european pharmacopoeia 5 with all supplements 3

Nitrous oxide

EUROPEAN PHARMACOPOEIA 5.0

Carbon monoxide. Gas chromatography (2.2.28). When the
test is carried out on a cylinder, use the first portion of gas
to be withdrawn.
Gas to be examined. The substance to be examined.
Reference gas. A mixture containing 5 ppm V/V of carbon
IMPURITIES
monoxide R in nitrous oxide R.
Column :
A. oxygen,
— material : stainless steel,
B. argon.
— size : l = 2 m, Ø = 4 mm,
— stationary phase : suitable molecular sieve for
chromatography (0.5 nm).
01/2005:0416
Carrier gas : helium for chromatography R.
NITROUS OXIDE
Flow rate : 60 ml/min.
Temperature :
Dinitrogenii oxidum
— column : 50 °C,
— injection port and detector : 130 °C.
N 2O
Mr 44.01 Detection : flame ionisation with methaniser.
Injection : loop injector.
DEFINITION
Content : minimum 98.0 per cent V/V of N2O in the gaseous Adjust the injected volumes and the operating conditions so

that the height of the peak due to carbon monoxide in the
phase, when sampled at 15 °C.
chromatogram obtained with the reference gas is at least
This monograph applies to nitrous oxide for medicinal use.
35 per cent of the full scale of the recorder.
Limit :
CHARACTERS
— carbon monoxide : not more than the area of the
Appearance : colourless gas.
corresponding peak in the chromatogram obtained with
Solubility : at 20 °C and at a pressure of 101 kPa, 1 volume
the reference gas (5 ppm V/V).
dissolves in about 1.5 volumes of water.
Nitrogen monoxide and nitrogen dioxide : maximum
PRODUCTION
2 ppm V/V in total in the gaseous and liquid phases,
determined using a chemiluminescence analyser (2.5.26).
Nitrous oxide is produced from ammonium nitrate by
Gas to be examined. The substance to be examined.
thermic decomposition.
Reference gas (a). Nitrous oxide R.
Examine the gaseous phase.
If the test is performed on a cylinder, keep the cylinder at Reference gas (b). A mixture containing 2 ppm V/V of
nitrogen monoxide R in nitrogen R1.
room temperature for at least 6 h before carrying out the
tests. Keep the cylinder in the vertical position with the
Calibrate the apparatus and set the sensitivity using
outlet valve uppermost.
reference gases (a) and (b). Measure the content of nitrogen
monoxide and nitrogen dioxide, separately examining the

Carbon dioxide. Gas chromatography (2.2.28).
samples collected from the gaseous phase and the liquid
Gas to be examined. The substance to be examined.
phase of the gas to be examined.
Reference gas. A mixture containing 300 ppm V/V of carbon Multiply the result obtained by the quenching correction
dioxide R1 in nitrous oxide R.
factor in order to correct the quenching effect on the
Column :
analyser response caused by the nitrous oxide matrix effect.
— material : stainless steel,
The quenching correction factor is determined by applying
a known reference mixture of nitrogen monoxide in nitrous
— size : l = 3.5 m, Ø = 2 mm,
oxide and comparing the actual content with the content
— stationary phase: ethylvinylbenzene-divinylbenzene
indicated by the analyser which has been calibrated with
copolymer R.
a NO/N2 reference mixture.
Carrier gas : helium for chromatography R.
=
Flow rate : 15 ml/min.
Temperature :
Water : maximum 67 ppm V/V, determined using an
— column : 40 °C,
electrolytic hygrometer (2.5.28).
— detector : 90 °C.
Assay. Gas chromatography (2.2.28).
Detection : thermal conductivity.
Gas to be examined. The substance to be examined.
Injection : loop injector.

Reference gas. Nitrous oxide R.
Adjust the injected volumes and operating conditions so
Column :
that the height of the peak due to carbon dioxide in the
— material : stainless steel,
chromatogram obtained with the reference gas is at least
— size : l = 2 m, Ø = 2 mm,
35 per cent of the full scale of the recorder. The test is
— stationary phase : silica gel for chromatography R
not valid unless the chromatograms obtained show a clear
(250-355 µm).
separation of carbon dioxide from nitrous oxide.
Carrier
gas : helium for chromatography R.
Limit :
Flow
rate
: 50 ml/min.
— carbon dioxide : not more than the area of the
Temperature
:
corresponding peak in the chromatogram obtained with
— column and injection port : 60 °C,
the reference gas (300 ppm V/V).
STORAGE
Where the gas has to be stored, store as a compressed gas
or a liquid in appropriate containers complying with the
legal regulations.

2110

See the information section on general monographs (cover pages)


Nizatidine

EUROPEAN PHARMACOPOEIA 5.0

01/2005:1453

— detector : 130 °C.
Detection : thermal conductivity.

NIZATIDINE

Injection : loop injector.
Adjust the injected volumes and the operating conditions
so that the height of the peak due to nitrous oxide in the
chromatogram obtained with the reference gas is at least
35 per cent of the full scale of the recorder.

Nizatidinum

The area of the peak due to nitrous oxide in the
chromatogram obtained with the gas to be examined is at
least 98.0 per cent of the area of the peak due to nitrous
oxide in the chromatogram obtained with the reference gas.
IDENTIFICATION
First identification : A.
Second identification : B, C.

A. Infrared absorption spectrophotometry (2.2.24).
Comparison : Ph. Eur. reference spectrum of nitrous
oxide.
B. Place a glowing splinter of wood in the substance to be
examined. The splinter bursts into flame.
C. Introduce the substance to be examined into alkaline
pyrogallol solution R. A brown colour does not develop.
TESTS
Examine the gaseous phase.
If the test is performed on a cylinder, keep the cylinder of
the substance to be examined at room temperature for at
least 6 h before carrying out the tests. Keep the cylinder in
the vertical position with the outlet valve uppermost.
Carbon dioxide : maximum 300 ppm V/V, determined using
a carbon dioxide detector tube (2.1.6).
Carbon monoxide : maximum 5 ppm V/V, determined using
a carbon monoxide detector tube (2.1.6). When the test is
carried out on a cylinder, use the first portion of the gas
to be withdrawn.
Nitrogen monoxide and nitrogen dioxide : maximum
2 ppm V/V, determined using a nitrogen monoxide and
nitrogen dioxide detector tube (2.1.6).
Water vapour : maximum 67 ppm V/V, determined using a
water vapour detector tube (2.1.6).
STORAGE
Store liquefied under pressure in suitable containers
complying with the legal regulations. The taps and valves
are not greased or oiled.
IMPURITIES
A. carbon dioxide,

B. carbon monoxide,
C. nitrogen monoxide,
D. nitrogen dioxide,
E. water.
General Notices (1) apply to all monographs and other texts

C12H21N5O2S2

Mr 331.5

DEFINITION
Nizatidine contains not less than 97.0 per cent and not
more than the equivalent of 101.0 per cent of (EZ)-N-[2-[[[2[(dimethylamino)methyl]thiazol-4-yl]methyl]sulphanyl]ethyl]N′-methyl-2-nitroethene-1,1-diamine, calculated with
reference to the dried substance.
CHARACTERS
An almost white or slightly brownish, crystalline powder,
sparingly soluble in water, soluble in methanol.
IDENTIFICATION
First identification : C.
Second identification : A, B, D.
A. Melting point (2.2.14) : 131 °C to 134 °C.
B. Dissolve 0.10 g of the substance to be examined in
methanol R and dilute to 100.0 ml with the same solvent.
Dilute 2.0 ml of the solution to 100.0 ml with methanol R.
Examined between 220 nm and 350 nm (2.2.25), the
solution shows two absorption maxima, at 242 nm and
325 nm. The ratio of the absorbance measured at the
maximum at 325 nm to that measured at the maximum at
242 nm is 2.2 to 2.5.
C. Examine by infrared absorption spectrophotometry

(2.2.24), comparing with the spectrum obtained with
nizatidine CRS. Examine the substance prepared as discs.
D. Examine by thin-layer chromatography (2.2.27), using a
TLC silica gel plate R.
Test solution. Dissolve 50 mg of the substance to be
examined in methanol R and dilute to 10 ml with the
same solvent.
Reference solution (a). Dissolve 50 mg of nizatidine CRS
in methanol R and dilute to 10 ml with the same solvent.
Reference solution (b). Dissolve 50 mg of nizatidine CRS
and 50 mg of ranitidine hydrochloride CRS in
methanol R and dilute to 10 ml with the same solvent.
Apply to the plate 5 µl of each solution. Develop over a
path corresponding to two thirds of the height of the plate
using a mixture of 2 volumes of water R, 4 volumes of
concentrated ammonia R1, 15 volumes of 2-propanol R
and 25 volumes of ethyl acetate R. Allow the plate to dry
in air and expose to iodine vapour until the spots are
clearly visible. Examine in daylight. The principal spot
in the chromatogram obtained with the test solution is
similar in position and size to the principal spot in the
chromatogram obtained with reference solution (a). The
test is not valid unless the chromatogram obtained with
reference solution (b) shows two clearly separated spots.
2111


Nizatidine

EUROPEAN PHARMACOPOEIA 5.0

TESTS
Appearance of solution. Dissolve 0.2 g of the substance to
be examined in a 10 g/l solution of hydrochloric acid R and
dilute to 20 ml with the same acid solution. The solution is
clear (2.2.1) and not more intensely coloured than reference
solution Y5 (2.2.2, Method II).
pH (2.2.3). Dissolve 0.2 g of the substance to be examined
in carbon dioxide-free water R and dilute to 20 ml with the
same solvent. The pH of the solution is 8.5 to 10.0.
Related substances. Examine by liquid chromatography
(2.2.29) as described under Assay, replacing the mixture of
mobile phases by the following elution programme :

Reference solution (a). Dilute 1.0 ml of test solution (a) to
100.0 ml with a mixture of 24 volumes of mobile phase B
and 76 volumes of mobile phase A.
Reference solution (b). Dissolve 15.0 mg of nizatidine CRS
in a mixture of 24 volumes of mobile phase B and 76 volumes
of mobile phase A and dilute to 50.0 ml with the same
mixture of mobile phases.
Reference solution (c). Dissolve 5 mg of nizatidine CRS
and 0.5 mg of nizatidine impurity F CRS in a mixture of
24 volumes of mobile phase B and 76 volumes of mobile
phase A and dilute to 100.0 ml with the same mixture of
mobile phases.
The chromatographic procedure may be carried out using :

Time
(min)

Mobile phase A
(per cent V/V)

Comment

Mobile phase B
(per cent V/V)

— a stainless steel column 0.25 m long and 4.6 mm in
internal diameter packed with octadecylsilyl silica gel for
chromatography R (5 µm),

0-3

76

24

isocratic

3 - 20

76 → 50

24 → 50

linear gradient

20 - 45

50

50

isocratic

45 - 50

50 → 76

50 → 24

linear gradient

50 - 60

76

24

re-equilibration

Inject 20 µl of reference solution (a). Adjust the sensitivity
of the system so that the height of the principal peak in
the chromatogram obtained with the reference solution is
at least 50 per cent of the full scale of the recorder. The
test is not valid unless the retention time of nizatidine is
between 10 min and 20 min and the symmetry factor of the
peak due to nizatidine is not greater than 2.0. Inject 20 µl

of reference solution (c). The test is not valid unless the
resolution between the peak due to nizatidine (first peak)
and impurity F (second peak) is at least 2.0.
Inject 20 µl of test solution (a). In the chromatogram
obtained, the area of any peak apart from the principal peak,
is not greater than 0.3 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.3 per cent) and the sum of the areas of all these peaks
is not greater than 1.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(1.5 per cent). Disregard any peak with an area less
than 0.03 times the area of the principal peak in the
chromatogram obtained with reference solution (a).

