 273 nm and a
shoulder at 300 nm to 310 nm. The specific absorbance
at the maximum is 530 to 570.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: Ph. Eur. reference spectrum of
apomorphine hydrochloride.
C. To 5 ml of solution S (see Tests) add a few millilitres
of sodium hydrogen carbonate solution R until a
permanent, white precipitate is formed. The precipitate
slowly becomes greenish. Add 0.25 ml of 0.05 M iodine
and shake. The precipitate becomes greyish-green.
Collect the precipitate. The precipitate dissolves in
ether R giving a purple solution, in methylene chloride R
giving a violet-blue solution and in alcohol R giving a
blue solution.
D. To 2 ml of solution S add 0.1 ml of nitric acid R. Mix and
filter. The filtrate gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S. Dissolve 0.25 g without heating in carbon
dioxide-free water R and dilute to 25 ml with the same
solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution BY
5
or
GY
5
(2.2.2, Method II).
pH (2.2.3) : 4.0 to 5.0 for solution S.
Specific optical rotation (2.2.7) : 48 to 52 (dried
substance).
Dissolve 0.25 g in 0.02 M hydrochloric acid and dilute to
25.0 ml with the same acid.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.25 g of the substance to be
examined in a 1 per cent V/V solution of glacial acetic
acid R and dilute to 100.0 ml with the same solution.
Reference solution (a). Dilute 1.0 ml of the test solution
to 10.0 ml with a 1 per cent V/V solution of glacial acetic
acid R. Dilute 1.0 ml to 100.0 ml with a 1 per cent V/V
solution of glacial acetic acid R.
Reference solution (b). Dissolve 25 mg of boldine R in a
1 per cent V/V solution of glacial acetic acid R and dilute to
10.0 ml with the same solvent. To 1 ml of this solution, add
1 ml of the test solution and dilute to 10.0 ml with a 1 per
cent V/V solution of glacial acetic acid R.
Column:
size: l = 0.15 m, = 4.6 mm,
stationary phase: octadecylsilyl silica gel for
chromatography R (5 m),
temperature: 35 C.
Mobile phase:
mobile phase A: 1.1 g/l solution of sodium
octanesulphonate R, adjusted to pH 2.2 using a 50 per
cent m/m solution of phosphoric acid R,
mobile phase B: acetonitrile R,
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 - 30 85 68 15 32
30 - 35 68 32
35 - 45 68 85 32 15
Flow rate: 1.5 ml/min.
Detection: spectrophotometer at 280 nm.
Injection: 10 l.
System suitability: reference solution (b) :
resolution: minimum 2.5 between the peaks due to
boldine and apomorphine.
Limits:
any 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 8 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.8 per cent),
disregard limit : 0.2 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.02 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with limit test C. Prepare the standard using
2 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : 2.5 per cent to 4.2 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 0.250 g in a mixture of 5.0 ml of 0.01 M hydrochloric
acid and 50 ml of alcohol R. Carry out a potentiometric
titration (2.2.20), using 0.1 M sodium hydroxide. Read the
volume added between the first 2 points of inflexion.
General Notices (1) apply to all monographs and other texts 1207
Aprotinin EUROPEAN PHARMACOPOEIA 6.0
1 ml of 0.1 M sodium hydroxide is equivalent to 30.38 mg of
C
17
H
18
ClNO
2
.
STORAGE
In an airtight container, protected from light.
IMPURITIES
A. (6aR)-10-methoxy-6-methyl-5,6,6a,7-tetrahydro-4H-
dibenzo[de,g]quinolin-11-ol (apocodeine),
B. morphine.
01/2008:0580
APROTININ
Aprotininum
DEFINITION
Aprotinin is a polypeptide consisting of a chain of 58 amino
acids. It inhibits stoichiometrically the activity of several
proteolytic enzymes such as chymotrypsin, kallikrein,
plasmin and trypsin. It contains not less than 3.0 Ph. Eur. U.
of aprotinin activity per milligram, calculated with reference
to the dried substance.
PRODUCTION
The animals from which aprotinin is derived must fulfil the
requirements for the health of animals suitable for human
consumption to the satisfaction of the competent authority.
The manufacturing process is validated to demonstrate
suitable inactivation or removal of any contamination by
viruses or other infectious agents.
The method of manufacture is validated to demonstrate that
the product, if tested, would comply with the following tests.
Abnormal toxicity (2.6.9). Inject into each mouse a quantity
of the substance to be examined containing 2 Ph. Eur. U.
dissolved in a sufficient quantity of water for injections R to
give a volume of 0.5 ml.
Histamine (2.6.10) : maximum 0.2 g of histamine base per
3 Ph. Eur. U.
CHARACTERS
Appearance: almost white powder, hygroscopic.
Solubility: soluble in water and in isotonic solutions,
practically insoluble in organic solvents.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Test solution. Solution S (see Tests).
Reference solution. Aprotinin solution BRP.
Plate: TLC silica gel G plate R.
Mobile phase: water R, glacial acetic acid R (80:100 V/V)
containing 100 g/l of sodium acetate R.
Application: 10 l.
Development : over a path of 12 cm.
Drying: in air.
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
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 substance to be examined to
inhibit trypsin activity using the method described below.
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
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. Mix 1 volume of glacial acetic
acid R, 49 volumes of water R and 50 volumes of
ethanol R.
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.
The solution is cloudy. Carry out a blank test under the
same conditions using buffer solution pH 7.2 R instead of
the test solution. The solution is not cloudy.
TESTS
Solution S. Prepare a solution of the substance to be
examined containing 15 Ph. Eur. U./ml, calculated from the
activity stated on the label.
Appearance of solution. Solution S is clear (2.2.1).
Absorbance (2.2.25) : maximum 0.80 by measuring at the
absorption maximum at 277 nm.
Prepare a solution of the substance to be examined
containing 3.0 Ph. Eur. U./ml.
Protein impurities of higher molecular mass. Size-exclusion
chromatography (2.2.30).
Use cross-linked dextran for chromatography R2. Use a
180 g/l solution of anhydrous acetic acid R to swell the
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
introduction of air bubbles. Place at the top of the column
a quantity of the substance to be examined containing
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
eluate in fractions of 2 ml. Measure the absorbance (2.2.25)
of each fraction at the absorption maximum at 277 nm and
plot the values on a graph. The chromatogram obtained
does not present an absorption maximum before the elution
of the aprotinin.
Loss on drying (2.2.32) : maximum 6.0 per cent, determined
on 0.100 g by drying in vacuo.
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.
1208 See the information section on general monographs (cover pages)