— as mobile phase at a flow rate of 1.0 ml/min a mixture of
35 volumes of mobile phase B and 65 volumes of mobile
phase A :
Mobile phase A. Dissolve 5.9 g of ammonium acetate R
in 760 ml of water R, add 1 ml of diethylamine R, and
adjust the pH to 7.5 with acetic acid R,
Mobile phase B. Methanol R,
— as detector a spectrophotometer set at 254 nm.
Inject 20 µl of reference solution (b). The test is not valid
unless the retention time of nizatidine is between 8 min and
10 min and the symmetry of the peak due to nizatidine is
not greater than 2.0.
Inject reference solution (b) six times. The test is not valid
unless the relative standard deviation of the peak area for
nizatidine is at most 2.0 per cent.
Inject 20 µl of test solution (b) and 20 µl of reference

solution (b). Calculate the percentage content of nizatidine
from the areas of the peaks and the declared content of
nizatidine CRS.
IMPURITIES

Heavy metals (2.4.8). 1.0 g complies with limit test C for
heavy metals (20 ppm). Prepare the standard using 2 ml of
lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32). Not more than 0.5 per cent,
determined on 1.000 g by drying in an oven at 100 °C to
105 °C.
Sulphated ash (2.4.14). Not more than 0.1 per cent,
determined on 1.0 g.

A. X = NH : N,N′-dimethyl-2-nitroethene-1,1-diamine,
B. X = S :(EZ)-N-methyl-1-(methylsulphanyl)-2-nitroethen-1amine,

ASSAY
Examine by liquid chromatography (2.2.29).
Test solution (a). Dissolve 50.0 mg of the substance to be
examined in a mixture of 24 volumes of mobile phase B and
76 volumes of mobile phase A and dilute to 10.0 ml with the
same mixture of mobile phases.
Test solution (b). Dissolve 15.0 mg of the substance to be
examined in a mixture of 24 volumes of mobile phase B and C. (EZ)-N-[2-[[[2-[(dimethylamino)methyl]thiazol-476 volumes of mobile phase A and dilute to 50.0 ml with the
yl]methyl]sulphinyl]ethyl]-N′-methyl-2-nitroethene-1,1same mixture of mobile phases.
diamine,
2112

See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 5.0

Nomegestrol acetate

DEFINITION
Nomegestrol acetate contains not less than 97.0 per cent
and not more than the equivalent of 103.0 per cent of
6-methyl-3,20-dioxo-19-norpregna-4,6-dien-17-yl acetate,
calculated with reference to the dried substance.
D. R = S-CH2-CH2-NH2 : 2-[[[2-[(dimethylamino)methyl]thiazol-4-yl]methyl]sulphanyl]ethanamine,
E. R = S-CH2-CH2-NH-CO-CH2-NO2 :N-[2-[[[2-[(dimethylamino)methyl]thiazol-4-yl]methyl]sulphanyl]ethyl]-2-nitroacetamide,
I. R = S-CH2-CH2-NH-CO-NH-CH3 :N-[2-[[[2-[(dimethylamino)methyl]thiazol-4-yl]methyl]sulphanyl]ethyl]-N′-methylurea,

CHARACTERS
A white or almost white, crystalline powder, practically
insoluble in water, freely soluble in acetone, soluble in
alcohol.
IDENTIFICATION
Examine by infrared absorption spectrophotometry (2.2.24),
comparing with the spectrum obtained with nomegestrol
acetate CRS.

J. R = OH : [2-[(dimethylamino)methyl]thiazol-4-yl]methanol, TESTS
Appearance of solution. Dissolve 1.0 g in methylene
chloride R and dilute to 10 ml with the same solvent. The
solution is clear (2.2.1) and not more intensely coloured
than reference solution Y5 (2.2.2, Method II).
Specific optical rotation (2.2.7). Dissolve 0.500 g in

ethanol R and dilute to 25.0 ml with the same solvent. The
1 1
F. (EZ)-N ,N ′-[thiazole-2,4-diylbis(methylenesulphanediylspecific optical rotation is − 60.0 to − 64.0, calculated with
ethylene)]bis(N′-methyl-2-nitroethene-1,1-diamine),
reference to the dried substance.
Related substances. Examine by liquid chromatography
(2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be
examined in methanol R and dilute to 50.0 ml with the same
solvent.
Reference solution (a). Dilute 1.0 ml of the test solution to
200.0 ml with the mobile phase.
Reference solution (b). Dissolve 25.0 mg of nomegestrol
G. N,N′-bis[2-[[[2-[(dimethylamino)methyl]thiazol-4acetate impurity A CRS in methanol R and dilute to 50.0 ml
yl]methyl]sulphanyl]ethyl]-2-nitroethene-1,1-diamine,
with the same solvent.
Reference solution (c). Dissolve 25.0 mg of nomegestrol
acetate CRS in 20 ml of methanol R, add 0.25 ml of reference
solution (b) and dilute to 50.0 ml with the mobile phase.
The chromatographic procedure may be carried out using :
H. 2-(dimethylamino)thioacetamide,
— a stainless steel column 0.25 m long and 4.6 mm in
internal diameter packed with octadecylsilyl silica gel for
chromatography R (5 µm),
— as mobile phase at a flow rate of 1.3 ml/min a mixture of
24 volumes of acetonitrile R, 38 volumes of methanol R
and 38 volumes of water R,
K. 3-(methylamino)-5,6-dihydro-2H-1,4-thiazin-2-one oxime.
— as detector, a variable wavelength spectrophotometer
capable of operating at 245 nm and at 290 nm.

01/2005:1551 Inject 10 µl of reference solution (c) and record the
chromatogram with the detector set at 245 nm.
When the chromatogram is recorded in the prescribed
NOMEGESTROL ACETATE
conditions, the retention times are : nomegestrol acetate
about 17 min and impurity A about 18.5 min. Adjust the
Nomegestroli acetas
sensitivity of the system at 245 nm so that the height of the
peak due to impurity A in the chromatogram obtained with
reference solution (c) is at least 50 per cent of the full scale
of the recorder.
Measure the height Hp above the baseline of the peak due
to impurity A and the height Hv above the baseline of the
lowest point of the curve separating this peak from the peak
due to nomegestrol acetate. The test is not valid unless Hp is
greater than 5 times Hv.
Inject 10 µl of reference solution (a) and record the
chromatogram with the detector set at 290 nm. Adjust the
C23H30O4
Mr 370.5 sensitivity of the system at 290 nm so that the height of the
General Notices (1) apply to all monographs and other texts

2113


Nonoxinol 9

EUROPEAN PHARMACOPOEIA 5.0

principal peak in the chromatogram obtained with reference

solution (a) is at least 50 per cent of the full scale of the
recorder.
Inject 10 µl of the test solution and record the chromatograms
at 245 nm and 290 nm for 1.5 times the retention time of
the principal peak.
In the chromatogram obtained with the test solution at
290 nm : the area of any peak, apart from the principal peak,
is not greater than 0.2 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.1 per cent). Disregard any peak with an area less than
0.04 times that of the principal peak in the chromatogram
obtained with reference solution (a) (0.02 per cent). In the
chromatogram obtained with the test solution at 245 nm : the
area of any peak corresponding to impurity A is not greater
than 0.4 times the area of the peak due to impurity A in the
chromatogram obtained with reference solution (c) (0.2 per
cent) ; the area of any peak, apart from the principal peak
and any peak corresponding to impurity A, is not greater
than 0.2 times the area of the peak due to impurity A in the
chromatogram obtained with reference solution (c) (0.1 per
cent). Disregard any peak with an area less than 0.1 times
that of the peak due to impurity A in the chromatogram
obtained with reference solution (c) (0.05 per cent).
In the chromatograms obtained at 290 nm and 245 nm, the
sum of the related substances apart from impurity A is not
greater than 0.3 per cent.
Loss on drying (2.2.32). Not more than 0.5 per cent,
determined on 1.000 g by drying in an oven at 100-105 °C.
ASSAY
Dissolve 50.0 mg in ethanol R and dilute to 100.0 ml with

the same solvent. Dilute 2.0 ml of the solution to 100.0 ml
with ethanol R. Measure the absorbance (2.2.25) at the
maximum at 287 nm.
Calculate the content of C23H30O4 taking the specific
absorbance to be 685.
STORAGE
Store protected from light.
IMPURITIES

CHARACTERS
Appearance : clear, colourless or light yellow, viscous liquid.
Solubility : miscible with water, with alcohol and with
vegetable oils.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : Ph. Eur. reference spectrum of
nonoxinol 9.
Preparation : film between sodium chloride R plates.
B. It complies with the test for cloud point (see Tests).
TESTS
Acidity or alkalinity. Boil 1.0 g with 20 ml of carbon
dioxide-free water R for 1 min, with constant stirring.
Cool and filter. To 10 ml of the filtrate, add 0.05 ml of
bromothymol blue solution R1. Not more than 0.5 ml of
0.01 M hydrochloric acid or 0.01 M sodium hydroxide is
required to change the colour of the indicator.
Hydroxyl value (2.5.3, Method A) : 84 to 94.
Cloud point : 52 °C to 58 °C.
Dissolve 1.0 g in 99 g of water R. Transfer about 30 ml of
this solution into a test-tube, heat on a water-bath and stir

continuously until the solution becomes cloudy. Remove the
test-tube from the water-bath (ensuring that the temperature
does not increase more than 2 °C) and continue to stir.
The cloud point is the temperature at which the solution
becomes sufficiently clear that the entire thermometer bulb
is plainly seen.
Ethylene oxide and dioxan (2.4.25) : maximum 1 ppm of
ethylene oxide and maximum 10 ppm of dioxan.
Heavy metals (2.4.8) : maximum 10 ppm.
Dissolve 2.0 g in distilled water R and dilute to 20.0 ml with
the same solvent. 12 ml of this solution complies with limit
test A. Prepare the standard using lead standard solution
(1 ppm Pb) R.
Water (2.5.12) : maximum 0.5 per cent, determined on 2.00 g.
Total ash (2.4.16) : maximum 0.4 per cent, determined on
1.0 g.
STORAGE
In an airtight container.
01/2005:0732

NORADRENALINE HYDROCHLORIDE
Noradrenalini hydrochloridum
A. 6α-methyl-3,20-dioxo-19-norpregn-4-en-17-yl acetate.
01/2005:1454
C8H12ClNO3

NONOXINOL 9
Nonoxinolum 9
DEFINITION
α-(4-Nonylphenyl)-ω-hydroxynona(oxyethylene).

Mixture consisting mainly of monononylphenyl
ethers of macrogols corresponding to the formula :
C9H19C6H4-[OCH2-CH2]n-OH where the average value of n is 9.
It may contain free macrogols.
2114

Mr 205.6

DEFINITION
(R)-2-Amino-1-(3,4-dihydroxyphenyl)ethanol hydrochloride.
Content : 98.5 per cent to 101.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or brownish-white, crystalline powder.
Solubility : very soluble in water, slightly soluble in alcohol.
It becomes coloured on exposure to air and light.

See the information section on general monographs (cover pages)


Noradrenaline tartrate

EUROPEAN PHARMACOPOEIA 5.0

IDENTIFICATION
A. Specific optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Dissolve 2 g in 20 ml of a 5 g/l solution of sodium
metabisulphite R and make alkaline by addition of
ammonia R. Keep in iced water for 1 h and filter. Wash

the precipitate with 3 quantities, each of 2 ml, of water R,
with 5 ml of alcohol R and finally with 5 ml of ether R
and dry in vacuo for 3 h. Examine the noradrenaline base
thus prepared, comparing with the spectrum obtained
with noradrenaline base prepared by the same method
from a suitable amount of noradrenaline tartrate CRS.
Examine the substances prepared as discs.
C. 0.2 ml of solution S (see Tests) 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. The solution is clear (2.2.1) and
not more intensely coloured than a mixture of 0.2 ml of blue
primary solution, 0.4 ml of yellow primary solution, 0.4 ml of
red primary solution and 9 ml of a 13.7 per cent V/V solution
of dilute hydrochloric acid R (2.2.2, Method II).
Dissolve 0.2 g in carbon dioxide-free water R and dilute
to 10 ml with the same solvent. Examine the solution
immediately.
pH (2.2.3) : 3.5 to 4.5 for solution S.
Specific optical rotation (2.2.7) : − 37 to − 41, determined
on solution S (anhydrous substance).
Noradrenalone : maximum 0.12 per cent.
Dissolve 30.0 mg in 0.01 M hydrochloric acid and dilute to
25.0 ml with the same acid. The absorbance (2.2.25) of the
solution measured at 310 nm is not greater than 0.20.
Adrenaline. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 0.15 g of the substance to be
examined in water R and dilute to 10 ml with the same

solvent. Prepare immediately before use.
Reference solution (a). Dissolve 12.5 mg of adrenaline
tartrate CRS in water R and dilute to 10 ml with the same
solvent. Prepare immediately before use.
Reference solution (b). Dilute 2 ml of reference solution (a)
to 10 ml with water R.
Reference solution (c). Mix 2 ml of the test solution and
2 ml of reference solution (b).
Plate : TLC silica gel G plate R.
Mobile phase : anhydrous formic acid R, acetone R,
methylene chloride R (0.5:50:50 V/V/V).
Application : apply as bands 20 mm by 2 mm, 6 µl of the
test solution, 6 µl of reference solution (a), 6 µl of reference
solution (b) and 12 µl of reference solution (c). Allow to
dry in air and spray the bands with a saturated solution of
sodium hydrogen carbonate R. Allow the plate to dry in air
and spray the bands twice with acetic anhydride R, drying
between the 2 sprayings. Heat the plate at 50 °C for 90 min.
Development : over a path of 15 cm.
Drying : in air.
Detection : spray with a solution freshly prepared by
mixing 2 volumes of ethylenediamine R and 8 volumes of
methanol R and adding 2 volumes of a 5 g/l solution of
potassium ferricyanide R. Dry the plate at 60 °C for 10 min
and examine in ultraviolet light at 254 nm and 365 nm.
General Notices (1) apply to all monographs and other texts

System suitability : the chromatogram obtained with
reference solution (c) shows above the most intense
zone a clearly separated zone corresponding to the most

intense zone in the chromatogram obtained with reference
solution (a).
Limits : any zone situated immediately above the most
intense zone is not more intense than the corresponding
zone in the chromatogram obtained with reference
solution (b) (1.0 per cent).
Water (2.5.12) : maximum 0.5 per cent, determined on
1.000 g.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 0.50 g.
ASSAY
Dissolve 0.180 g in 50 ml of acetic anhydride R and add 10 ml
of anhydrous formic 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 20.56 mg of
C8H12ClNO3.
STORAGE
Store in an airtight container, or preferably in a sealed tube
under vacuum or under an inert gas, protected from light.
01/2005:0285

NORADRENALINE TARTRATE
Noradrenalini tartras

C12H17NO9,H2O

Mr 337.3

DEFINITION
(1R)-2-Amino-1-(3,4-dihydroxyphenyl)ethanol hydrogen

(2R,3R)-2,3-dihydroxybutanedioate monohydrate.
Content : 98.5 per cent to 101.0 per cent (anhydrous
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : freely soluble in water, slightly soluble in alcohol.
IDENTIFICATION
A. Dissolve 2 g in 20 ml of a 5 g/l solution of sodium
metabisulphite R and make alkaline by addition of
ammonia R. Keep in iced water for 1 h and filter.
Reserve the filtrate for identification test C. Wash the
precipitate with 3 quantities, each of 2 ml, of water R,
with 5 ml of alcohol R and finally with 5 ml of ether R
and dry in vacuo for 3 h. The specific optical rotation
(2.2.7) of the precipitate (noradrenaline base) is − 44
to − 48, determined using a 20.0 g/l solution in 0.5 M
hydrochloric acid.
B. Infrared absorption spectrophotometry (2.2.24).
Use noradrenaline base prepared as described under
identification test A and compare with the spectrum
obtained with noradrenaline base prepared by the
same method from a suitable amount of noradrenaline
tartrate CRS. Examine the substances prepared as discs.
C. 0.2 ml of the filtrate obtained in identification test A gives
reaction (b) of tartrates (2.3.1).
2115


Norethisterone

EUROPEAN PHARMACOPOEIA 5.0

TESTS
Appearance of solution. The solution is clear (2.2.1) and
not more intensely coloured than reference solution BY5
(2.2.2, Method II).
Dissolve 0.2 g in water R and dilute to 10 ml with the same
solvent. Examine the solution immediately.
Noradrenalone : the absorbance (2.2.25) of the solution
measured at 310 nm is not greater than 0.20.
Dissolve 50.0 mg in 0.01 M hydrochloric acid and dilute to
25.0 ml with the same acid.
Adrenaline. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 0.25 g of the substance to be
examined in water R and dilute to 10 ml with the same
solvent. Prepare immediately before use.
Reference solution (a). Dissolve 12.5 mg of adrenaline
tartrate CRS in water R and dilute to 10 ml with the same
solvent. Prepare immediately before use.
Reference solution (b). Dilute 2 ml of reference solution (a)
to 10 ml with water R.
Reference solution (c). Mix 2 ml of the test solution with
2 ml of reference solution (b).
Plate : TLC silica gel G plate R.
Mobile phase : anhydrous formic acid R, acetone R,
methylene chloride R (0.5:50:50 V/V/V).
Application : apply as bands 20 mm by 2 mm, 6 µl of the
test solution, 6 µl of reference solution (a), 6 µl of reference
solution (b) and 12 µl of reference solution (c). Allow to
dry in air and spray the bands with a saturated solution of

sodium hydrogen carbonate R. Allow the plate to dry in air
and spray the bands twice with acetic anhydride R, drying
between the 2 sprayings. Heat the plate at 50 °C for 90 min.
Development : over a path of 15 cm.
Drying : in air.
Detection : spray with a solution freshly prepared by
mixing 2 volumes of ethylenediamine R and 8 volumes of
methanol R and adding 2 volumes of a 5 g/l solution of
potassium ferricyanide R. Dry the plate at 60 °C for 10 min
and examine in ultraviolet light at 254 nm and 365 nm.
System suitability : the chromatogram obtained with
reference solution (c) shows above the most intense
zone a clearly separated zone corresponding to the most
intense zone in the chromatogram obtained with reference
solution (a).
Limit :
— adrenaline : any zone situated immediately above
the most intense zone is not more intense than the
corresponding zone in the chromatogram obtained with
reference solution (b) (1.0 per cent).
Water (2.5.12) : 4.5 per cent to 5.8 per cent, determined on
0.500 g.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 0.5 g.

01/2005:0234

NORETHISTERONE
Norethisteronum

C20H26O2

Mr 298.4

DEFINITION
Norethisterone contains not less than 98.0 per cent
and not more than the equivalent of 102.0 per cent of
17-hydroxy-19-nor-17α-pregn-4-en-20-yn-3-one, calculated
with reference to the dried substance.
CHARACTERS
A white or yellowish-white, crystalline powder, practically
insoluble in water, slightly soluble in alcohol.
It melts at about 206 °C, with decomposition.

IDENTIFICATION
First identification : A, B, E.
Second identification : B, C, D, E.
A. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
norethisterone CRS. Examine the substances prepared
in the form of discs. If the spectra obtained with the
substance to be examined and the reference substance
show differences, dissolve the substances in chloroform R,
evaporate to dryness on a water-bath and then record
the spectra.
B. Examine by thin-layer chromatography (2.2.27), using
kieselguhr G R as the coating substance. Impregnate the
plate by placing it in a tank containing the necessary
quantity of a mixture of 10 volumes of formamide R
and 90 volumes of acetone R so that the plate dips

about 5 mm into the liquid. When the front of the
impregnation mixture has risen at least 1 cm above
the level prescribed for the mobile phase, remove the
plate and allow it to stand at room temperature until
the solvent has completely evaporated (about 2 min to
5 min). Use the impregnated plate within 2 h and carry
out the chromatography in the same direction as the
impregnation.
Test solution. Dissolve 10 mg of the substance to be
examined in chloroform R and dilute to 10 ml with the
same solvent.
Reference solution. Dissolve 10 mg of norethisterone CRS
in chloroform R and dilute to 10 ml with the same solvent.
ASSAY
Apply separately to the plate 2 µl of each solution.
Dissolve 0.300 g in 50 ml of anhydrous acetic acid R,
Develop over a path of 15 cm using a mixture of
heating gently if necessary. Titrate with 0.1 M perchloric
20 volumes of dioxan R and 80 volumes of hexane R.
acid using 0.1 ml of crystal violet solution R as indicator,
Heat the plate at 120 °C for 15 min and spray with
until a bluish-green colour is obtained.
alcoholic solution of sulphuric acid R. Heat at 120 °C
for 10 min to 15 min or until the spots appear. Allow
1 ml of 0.1 M perchloric acid is equivalent to 31.93 mg of
to cool and examine in daylight and in ultraviolet light
C12H17NO9.
at 365 nm. The principal spot in the chromatogram
STORAGE
obtained with the test solution is similar in position,

colour, fluorescence and size to the principal spot in the
In an airtight container, or preferably in a sealed tube under
chromatogram obtained with the reference solution.
vacuum or under an inert gas, protected from light.

2116

See the information section on general monographs (cover pages)


EUROPEAN PHARMACOPOEIA 5.0

Norethisterone acetate

ASSAY
C. Dissolve about 2 mg in 2 ml of alcohol R, add 1 ml of
ammoniacal silver nitrate solution R and heat on a
Dissolve 0.200 g in 40 ml of tetrahydrofuran R. Add 10 ml
water-bath. The solution becomes turbid and a white
of a 100 g/l solution of silver nitrate R. Using 2 ml of
precipitate is formed which becomes grey when heated. A bromocresol green solution R as indicator, titrate with 0.1 M
silver mirror is deposited on the walls of the tube.
sodium hydroxide until a violet colour is obtained. Carry
out a blank titration.
D. Dissolve about 2 mg in a cooled mixture of 2 ml of
ethanol R and 2 ml of sulphuric acid R and heat to 70 °C. 1 ml of 0.1 M sodium hydroxide is equivalent to 29.84 mg of
The resulting solution is dichroic, appearing blue-violet in C20H26O2.
transmitted light and red in reflected light. The solution STORAGE
shows a bright-red fluorescence in ultraviolet light at
Store protected from light.

365 nm.
E. Dissolve about 2 mg in 2 ml of alcohol R, add 1 ml of a
10 g/l solution of butylhydroxytoluene R in alcohol R
and 2 ml of 1 M sodium hydroxide. Heat in a water-bath
at 80 °C for 30 min and cool to room temperature. A
yellowish-pink colour is produced.

01/2005:0850

NORETHISTERONE ACETATE
Norethisteroni acetas

TESTS
Solution S. Dissolve 0.200 g in dioxan R and dilute to
10.0 ml with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y6 (2.2.2,
Method I).
Specific optical rotation (2.2.7). Dilute 5.0 ml of solution S C H O
Mr 340.5
to 10.0 ml with dioxan R. The specific optical rotation is − 33 22 28 3
to − 37, calculated with reference to the dried substance.
DEFINITION
3-Oxo-19-nor-17α-pregn-4-en-20-yn-17-yl acetate.
Absorbance (2.2.25). Dissolve 10.0 mg in alcohol R and
dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of
Content : 98.0 per cent to 101.0 per cent (dried substance).
this solution to 100.0 ml with alcohol R. The solution shows
an absorption maximum at 240 nm. The specific absorbance CHARACTERS
at the maximum is 550 to 590, calculated with reference to

Appearance : white or yellowish-white, crystalline powder.
the dried substance.
Solubility : practically insoluble in water, freely soluble in
Related substances. Examine by thin-layer chromatography methylene chloride, soluble in alcohol.
(2.2.27), using a suitable silica gel as the coating substance. It shows polymorphism.
IDENTIFICATION
Test solution. Dissolve 50 mg of the substance to be
examined in a mixture of 1 volume of methanol R and
Infrared absorption spectrophotometry (2.2.24).
9 volumes of chloroform R and dilute to 10 ml with the same Preparation : discs.
mixture of solvents.
Comparison : norethisterone acetate CRS.
Reference solution (a). Dilute 1.0 ml of the test solution
If the spectra show differences, dissolve the substance to
to 200 ml with a mixture of 1 volume of methanol R and
be examined and the reference substance separately in
9 volumes of chloroform R.
methylene chloride R, evaporate to dryness on a water-bath
and record new spectra using the residues.
Reference solution (b). Dissolve 25 mg of ethisterone CRS
TESTS
in a mixture of 1 volume of methanol R and 9 volumes of
chloroform R, add 5 ml of the test solution and dilute to
Specific optical rotation (2.2.7) − 30 to − 35 (dried
100 ml with the same mixture of solvents.
substance).
Dissolve 0.500 g in ethanol R and dilute to 25.0 ml with the
Apply separately to the plate, as two applications of 5 µl,
same
solvent.

10 µl of each solution. Develop over a path of 15 cm using
a mixture of 10 volumes of acetone R and 90 volumes
Related substances. Liquid chromatography (2.2.29).
of chloroform R. Allow the plate to dry in air, spray with
Test solution. Dissolve 25.0 mg of the substance to be
alcoholic solution of sulphuric acid R and heat at 100 °C to examined in the mobile phase and dilute to 10.0 ml with the
105 °C for 5 min. Examine in ultraviolet light at 365 nm.
mobile phase.
Reference solution (a). Dissolve 2 mg of desoxycortone
Any spot in the chromatogram obtained with the test
acetate CRS and 2 mg of norethisterone acetate CRS in the
solution, apart from the principal spot, is not more intense
than the spot in the chromatogram obtained with reference mobile phase and dilute to 50.0 ml with the mobile phase.
Reference solution (b). Dilute 1.0 ml of the test solution to
solution (a) (0.5 per cent). The test is not valid unless the
100.0 ml with the mobile phase.
chromatogram obtained with reference solution (b) shows
two clearly separated spots of approximately equal intensity. Column :
— size : l = 0.25 m, Ø = 4.6 mm,
Loss on drying (2.2.32). Not more than 0.5 per cent,
determined on 1.00 g by drying in an oven at 100 °C to
— stationary phase : octadecylsilyl silica gel for
105 °C for 3 h.
chromatography R (5 µm).
General Notices (1) apply to all monographs and other texts

2117


Norfloxacin

EUROPEAN PHARMACOPOEIA 5.0

Mobile phase : acetonitrile R, water R (60:40 V/V).
Flow rate : 1.0 ml/min.
Detection : variable wavelength spectrophotometer capable
of operating at 254 nm and at 210 nm.
Injection : 20 µl.
Run time : 3 times the retention time of norethisterone
acetate.
Relative retention with reference to norethisterone acetate
(retention time = about 10 min) : impurity A = about 0.48 ;
impurity D = about 0.65 ; impurity E = about 0.83 ;
impurity C = about 1.35 ; impurity B = about 1.40.
System suitability : reference solution (a) at 254 nm :
— resolution : minimum of 3.5 between the peaks due to
norethisterone acetate and to desoxycortone acetate.
Limits : spectrophotometer at 254 nm :
— any impurity : not more than 0.5 times the area of
the principal peak in the chromatogram obtained with
reference solution (b) (0.5 per cent),
— total: not more than 0.75 times the area of the principal
peak in the chromatogram obtained with reference
solution (b) (0.75 per cent),
— disregard limit : 0.05 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.05 per cent).
Limits : spectrophotometer at 210 nm :
— any impurity with a relative retention between 1.0 and
1.6, with reference to norethisterone acetate (retention

time = about 10 min) : not more than 0.3 times the area
of the principal peak in the chromatogram obtained with
reference solution (b) (0.3 per cent),
— total of these impurities: not more than 0.5 times the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.5 per cent),
— disregard limit : 0.05 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.05 per cent).
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.
ASSAY
Dissolve 0.200 g in 40 ml of tetrahydrofuran R. Add
10 ml of a 100 g/l solution of silver nitrate R and titrate
with 0.1 M sodium hydroxide, determining the end-point
potentiometrically (2.2.20). Carry out a blank titration.
1 ml of 0.1 M sodium hydroxide is equivalent to 34.05 mg of
C22H28O3.
IMPURITIES
Specified impurities : A, B, C, D, E.
Other detectable impurities : F, G.
A. norethisterone,

B. 3-oxo-19-nor-17α-pregn-5(10)-en-20-yn-17-yl acetate,
2118

C. 3-oxo-19-nor-17α-pregn-5-en-20-yn-17-yl acetate,

D. R1 = H, R2 = CO-CH3, R3 = C≡CH : 6β-acetyl-3-oxo-19-nor17α-pregn-4-en-20-yn-17-yl acetate,
E. R1 = R2 = H, R3 = CO-CH3 : 3,20-dioxo-19-nor-17α-pregn4-en-17-yl acetate,

F. R1 = H, R2 = OH, R3 = C≡CH : 6β-hydroxy-3-oxo-19-nor17α-pregn-4-en-20-yn-17-yl acetate,
G. R1 + R2 = O, R3 = C≡CH : 3,6-dioxo-19-nor-17α-pregn-4en-20-yn-17-yl acetate.
01/2005:1248

NORFLOXACIN
Norfloxacinum

C16H18FN3O3

Mr 319.3

DEFINITION
Norfloxacin contains not less than 99.0 per cent and not
more than the equivalent of 101.0 per cent of 1-ethyl-6-fluoro4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic
acid, calculated with reference to the dried substance.
CHARACTERS
A white or pale-yellow, hygroscopic, photosensitive,
crystalline powder, very slightly soluble in water, slightly
soluble in acetone and in alcohol.
IDENTIFICATION
Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
norfloxacin CRS. Examine the substances prepared as discs.
TESTS
Appearance of solution. Dissolve 0.5 g in a previously
filtered 4 g/l solution of sodium hydroxide R in methanol R
and dilute to 50 ml with the same solution. The solution
is not more opalescent than reference suspension II (2.2.1)
and not more intensely coloured than reference solution B7
(2.2.2, Method II).

See the information section on general monographs (cover pages)


Norgestrel

EUROPEAN PHARMACOPOEIA 5.0

Related substances. Examine by thin-layer chromatography
(2.2.27), using a TLC silica gel GF254 plate R, previously
washed with methanol R and dried in air.
Test solution (a). Dissolve 40 mg of the substance to be
examined in a mixture of equal volumes of methanol R and
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 equal volumes of methanol R and methylene
chloride R.
Reference solution (a). Dilute 1 ml of test solution (b) to
50 ml with a mixture of equal volumes of methanol R and
methylene chloride R.
Reference solution (b). Dissolve 4.0 mg of norfloxacin
impurity A CRS in a mixture of equal volumes of methanol R
and methylene chloride R and dilute to 5 ml with the same
mixture of solvents. Dilute 1 ml of this solution to 2 ml with
test solution (b).
Apply to the plate 5 µl of test solution (a) and 5 µl of
each reference solution. Develop over a path of 18 cm
using a mixture of 8 volumes of water R, 14 volumes of
diethylamine R, 20 volumes of toluene R, 40 volumes of

chloroform R and 40 volumes of methanol R. Dry the plate
in a current of air and examine in ultraviolet light at 254 nm
and then 365 nm. Any spot in the chromatogram obtained
with test solution (a), apart from the principal spot, is not
more intense than the principal spot in the chromatogram
obtained with reference solution (a) (0.2 per cent) and
there are no more than three such spots. The test is not
valid unless, in the chromatogram obtained with reference
solution (b), the ratio of the Rf value of impurity A to the Rf
value of norfloxacin is at least 1.2.
Heavy metals (2.4.8). 2.0 g complies with limit test D for
heavy metals (15 ppm). Prepare the standard using 3 ml of
lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32). Not more than 1.0 per cent,
determined on 1.000 g by drying in an oven at 100-105 °C
under high vacuum for 2 h.
Sulphated ash (2.4.14). Not more than 0.1 per cent,
determined on 1.0 g in a platinum crucible.

01/2005:0940

NORGESTREL
Norgestrelum

C21H28O2

Mr 312.5

DEFINITION
Norgestrel contains not less than 98.0 per cent and not more

than the equivalent of 102.0 per cent of rac-13-ethyl-17hydroxy-18,19-dinor-17α-pregn-4-en-20-yn-3-one, calculated
with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, practically
insoluble in water, sparingly soluble in methylene chloride,
slightly soluble in alcohol.
IDENTIFICATION
A. Dissolve 0.5 g in methylene chloride R and dilute to
10.0 ml with the same solvent. The angle of optical
rotation (2.2.7) is + 0.05° to − 0.05°.
B. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
norgestrel CRS.

TESTS
Related substances. 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 methylene chloride R and dilute to 10 ml with the same
solvent.
Reference solution (a). Dilute 1 ml of the test solution to
10 ml with methylene chloride R. Dilute 1 ml of this solution
ASSAY
to 20 ml with methylene chloride R.
Dissolve 0.240 g in 80 ml of anhydrous acetic acid R. Titrate Reference solution (b). Dilute 4 ml of reference solution (a)
with 0.1 M perchloric acid, determining the end-point
to 10 ml with methylene chloride R.
potentiometrically (2.2.20).
Reference solution (c). Dissolve 5 mg of norgestrel CRS and
1 ml of 0.1 M perchloric acid is equivalent to 31.93 mg of

5 mg of ethinylestradiol CRS in methylene chloride R and
C16H18FN3O3.
dilute to 50 ml with the same solvent.
Apply to the plate 10 µl of each solution. Develop over
STORAGE
a path of 15 cm using a mixture of 20 volumes of ethyl
Store in an airtight container, protected from light.
acetate R and 80 volumes of methylene chloride R. Allow
the plate to dry in air, spray with a 100 g/l solution of
IMPURITIES
phosphomolybdic acid R in alcohol R, heat at 100-105 °C
for 15 min and examine immediately. Any spot in the
chromatogram obtained with the test solution, apart from
the principal spot, is not more intense than the principal spot
in the chromatogram obtained with reference solution (a)
(0.5 per cent) and at most two such spots are more intense
than the spot in the chromatogram obtained with reference
solution (b) (0.2 per cent). The test is not valid unless the
chromatogram obtained with reference solution (c) shows
two clearly separated spots.
A. R = Cl : 7-chloro-1-ethyl-6-fluoro-4-oxo-1,4Loss on drying (2.2.32). Not more than 0.5 per cent,
dihydroquinoline-3-carboxylic acid,
determined on 1.000 g by drying in an oven at 100-105 °C.
Sulphated ash (2.4.14). Not more than 0.1 per cent,
B. R = NH-CH2-CH2-NH2 : 7-[(2-aminoethyl)amino]-1-ethyl-6determined on 1.0 g.
fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid.
General Notices (1) apply to all monographs and other texts

2119

Nortriptyline hydrochloride

EUROPEAN PHARMACOPOEIA 5.0

Related substances. Prepare the solutions in subdued
light and develop the chromatograms protected from light.
Examine by thin-layer chromatography (2.2.27), using a TLC
silica gel plate R.
Test solution (a). Dissolve 0.20 g of the substance to be
examined in alcohol R and dilute to 10 ml with the same
solvent.
Test solution (b). Dilute 1 ml of test solution (a) to 2 ml with
STORAGE
alcohol R.
Store protected from light.
Reference solution (a). Dissolve 10 mg of
dibenzosuberone CRS in alcohol R and dilute to
10 ml with the same solvent. Dilute 1 ml of the solution to
01/2005:0941 100 ml with alcohol R.
solution (b). Dissolve 10 mg of
NORTRIPTYLINE HYDROCHLORIDE Reference
norcyclobenzaprine CRS in alcohol R and dilute to
10 ml with the same solvent. Dilute 1 ml of the solution to
Nortriptylini hydrochloridum
100 ml with alcohol R.
Reference solution (c). To 0.1 ml of test solution (b) add
10 ml of reference solution (b).
Apply to the plate 5 µl of each solution. Develop over a
path of 15 cm in an unsaturated tank using a mixture of

3 volumes of diethylamine R, 15 volumes of ethyl acetate R
and 85 volumes of cyclohexane R. Allow the plate to dry in
air and spray with a freshly prepared mixture of 4 volumes
of formaldehyde solution R and 96 volumes of sulphuric
acid R. Examine immediately in ultraviolet light at 365 nm
C19H22ClN
Mr 299.8 and then at 254 nm. In the chromatogram obtained with test
solution (a) : any spot corresponding to dibenzosuberone,
DEFINITION
is not more intense than the spot in the chromatogram
Nortriptyline hydrochloride contains not less than 98.0 per
obtained with reference solution (a) (0.05 per cent) ; and any
cent and not more than the equivalent of 101.0 per cent
spot in the chromatogram obtained with test solution (b),
of 3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ylidene)apart from the principal spot and any spot corresponding to
N-methylpropan-1-amine hydrochloride, calculated with
dibenzosuberone, is not more intense than the spot in the
reference to the dried substance.
chromatogram obtained with reference solution (b) (0.1 per
cent). The test is not valid unless the chromatogram obtained
CHARACTERS
A white or almost white powder, sparingly soluble in water, with reference solution (c) shows two clearly separated spots.
Heavy metals (2.4.8). 1.0 g complies with limit test C for
soluble in alcohol and in methylene chloride.
heavy metals (20 ppm). Prepare the standard using 2 ml of
IDENTIFICATION
lead standard solution (10 ppm Pb) R.
First identification : C, E.
Loss on drying (2.2.32). Not more than 0.5 per cent,
Second identification : A, B, D, E.

determined on 1.000 g by drying in an oven at 100-105 °C
for 2 h.
A. Melting point (2.2.14) : 216 °C to 220 °C.
Sulphated ash (2.4.14). Not more than 0.1 per cent,
B. Dissolve 20.0 mg in methanol R and dilute to 100.0 ml
determined on 1.0 g.
with the same solvent. Dilute 5.0 ml of this solution to
100.0 ml with methanol R. Examined between 230 nm
ASSAY
and 350 nm (2.2.25), the solution shows an absorption
Dissolve 0.250 g in 30 ml of alcohol R. Add 1.0 ml of 0.1 M
maximum at 239 nm. The specific absorbance at the
hydrochloric acid. Carry out a potentiometric titration
maximum is 465 to 495.
(2.2.20), using 0.1 M sodium hydroxide. Read the volume
C. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the Ph. Eur. reference spectrum added between the two points of inflexion.
1 ml of 0.1 M sodium hydroxide is equivalent to 29.98 mg
of nortriptyline hydrochloride.
of C19H22ClN.
D. Dissolve 50 mg in 3 ml of warm water R, cool and
add 0.05 ml of a 25 g/l solution of quinhydrone R in
STORAGE
methanol R. A red colour develops slowly.
Store protected from light.
E. 50 mg gives reaction (b) of chlorides (2.3.1).
IMPURITIES
TESTS
Appearance of solution. Dissolve 0.5 g in water R with
gentle heating and dilute to 25 ml with the same solvent.

The solution is clear (2.2.1) and not more intensely coloured
than reference solution B7 (2.2.2, Method II).
Acidity or alkalinity. Dissolve 0.2 g with gentle heating
in carbon dioxide-free water R and dilute to 10 ml with
the same solvent. Add 0.1 ml of methyl red solution R and
0.2 ml of 0.01 M sodium hydroxide. The solution is yellow. A. 10,11-dihydro-5H-dibenzo[a,d][7]annulen-5one(dibenzosuberone),
Add 0.4 ml of 0.01 M hydrochloric acid. The solution is red.

ASSAY
Dissolve 0.200 g in 45 ml of tetrahydrofuran R. Add 10 ml
of a 100 g/l solution of silver nitrate R. After 1 min, titrate
with 0.1 M sodium hydroxide determining the end-point
potentiometrically (2.2.20). Carry out a blank titration.
1 ml of 0.1 M sodium hydroxide is equivalent to 31.25 mg of
C21H28O2.

2120

See the information section on general monographs (cover pages)


Noscapine

EUROPEAN PHARMACOPOEIA 5.0

B. 3-(5H-dibenzo[a,d][7]annulen-5-ylidene)-N-methylpropan1-amine (norcyclobenzaprine),

C. 10,11-dihydro-5-[3-(methylamino)propylidene]-5Hdibenzo[a,d][7]annulen-10-ol.
01/2005:0516

NOSCAPINE
Noscapinum

C22H23NO7

Mr 413.4

DEFINITION
(3S)-6,7-Dimethoxy-3-[(5R)-4-methoxy-6-methyl-5,6,7,8tetrahydro-1,3-dioxolo[4,5-g]isoquinolin-5-yl]isobenzofuran1(3H)-one.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white, crystalline powder or colourless crystals.
Solubility : practically insoluble in water, soluble in acetone,
slightly soluble in alcohol. It dissolves in strong acids ;
on dilution of the solution with water, the base may be
precipitated.
IDENTIFICATION
First identification : C, E.
Second identification : A, B, D, E.
A. It complies with the test for specific optical rotation (see
Tests).
B. Melting point (2.2.14) : 174 °C to 177 °C.
C. Infrared absorption spectrophotometry (2.2.24).
Comparison : noscapine CRS.
D. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 25 mg of the substance to be
examined in acetone R and dilute to 100 ml with the
same solvent.
Reference solution. Dissolve 25 mg of noscapine CRS in
acetone R and dilute to 100 ml with the same solvent.

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

Plate : TLC silica gel plate R.
Mobile phase : concentrated ammonia R, alcohol R,
acetone R, toluene R (1:3:20:20 V/V/V/V).
Application : 10 µl.
Development : over 2/3 of the plate.
Drying : in air.
Detection : 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.
E. To 20 mg add 10 ml of water R and shake. It does not
dissolve.
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution Y6 (2.2.2,
Method II).
Dissolve 0.2 g in acetone R and dilute to 10 ml with the same
solvent. Examine immediately after preparation.
Specific optical rotation (2.2.7) : + 42 to + 48 (dried
substance).
Dissolve 0.500 g in 0.1 M hydrochloric acid and dilute to
25.0 ml with the same acid.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 20.0 mg of the substance to be
examined with gentle heating in 14 ml of methanol R, cool
the solution and dilute to 20.0 ml with phosphate buffer

solution pH 6.0 R1.
Reference solution (a). Dilute 1.0 ml of the test solution to
20.0 ml with the mobile phase. Dilute 1.0 ml of the solution
to 10.0 ml with the mobile phase.
Reference solution (b). Dissolve 5 mg of papaverine
hydrochloride R in the mobile phase and dilute to 50.0 ml
with the mobile phase. Dilute 1.0 ml of the solution to
20.0 ml with the mobile phase.
Reference solution (c). Dissolve 1.5 mg of papaverine
hydrochloride R in 10 ml of the test solution and dilute to
50 ml with the mobile phase.
Column :
— size : l = 0.125 m, Ø = 4.6 mm,
— stationary phase : nitrile silica gel for chromatography R
(5 µm).
Mobile phase : methanol R, phosphate buffer solution
pH 6.0 R1 (350:650 V/V).
Flow rate : 1 ml/min.
Detection : spectrophotometer at 240 nm.
Injection : 20 µl.
Run time : 2.5 times the retention time of noscapine.
Relative retention with reference to noscapine (retention
time = about 10 min) : impurity A = about 1.3.
System suitability : reference solution (c) :
— resolution : minimum 2 between the peaks due to
noscapine and to impurity A.
Limits :
— impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.5 per cent),

— any other impurity : not more than 0.4 times the area of
the principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent),
2121


Noscapine hydrochloride

EUROPEAN PHARMACOPOEIA 5.0

Comparison : noscapine CRS.
— total of other impurities : not more than the area of
the principal peak in the chromatogram obtained with
D. Thin-layer chromatography (2.2.27).
reference solution (a) (0.5 per cent),
Test solution. Dissolve 25 mg of the substance to be
— disregard limit : 0.1 times the area of the principal peak
examined in alcohol R and dilute to 100 ml with the same
in the chromatogram obtained with reference solution (a)
solvent.
(0.05 per cent).
Reference solution. Dissolve 22 mg of noscapine CRS in
Loss on drying (2.2.32) : maximum 1.0 per cent, determined
acetone R and dilute to 100 ml with the same solvent.
on 0.500 g by drying in an oven at 100-105 °C.
Plate : TLC silica gel plate R.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
Mobile phase : concentrated ammonia R, alcohol R,
on 1.0 g.
acetone R, toluene R (1:3:20:20 V/V/V/V).

Application : 10 µl.
ASSAY
Development : over 2/3 of the plate.
Dissolve 0.350 g in 40 ml of anhydrous acetic acid R,
Drying : in air.
warming gently. Titrate with 0.1 M perchloric acid,
determining the end-point potentiometrically (2.2.20).
Detection : spray with dilute potassium iodobismuthate
solution R.
1 ml of 0.1 M perchloric acid is equivalent to 41.34 mg of
C22H23NO7.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
STORAGE
size to the principal spot in the chromatogram obtained
Protected from light.
with the reference solution.
E. Dissolve about 40 mg in a mixture of 2 ml of water R and
IMPURITIES
3 ml of alcohol R and add 1 ml of dilute ammonia R2.
A. papaverine.
Heat until dissolution is complete. Allow to cool,
scratching the wall of the tube with a glass rod. Filter.
01/2005:0515
The filtrate gives reaction (a) of chlorides (2.3.1). Wash
the precipitate with water R, dry at 100-105 °C and
reserve for identification tests B and C.
NOSCAPINE HYDROCHLORIDE
TESTS
Appearance of solution. The solution is clear (2.2.1) and not

more intensely coloured than reference solution Y6 or BY6
(2.2.2, Method II).
Dissolve 0.5 g in water R, add 0.3 ml of 0.1 M hydrochloric
acid and dilute to 25 ml with water R.
pH (2.2.3) : minimum 3.0.
Dissolve 0.2 g in 10 ml of carbon dioxide-free water R.
Specific optical rotation (2.2.7) : + 38.5 to + 44.0 (dried
substance).
Dissolve 0.500 g in 0.01 M hydrochloric acid and dilute to
C22H24ClNO7,H2O
Mr 467.9 25.0 ml with the same acid.
Related substances. Liquid chromatography (2.2.29).
DEFINITION
Test solution. Dissolve 20.0 mg of the substance to be
(3S)-6,7-Dimethoxy-3-[(5R)-4-methoxy-6-methyl-5,6,7,8examined with gentle heating in 14 ml of methanol R, cool
tetrahydro-1,3-dioxolo[4,5-g]isoquinolin-5-yl]isobenzofuranthe solution and dilute to 20.0 ml with phosphate buffer
1(3H)-one hydrochloride monohydrate.
solution pH 6.0 R1.
Content : 99.0 per cent to 101.0 per cent (dried substance).
Reference solution (a). Dilute 1.0 ml of the test solution to
20.0 ml with the mobile phase. Dilute 1.0 ml of the solution
CHARACTERS
to 10.0 ml with the mobile phase.
Appearance : white, crystalline powder or colourless
Reference solution (b). Dissolve 5 mg of papaverine
crystals, hygroscopic.
hydrochloride R in the mobile phase and dilute to 50.0 ml
Solubility : freely soluble in water and in alcohol. Aqueous
with the mobile phase. Dilute 1.0 ml of the solution to
solutions are faintly acid ; the base may be precipitated when 20.0 ml with the mobile phase.

the solutions are allowed to stand.
Reference solution (c). Dissolve 1.5 mg of papaverine
mp : about 200 °C, with decomposition.
hydrochloride R in 10 ml of the test solution and dilute to
50 ml with the mobile phase.
IDENTIFICATION
Column :
First identification : C, E.
— size : l = 0.125 m, Ø = 4.6 mm,
Second identification : A, B, D, E.
A. It complies with the test for specific optical rotation (see — stationary phase : nitrile silica gel for chromatography R
(5 µm).
Tests).
Mobile
phase : methanol R, phosphate buffer solution
B. Melting point (2.2.14) of the precipitate obtained in
pH 6.0 R1 (350:650 V/V).
identification test E : 174 °C to 177 °C.
Flow rate : 1 ml/min.
C. Infrared absorption spectrophotometry (2.2.24).
Detection : spectrophotometer at 240 nm.
Preparation : examine the precipitate obtained in
Injection : 20 µl.
identification test E.

Noscapini hydrochloridum

2122

See the information section on general monographs (cover pages)

Nutmeg oil

EUROPEAN PHARMACOPOEIA 5.0

Run time : 2.5 times the retention time of noscapine.
Relative retention with reference to noscapine (retention
time = about 10 min) : impurity A = about 1.3.
System suitability : reference solution (c) :
— resolution : minimum 2 between the peaks due to
noscapine and to impurity A.
Limits :
— impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.5 per cent),
— any other impurity : not more than 0.4 times the area of
the principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent),
— total of other impurities : 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) : 2.5 per cent to 6.5 per cent,
determined on 0.200 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.

The chromatogram obtained with the reference solution
shows in the upper third a pink to reddish-brown zone
(myristicine). The chromatogram obtained with the test
solution shows a series of zones of which one is similar
in position and colour to the zone in the chromatogram
obtained with the reference solution. Above this zone a
brownish zone (safrole) and a violet zone (hydrocarbons)
are present. Below the myristicine zone, 5 blue zones of
variable intensity are present.
B. Examine the chromatograms obtained in the test for
chromatographic profile. The retention times of the
principal peaks in the chromatogram obtained with the
test solution are similar to those in the chromatogram
obtained with the reference solution.

TESTS
Relative density (2.2.5) : 0.885 to 0.905.
Refractive index (2.2.6) : 1.475 to 1.485.
Optical rotation (2.2.7) : + 8° to + 18°.
Chromatographic profile. Examine by gas chromatography
(2.2.28).
Test solution. The substance to be examined.
Reference solution. Dissolve 15 µl of α-pinene R, 15 µl of
β-pinene R, 15 µl of sabinene R, 5 µl of car-3-ene R, 5 µl of
limonene R, 5 µl of γ-terpinene R, 5 µl of terpinen-4-ol R, 5 µl
ASSAY
of safrole R and 10 µl of myristicine R in 1 ml of hexane R.
Dissolve 0.400 g in a mixture of 5.0 ml of 0.01 M hydrochloric
The chromatographic procedure may be carried out using :
acid and 50 ml of alcohol R. Carry out a potentiometric

titration (2.2.20), using 0.1 M sodium hydroxide. Read the — a fused-silica column 25 m to 60 m long and about 0.3 mm
in internal diameter, coated with macrogol 20 000 R as
volume added between the 2 points of inflexion.
the bonded phase,
1 ml of 0.1 M sodium hydroxide is equivalent to 44.99 mg
—
helium
for chromatography R as the carrier gas at a flow
of C22H24ClNO7.
rate of 1.5 ml/min,
STORAGE
— a flame-ionisation detector,
In an airtight container, protected from light.
— a split ratio of 1:100.
— with the following temperature programme :
IMPURITIES
Time
(min)

A. papaverine.
01/2005:1552

Column

NUTMEG OIL
Myristicae fragrantis aetheroleum
DEFINITION
Nutmeg oil is obtained by steam distillation of the dried and
crushed kernels of Myristica fragrans Houtt.

0 - 10

Temperature
Rate
(°C)
(°C/min)
–
50

10 - 75

50 → 180

75 - 130

180

Injection port

200 - 220

Detector

240 - 250

2

Comment
isothermal
linear gradient

isothermal

Inject 0.2 µl of the reference solution. When the
chromatogram is recorded in the prescribed conditions, the
components elute in the order indicated in the composition
CHARACTERS
of the reference solution. Record the retention times of
A colourless or pale yellow liquid, with a spicy odour.
these substances.
The test is not valid unless the resolution between the peaks
IDENTIFICATION
corresponding to β-pinene and sabinene is at least 1.5.
First identification : B.
Inject 0.2 µl of the test solution. Using the retention times
Second identification : A.
determined from the chromatogram obtained with the
A. Examine by thin-layer chromatography (2.2.27), using a
reference solution, locate the components of the reference
TLC silica gel plate R.
solution in the chromatogram obtained with the test
Test solution. Dissolve 1 ml of the substance to be
solution. Determine the percentage content of each of these
examined in toluene R and dilute to 10 ml with the same components by the normalisation procedure.
solvent.
The percentages are within the following ranges :
Reference solution. Dissolve 20 µl of myristicine R in
— α-pinene : 15 per cent to 28 per cent,
10 ml of toluene R.
— β-pinene : 13 per cent to 18 per cent,
Apply to the plate as bands 10 µl of each solution. Develop — sabinene : 14 per cent to 29 per cent,

over a path of 15 cm using a mixture of 5 volumes of ethyl
— car-3-ene : 0.5 per cent to 2.0 per cent,
acetate R and 95 volumes of toluene R. Allow the plate
to dry in air and spray with vanillin reagent R. Heat the — limonene : 2.0 per cent to 7.0 per cent,
plate at 100 °C to 105 °C for 10 min. Examine in daylight. — γ-terpinene : 2.0 per cent to 6.0 per cent,
General Notices (1) apply to all monographs and other texts

2123


Nystatin

EUROPEAN PHARMACOPOEIA 5.0

— terpinen-4-ol: 2.0 per cent to 6.0 per cent,
— safrole : less than 2.5 per cent,
— myristicine : 5.0 per cent to 12.0 per cent.
STORAGE
Store in a well-filled, airtight container, protected from light
and heat.

B.

01/2005:0517
C.

NYSTATIN

D.

Nystatinum

E.

and 319 nm, and a shoulder at 280 nm. The ratios of the
absorbances at the absorption maxima at 291 nm and
319 nm to the absorbance at the absorption maximum at
305 nm are 0.61 to 0.73 and 0.83 to 0.96, respectively.
The ratio of the absorbance measured at the absorption
maximum at 230 nm to that measured at the shoulder
at 280 nm is 0.83 to 1.25.
Infrared absorption spectrophotometry (2.2.24).
Comparison : nystatin CRS.
To about 2 mg add 0.1 ml of hydrochloric acid R. A
brown colour develops.
To about 2 mg add 0.1 ml of sulphuric acid R. A brown
colour develops that becomes violet on standing.
Examine the chromatograms obtained in the test for
composition.
Results : the principal peak in the chromatogram obtained
with the test solution is similar in retention time to
the principal peak in the chromatogram obtained with
reference solution (a).

TESTS
Absorbance (2.2.25). Dissolve 0.10 g in a mixture of 5.0 ml
of glacial acetic acid R and 50 ml of methanol R and dilute
to 100.0 ml with methanol R. Dilute 1.0 ml of the solution
to 100.0 ml with methanol R. Determined at the maximum
at 305 nm within 30 min of preparation of the solution, the

absorbance is not less than 0.60.
Composition. Liquid chromatography (2.2.29) : use the
normalisation procedure. Carry out the test protected from
light.
C47H75NO17
Mr 926 Test solution. Dissolve 20 mg of the substance to be
examined in dimethyl sulphoxide R and dilute to 50 ml with
DEFINITION
the same solvent.
Antifungal substance obtained by fermentation
Reference solution (a). Dissolve 20 mg of nystatin CRS in
using certain strains of Streptomyces noursei
dimethyl sulphoxide R and dilute to 50 ml with the same
as the production micro-organism. It contains
solvent.
mainly tetraenes, the principal component being
Reference solution (b). Dissolve 20 mg of the substance to
(1S,3R,4R,7R,9R,11R,15S,16R,17R,18S,19E,21E,25E,27E,29E,31E,33R,35S,36R,37S)-33-[(3-amino-3,6-dideoxy-β- be examined in 25 ml of methanol R and dilute to 50 ml
D-mannopyranosyl)oxy]-1,3,4,7,9,11,17,37-octahydroxy-15,16, with water R. To 10.0 ml of the solution add 2.0 ml of dilute
18-trimethyl-13-oxo-14,39-dioxabicyclo[33.3.1]nonatriaconta- hydrochloric acid R. Allow to stand at room temperature
for 1 h.
19,21,25,27,29,31-hexaene-36-carboxylic acid (nystatin A1).
Reference solution (c). Dilute 1.0 ml of reference solution (a)
Content : minimum 4400 IU/mg (dried substance) and
minimum 5000 IU/mg (dried substance) if intended for oral to 100.0 ml with dimethyl sulphoxide R. Dilute 1.0 ml of this
solution to 10.0 ml with dimethyl sulphoxide R.
administration.
Column :
PRODUCTION
— size : l = 0.15 m, Ø = 4.6 mm,

If nystatin is not intended for cutaneous administration, the
method of manufacture is validated to demonstrate that the — stationary phase : base-deactivated end-capped
octadecylsilyl silica gel for chromatography R (5 µm),
product, if tested, would comply with the following test.
—
temperature
: 30 °C.
Abnormal toxicity (2.6.9). Inject intraperitoneally into
Mobile
phase
:
each mouse a quantity equivalent to not less than 600 IU
suspended in 0.5 ml of a 5 g/l solution of acacia R.
— mobile phase A : acetonitrile R, 3.85 g/l solution of
ammonium acetate R (29:71 V/V),
CHARACTERS
— mobile phase B : 3.85 g/l solution of ammonium
Appearance : yellow or slightly brownish powder,
acetate R, acetonitrile R (40:60 V/V),
hygroscopic.
Time
Mobile phase A
Mobile phase B
Solubility : practically insoluble in water, freely soluble in
(min)
(per
cent
V/V)
(per cent V/V)
dimethylformamide and in dimethyl sulphoxide, slightly

0
0 - 25
100
soluble in methanol, practically insoluble in alcohol.
IDENTIFICATION
First identification : B, E.
Second identification : A, C, D.
A. Examine the solution prepared in the test for absorbance
between 220 nm and 350 nm (2.2.25). The solution
shows 4 absorption maxima at 230 nm, 291 nm, 305 nm
2124

25 - 35

100 → 0

0 → 100

35 - 45

0

100

45 - 50

0 → 100

100 → 0

50 - 55

100

0

Flow rate : 1.0 ml/min.

See the information section on general monographs (cover pages)


Nystatin

EUROPEAN PHARMACOPOEIA 5.0

Detection : spectrophotometer at 305 nm.
Injection : 20 µl
Retention time : nystatin A1 = about 14 min.
System suitability : reference solution (b) :
— resolution : minimum 3.5 between the 2 principal peaks
(retention time = about 13 min and 19 min).
Composition :
— nystatin A1 : minimum 85.0 per cent,
— any other compound : maximum 4.0 per cent,
— disregard limit: the area of the principal peak in the
chromatogram obtained with reference solution (c) ;
disregard any peak with a retention time of less than
2 min.
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.

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

Loss on drying (2.2.32) : maximum 5.0 per cent, determined
on 1.000 g by drying at 60 °C over diphosphorus
pentoxide R at a pressure not exceeding 0.1 kPa for 3 h.
Sulphated ash (2.4.14) : maximum 3.5 per cent, determined
on 1.0 g.
ASSAY
Carry out the microbiological assay of antibiotics (2.7.2).
Protect the solutions from light throughout the assay.
Dissolve the substance to be examined and nystatin CRS
separately in dimethylformamide R and dilute with a mixture
of 5 volumes of dimethylformamide R and 95 volumes of
buffer solution pH 6.0.
STORAGE
In an airtight container, protected from light.
LABELLING
The label states where applicable, that the substance is only
for cutaneous use.

2125


Octyl gallate

EUROPEAN PHARMACOPOEIA 5.0

OAK BARK

Quercus cortex

01/2005:1887 CHARACTERS
Appearance : clear, colourless or light yellow, viscous liquid.
Solubility : miscible with water, with ethanol and with
vegetable oils.

IDENTIFICATION
DEFINITION
A. Infrared absorption spectrophotometry (2.2.24).
Cut and dried bark from the fresh young branches of Quercus
Comparison : Ph. Eur. reference spectrum of
robur L., Q. petraea (Matt.) Liebl. and Q. pubescens Willd.
octoxinol 10.
Content : minimum 3.0 per cent of tannins, expressed as
Preparation : film between sodium chloride R plates.
pyrogallol (C6H6O3 ; Mr 126.1) (dried drug).
B. It complies with the test for cloud point (see Tests).
CHARACTERS
TESTS
Macroscopic and microscopic characters described under
Acidity or alkalinity. Boil 1.0 g with 20 ml of carbon
identification tests A and B.
dioxide-free water R for 1 min, with constant stirring.
Cool and filter. To 10 ml of the filtrate, add 0.05 ml of
IDENTIFICATION
bromothymol blue solution R1. Not more than 0.5 ml of
A. The bark occurs in channelled or quilled pieces, not
more than 3 mm thick. The outer surface is light grey or 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is
required to change the colour of the indicator.

greenish-grey, rather smooth, with occasional lenticels.
The inner surface is dull brown or reddish-brown and has Hydroxyl value (2.5.3, Method A) : 85 to 101.
slightly raised longitudinal striations about 0.5 mm to
Cloud point : 63 °C to 70 °C.
1 mm wide. The fracture is splintery and fibrous.
Dissolve 1.0 g in 99 g of water R. Transfer about 30 ml of
B. Reduce to a powder (355). The powder is light brown to this solution to a test-tube, heat on a water-bath and stir
reddish-brown and fibrous. Examine under a microscope continuously until the solution becomes cloudy. Remove the
using chloral hydrate solution R. The powder shows
test-tube from the water-bath (ensuring that the temperature
groups of thick-walled fibres surrounded by a moderately does not increase more than 2 °C), and continue to stir.
thickened parenchymatous sheath containing prism
The cloud point is the temperature at which the solution
crystals of calcium oxalate ; fragments of cork composed becomes sufficiently clear that the entire thermometer bulb
of thin-walled tabular cells filled with brownish or reddish is plainly seen.
contents ; abundant sclereids, isolated and in groups,
some large with thick, stratified walls and branching pits, Ethylene oxide and dioxan (2.4.25) : maximum 1 ppm of
others smaller and thinner-walled with simple pits, often ethylene oxide and maximum 10 ppm of dioxan.
with dense brown contents ; fragments of parenchyma
Heavy metals (2.4.8) : maximum 10 ppm.
containing cluster crystals of calcium oxalate ; occasional Dissolve 2.0 g in distilled water R and dilute to 20.0 ml with
fragments of sieve tissue, thin-walled, some showing sieve the same solvent. 12 ml of this solution complies with limit
areas on the oblique end-walls.
test A. Prepare the standard using lead standard solution
C. To 1 g of the powdered drug (710) add 10 ml of alcohol
(1 ppm Pb) R.
(30 per cent V/V) R and heat the mixture under a reflux
Water (2.5.12) : maximum 0.5 per cent, determined on 2.00 g.
condenser on a water-bath for 30 min. Cool and filter.
Total ash (2.4.16) : maximum 0.4 per cent, determined on

To 1 ml of the solution add 2 ml of a 10 g/l solution of
vanillin R in hydrochloric acid R. A red colour develops. 1.0 g.
STORAGE
TESTS
In an airtight container.
Foreign matter (2.8.2) : maximum 2 per cent.
Loss on drying (2.2.32) : maximum 10.0 per cent, determined
on 1.000 g of the powdered drug (710) by drying in an oven
at 100-105 °C for 2 h.
Total ash (2.4.16) : maximum 8.0 per cent.
OCTYL
ASSAY
Carry out the determination of tannins in herbal drugs
(2.8.14). Use 0.700 g of the powdered drug (710).

01/2005:2057

GALLATE

Octylis gallas

01/2005:1553

OCTOXINOL 10
Octoxinolum 10
DEFINITION
α-[4-(1,1,3,3-Tetramethylbutyl)phenyl]-ω-hydroxydeca(oxyethylene).
Mixture consisting mainly of mono-octylphenyl
ethers of macrogols corresponding to the formula
C8H17C6H4-[OCH2-CH2]n-OH where the average value of n is

10. It may contain free macrogols.
General Notices (1) apply to all monographs and other texts

C15H22O5

Mr 282.3

DEFINITION
Octyl 3,4,5-trihydroxybenzoate.
Content : 97.0 per cent to 103.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
2129


Octyldodecanol

EUROPEAN PHARMACOPOEIA 5.0

Solubility : practically insoluble in water, freely soluble in
ethanol (96 per cent), practically insoluble in methylene
chloride.
IDENTIFICATION
A. Melting point (2.2.14).
Determine the melting point of the substance to be
examined. Mix equal parts of the substance to be
examined and octyl gallate CRS and determine the
melting point of the mixture. The difference between the
melting points (which are about 101 °C) is not greater
than 2 °C.

B. Examine the chromatograms obtained in the test for
impurity A.
Results : 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).
TESTS
Impurity A. Thin-layer chromatography (2.2.27).
Test solution (a). Dissolve 0.20 g of the substance to be
examined in acetone R and dilute to 10 ml with the same
solvent.
Test solution (b). Dilute 1.0 ml of test solution (a) to 20 ml
with acetone R.
Reference solution (a). Dissolve 10 mg of octyl gallate CRS
in acetone R and dilute to 10 ml with the same solvent.
Reference solution (b). Dissolve 20 mg of gallic acid R in
acetone R and dilute to 20 ml with the same solvent.
Reference solution (c). Dilute 1.0 ml of reference solution (b)
to 10 ml with acetone R.
Reference solution (d). Dilute 1.0 ml of reference solution (b)
to 5 ml with test solution (a).
Plate : TLC silica gel plate R.
Mobile phase : anhydrous formic acid R, ethyl formate R,
toluene R (10:40:50 V/V/V).
Application : 5 µl of test solutions (a) and (b) and reference
solutions (a), (c) and (d).
Development : over 2/3 of the plate.
Drying : in air for 10 min.
Detection : spray with a mixture of 1 volume of ferric
chloride solution R1 and 9 volumes of ethanol (96 per

cent) R.
System suitability : reference solution (d) :
— the chromatogram shows 2 clearly separated principal
spots.
Limit : test solution (a) :
— impurity A : any spot due to impurity A is not more
intense than the spot in the chromatogram obtained with
reference solution (c) (0.5 per cent).
Chlorides (2.4.4) : maximum 100 ppm.
To 1.65 g add 50 ml of water R. Shake for 5 min. Filter.
15 ml of the filtrate complies with the test.
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with limit 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 70 °C.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
2130

ASSAY
Dissolve 0.100 g in methanol R and dilute to 250.0 ml with
the same solvent. Dilute 5.0 ml of the solution to 200.0 ml
with methanol R. Measure the absorbance (2.2.25) at the
absorption maximum at 275 nm.
Calculate the content of C15H22O5 taking the specific
absorbance to be 387.
STORAGE
In a non-metallic container, protected from light.
IMPURITIES

Specified impurities : A.

A. 3,4,5-trihydroxybenzoic acid (gallic acid).
01/2005:1136

OCTYLDODECANOL
Octyldodecanolum

DEFINITION
Condensation product of saturated liquid fatty alcohols.
Content : not less than 90 per cent of (2RS)-2-octyldodecan1-ol (C20H42O ; Mr 298.6), the remainder consisting mainly of
related alcohols.
CHARACTERS
Appearance : clear, colourless or yellowish, oily liquid.
Solubility : practically insoluble in water, miscible with
alcohol.
Relative density : about 0.840.
Refractive index : about 1.455.
IDENTIFICATION
A. It complies with the test for hydroxyl value (see Tests).
B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 0.20 g of the substance to be
examined in toluene R and dilute to 20 ml with the same
solvent.
Reference solution. Dissolve 0.20 g of
octyldodecanol CRS in toluene R and dilute to
20 ml with the same solvent.
Plate : suitable silica gel plate.
Mobile phase : ethyl acetate R, toluene R (5:95 V/V).
Application : 2 µl.

Development : over a path of 12 cm.
Drying : in air.
Detection : spray with about 7 ml of a mixture of 1 volume
of a 25 g/l solution of vanillin R in alcohol R and
4 volumes of sulphuric acid R and heat at 130 °C for
5-10 min.

See the information section on general monographs (cover pages)


Ofloxacin

EUROPEAN PHARMACOPOEIA 5.0

01/2005:1455

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
Acidity or alkalinity. Mix 5.0 g thoroughly for 1 min with
a mixture of 0.1 ml of bromothymol blue solution R1, 2 ml
of heptane R and 10 ml of water R. If the aqueous layer is
blue, not more than 0.15 ml of 0.01 M hydrochloric acid is
required to change the colour of the indicator to yellow. If
the aqueous layer is yellow, add 0.45 ml of 0.1 M sodium
hydroxide and shake vigorously. After standing to ensure
complete separation, the aqueous layer is blue.
Optical rotation (2.2.7) : − 0.10° to + 0.10°.

Dissolve 2.50 g in alcohol R and dilute to 25 ml with the
same solvent.
Hydroxyl value (2.5.3, Method A) : 175 to 190.
Iodine value (2.5.4) : maximum 8.0.
Peroxide value (2.5.5) : maximum 5.0.
Saponification value (2.5.6) : maximum 5.0.
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with limit test C. Prepare the standard using
2 ml of lead standard solution (10 ppm Pb) R.
Water (2.5.12) : maximum 0.5 per cent, determined on 2.00 g.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Gas chromatography (2.2.28).
Internal standard solution. Dissolve 0.4 g of tetradecane R
in hexane R and dilute to 100.0 ml with the same solvent.
Test solution. Dissolve 0.100 g of the substance to be
examined in the internal standard solution and dilute to
10.0 ml with the same solution.
Reference solution. Dissolve 0.100 g of octyldodecanol CRS
in the internal standard solution and dilute to 10.0 ml with
the same solution.
Column :
— material : stainless steel,
— size : l = 60 m, Ø = 0.25 mm,
— stationary phase : poly(dimethyl)(diphenyl)(divinyl)siloxane R (film thickness 0.25 µm).
Carrier gas : helium for chromatography R.
Flow rate : 0.68 ml/min.
Temperature :
Time

(min)
Column

Temperature
(°C)

0-2

180

2 - 22

180 → 280

22 - 52

280

Injection port

290

Detector

300

Detection : flame ionisation.
Injection : 1 µl.
Calculate the content of C20H42O in the substance to be
examined.

STORAGE
Protected from light.
General Notices (1) apply to all monographs and other texts

OFLOXACIN
Ofloxacinum

C18H20FN3O4

Mr 361.4

DEFINITION
Ofloxacin contains not less than 99.0 per cent and
not more than the equivalent of 101.0 per cent of
(RS)-9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,
3-dihydro-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic
acid, calculated with reference to the dried substance.
CHARACTERS
A pale yellow or bright yellow, crystalline powder, slightly
soluble in water, soluble in glacial acetic acid, slightly
soluble to soluble in methylene chloride, slightly soluble in
methanol.
IDENTIFICATION
Examine by infrared absorption spectrophotometry (2.2.24),
comparing with the spectrum obtained with ofloxacin CRS.
Examine the substances prepared as discs.
TESTS
Absorbance (2.2.25). Dissolve 0.5 g in 0.1 M hydrochloric
acid and dilute to 100 ml with the same solvent. The
absorbance of the solution measured at 440 nm is not

greater than 0.25.
Optical rotation (2.2.7). Dissolve 0.300 g in a mixture of
10 volumes of methanol R and 40 volumes of methylene
chloride R and dilute to 10 ml with the same mixture of
solvents. The angle of optical rotation is − 0.10° to + 0.10°.
Impurity A. Examine by thin-layer chromatography (2.2.27),
using a TLC silica gel GF254 plate R (2 µm to 10 µm).
Test solution. Dissolve 0.250 g of the substance to be
examined in a mixture of 10 volumes of methanol R and
40 volumes of methylene chloride R and dilute to 5.0 ml
with the same mixture of solvents.
Reference solution.Dissolve 10 mg of ofloxacin
impurity A CRS in a mixture of 10 volumes of methanol R
and 40 volumes of methylene chloride R and dilute to
100.0 ml with the same mixture of solvents.
Apply to the plate 10 µl of each solution. Develop over a path
of 10 cm using a mixture of 1 volume of glacial acetic acid R,
1 volume of water R and 2 volumes of ethyl acetate R. Allow
the plate to dry in air and examine in ultraviolet light at
254 nm. Any spot due to impurity A in the chromatogram
obtained with the test solution is not more intense than
the spot in the chromatogram obtained with the reference
solution (0.2 per cent).
Related substances. Examine by liquid chromatography
(2.2.29). Prepare the solutions immediately before use.
Test solution. Dissolve 10.0 mg of the substance to be
examined in a mixture of 10 volumes of acetonitrile R and
60 volumes of water R and dilute to 50.0 ml with the same
mixture of solvents.
2131

Oleic acid

EUROPEAN PHARMACOPOEIA 5.0

Reference solution (a). Dilute 1.0 ml of the test solution
to 50.0 ml with a mixture of 10 volumes of acetonitrile R
and 60 volumes of water R. Dilute 1.0 ml of this solution to
10.0 ml with a mixture of 10 volumes of acetonitrile R and
60 volumes of water R.

IMPURITIES

Reference solution (b). Dissolve 10.0 mg of ofloxacin
impurity E CRS in a mixture of 10 volumes of acetonitrile R
and 60 volumes of water R and dilute to 100.0 ml with the
same mixture of solvents. Mix 10.0 ml of this solution with
5.0 ml of the test solution. Dilute to 50.0 ml with a mixture A. (RS)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-pyrido[1,
2,3-de]-1,4-benzoxazine-6-carboxylic acid (FPA),
of 10 volumes of acetonitrile R and 60 volumes of water R.
Dilute 1.0 ml of this solution to 50.0 ml with a mixture of
10 volumes of acetonitrile R and 60 volumes of water R.
The chromatographic procedure may be carried out using :
— a stainless steel column 0.15 m long and 4.6 mm in
internal diameter packed with octadecylsilyl silica gel for
chromatography R (5 µm),
— as mobile phase a mixture prepared as follows : dissolve
B. R1 = H, R2 = F, R3 = CH3 :(RS)-9-fluoro-3-methyl-10-(44.0 g of ammonium acetate R and 7.0 g of sodium
methylpiperazin-1-yl)-2,3-dihydro-7H-pyrido[1,2,3-de]-1,

perchlorate R in 1300 ml of water R. Adjust to pH 2.2 with
4-benzoxazin-7-one,
phosphoric acid R. Add 240 ml of acetonitrile R. Adjust
C.
R1 = CO2H, R2 = H, R3 = CH3 :(RS)-3-methyl-10-(4the flow rate of the mobile phase so that a retention time
methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3of about 20 min is obtained for ofloxacin,
de]-1,4-benzoxazine-6-carboxylic acid,
— as detector a spectrophotometer set at 294 nm,
E. R1 = CO2H, R2 = F, R3 = H : (RS)-9-fluoro-3-methyl-7oxo-10-(piperazin-1-yl)-2,3-dihydro-7H-pyrido[1,2,3-de]-1,4maintaining the temperature of the column at 45 °C.
benzoxazine-6-carboxylic acid,
Inject 10 µl of reference solution (b). Adjust the sensitivity
of the system so that the heights of the two principal peaks
in the chromatogram obtained are at least 50 per cent of
the full scale of the recorder. The test is not valid unless :
in the chromatogram obtained, the resolution between the
peaks corresponding to impurity E and ofloxacin is at least
2.0. Inject 10 µl of the test solution and 10 µl of reference
solution (a). Continue the chromatography for 2.5 times the
retention time of the principal peak. In the chromatogram
obtained with the test solution, the area of any peak, apart
D. (RS)-10-fluoro-3-methyl-9-(4-methylpiperazin-1-yl)-7from the principal peak, is not greater than the area of
oxo-2,3-dihydro-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6the principal peak in the chromatogram obtained with
carboxylic acid,
reference solution (a) (0.2 per cent) ; the sum of the areas of
all the peaks is not greater than 2.5 times the area of the
principal peak in the chromatogram obtained with reference
solution (a) (0.5 per cent). Disregard any peak with an area
less than 0.1 times the area of the principal peak in the
chromatogram obtained with reference solution (a).
Heavy metals (2.4.8). 2.0 g complies with limit test C for

heavy metals (10 ppm). Prepare the standard using 2 ml of
lead standard solution (10 ppm Pb) R.

F. 4-[(RS)-6-carboxy-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7HLoss on drying (2.2.32). Not more than 0.2 per cent,
pyrido[1,2,3-de]-1,4-benzoxazine-10-yl]-1-methylpiperazine
determined on 1.000 g by drying at 100 °C to 105 °C for 4 h.
1-oxide.
Sulphated ash (2.4.14). Not more than 0.1 per cent,
determined on 1.0 g.
01/2005:0799
ASSAY

OLEIC ACID

Dissolve 0.300 g in 100 ml of anhydrous acetic acid R.
Acidum oleicum
Titrate with 0.1 M perchloric acid determining the end-point
DEFINITION
potentiometrically (2.2.20).
(Z)-Octadec-9-enoic acid (C18H34O2 ; Mr 282.5), together with
1 ml of 0.1 M perchloric acid is equivalent to 36.14 mg of
varying amounts of saturated and other unsaturated fatty
C18H20FN3O4.
acids. A suitable antioxidant may be added.
Content : 65.0 per cent to 88.0 per cent of C18H34O2.
STORAGE
CHARACTERS
Store in an airtight container, protected from light.
2132

Appearance : clear, yellowish or brownish, oily liquid.
See the information section on general monographs (cover pages)


EUROPEAN PHARMACOPOEIA 5.0

Solubility : practically insoluble in water, miscible with
alcohol and with methylene chloride.
Relative density : about 0.892.
IDENTIFICATION
A. It complies with the test for acid value (see Tests).
B. It complies with the test for iodine value (see Tests).
C. It complies with the test for composition of fatty acids
(see Tests).
Margaric acid : maximum 0.2 per cent for oleic acid of
vegetable origin and maximum 4.0 per cent for oleic acid
of animal origin.
TESTS
Appearance. The substance to be examined is not more
intensely coloured than reference solution Y1 or BY1 ( 2.2.2,
Method I).
Acid value (2.5.1) : 195 to 204, determined on 0.5 g.
Iodine value (2.5.4) : 89 to 105.
Peroxide value (2.5.5) : maximum 10.0.
Composition of fatty acids. Gas chromatography (2.4.22,
Method C).
Test solution. Prepare as described in the method but
omitting the initial hydrolysis.
Composition of the fatty acid fraction of the substance :
— myristic acid : maximum 5.0 per cent,

— palmitic acid : maximum 16.0 per cent,
— palmitoleic acid : maximum 8.0 per cent,
— stearic acid : maximum 6.0 per cent,
— oleic acid : 65.0 per cent to 88.0 per cent,
— linoleic acid : maximum 18.0 per cent,
— linolenic acid : maximum 4.0 per cent,
— fatty acids of chain length greater than C18 : maximum
4.0 per cent.
Total ash (2.4.16) : maximum 0.1 per cent, determined on
2.00 g.

Oleoyl macrogolglycerides

CHARACTERS
Amber oily liquids, which may give rise to a deposit after
prolonged periods at 20 °C, practically insoluble but
dispersible in water, freely soluble in methylene chloride.
The viscosity at 40 °C is about 35 mPa·s, the relative density
at 20 °C is about 0.95 and the refractive index at 20 °C is
about 1.47.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using a
suitable silica gel as the coating substance.
Test solution. Dissolve 1.0 g of the substance to be
examined in methylene chloride R and dilute to 20 ml
with the same solvent.
Apply to the plate 10 µl of the test solution. Develop
over a path of 15 cm using a mixture of 30 volumes of
hexane R and 70 volumes of ether R. Allow the plate to
dry in air. Spray with a 0.1 g/l solution of rhodamine B R

in alcohol R and examine in ultraviolet light at 365 nm.
The chromatogram shows a spot corresponding to
triglycerides with an Rf value of about 0.9 (Rst 1) and
spots corresponding to 1,3-diglycerides (Rst 0.7), to
1,2-diglycerides (Rst 0.6), to monoglycerides (Rst 0.1) and
to esters of macrogol (Rst 0).
B. They comply with the test for hydroxyl value (see Tests).
C. They comply with the test for fatty acid composition (see
Tests).
D. They comply with the test for saponification value (see
Tests).

TESTS
Acid value (2.5.1). Not more than 2.0, determined on 2.0 g.
Hydroxyl value (2.5.3, Method A) : 45 to 65, determined
on 1.0 g.
Iodine value (2.5.4) : 75 to 95.
Peroxide value (2.5.5). Not more than 12.0, determined on
2.0 g.
Saponification value (2.5.6) : 150 to 170, determined on
2.0 g.
STORAGE
Alkaline impurities. Introduce 5.0 g into a test-tube and
carefully add a mixture, neutralised if necessary with 0.01 M
In an airtight, well-filled container, protected from light.
hydrochloric acid or with 0.01 M sodium hydroxide,
of 0.05 ml of a 0.4 g/l solution of bromophenol blue R
LABELLING
in alcohol R, 0.3 ml of water R and 10 ml of alcohol R.
The label states :

Shake and allow to stand. Not more than 1.0 ml of 0.01 M
— the name and concentration of any added antioxidant,
hydrochloric acid is required to change the colour of the
— the origin of oleic acid (animal or vegetable).
upper layer to yellow.
Free glycerol. Not more than 3.0 per cent. Dissolve 1.20 g
in 25.0 ml of methylene chloride R. Heat if necessary. After
01/2005:1249 cooling, add 100 ml of water R. Shake and add 25.0 ml
of a 6 g/l solution of periodic acid R. Shake and allow
to stand for 30 min. Add 40 ml of a 75 g/l solution of
OLEOYL MACROGOLGLYCERIDES
potassium iodide R. Allow to stand for 1 min. Add 1 ml of
starch solution R. Titrate the iodine with 0.1 M sodium
Macrogolglyceridorum oleates
thiosulphate. Carry out a blank titration.
1 ml of 0.1 M sodium thiosulphate is equivalent to 2.3 mg
DEFINITION
of glycerol.
Oleoyl macrogolglycerides are mixtures of monoesters,
diesters and triesters of glycerol and monoesters and diesters Composition of fatty acids (2.4.22, Method A). The fatty-acid
fraction has the following composition :
of macrogols. They are obtained by partial alcoholysis of
an unsaturated oil mainly containing triglycerides of oleic
— palmitic acid : 4.0 per cent to 9.0 per cent,
acid using macrogol with a mean relative molecular mass
— stearic acid : not more than 6.0 per cent,
between 300 and 400 or by esterification of glycerol and
macrogol with unsaturated fatty acids or by mixing glycerol — oleic acid : 58.0 per cent to 80.0 per cent,
esters and condensates of ethylene oxide with the fatty acids — linoleic acid : 15.0 per cent to 35.0 per cent,
of this unsaturated oil.

— linolenic acid : not more than 2.0 per cent,
General Notices (1) apply to all monographs and other texts

2133


Oleyl alcohol

EUROPEAN PHARMACOPOEIA 5.0

— arachidic acid : not more than 2.0 per cent,
— eicosenoic acid : not more than 2.0 per cent.
Ethylene oxide and dioxan (2.4.25). Not more than 1 ppm
of ethylene oxide and 10 ppm of dioxan.
Heavy metals (2.4.8). 2.0 g complies with limit test C for
heavy metals (10 ppm). Prepare the standard using 2 ml of
lead standard solution (10 ppm Pb) R.
Water (2.5.12). Not more than 1.0 per cent, determined
on 1.0 g by the semi-micro determination of water. Use
a mixture of 30 volumes of anhydrous methanol R and
70 volumes of methylene chloride R as solvent.
Total ash (2.4.16). Not more than 0.1 per cent, determined
on 1.0 g.
STORAGE
Store protected from light and at room temperature.
LABELLING
The label states the type of macrogol used (mean relative
molecular mass) or the number of units of ethylene oxide
per molecule (nominal value).

Composition of fatty alcohols. Gas chromatography
(2.2.28) : use the normalisation procedure.
Test solution. Mix 25 mg of the substance to be examined
with 1.0 ml of methylene chloride R.
Reference solution (a). Dissolve 25 mg of each of arachidyl
alcohol R, linolenyl alcohol R, linoleyl alcohol R, oleyl
alcohol R, palmityl alcohol R and stearyl alcohol R in
methylene chloride R and dilute to 5 ml with the same
solvent. Dilute 1 ml of this solution to 5 ml with methylene
chloride R.
Reference solution (b). Dissolve 10 mg of linoleyl alcohol R
and 1 g of oleyl alcohol R in methylene chloride R and
dilute to 40 ml with the same solvent.
Column :
— size : l = 30 m, Ø = 0.32 mm,
— stationary phase : poly(dimethyl)siloxane R (film
thickness 1 µm).
Carrier gas : helium for chromatography R.
Flow rate : 1 ml/min.
Split ratio : 1:11.
Temperature :

01/2005:2073
Column

OLEYL ALCOHOL
Alcohol oleicus

Time
(min)

Temperature
(°C)

0-1

170

1-9

170 → 210

9 - 65

DEFINITION
Mixture of unsaturated and saturated long-chain fatty
alcohols consisting mainly of octadec-9-enol (oleyl alcohol
and elaidyl alcohol ; C18H36O ; Mr 268.5). It may be of
vegetable or animal origin.

210

Injection port

270

Detector

280

Detection : flame ionisation.
Injection : 1 µl.
Identify the peaks using the chromatogram obtained with
CHARACTERS
reference solution (a).
Appearance : colourless or light yellow liquid.
Relative retention with reference to oleyl alcohol (retention
time = about 30 min) : palmityl alcohol = about 0.6 ; linolenyl
IDENTIFICATION
alcohol = about 0.8 ; linoleyl alcohol = about 0.9 ; stearyl
A. It complies with the test for hydroxyl value (see Tests).
alcohol = about 1.1 ; arachidyl alcohol = about 1.9 (elaidyl
B. It complies with the test for composition of fatty alcohols alcohol co-elutes with oleyl alcohol).
(see Tests).
System suitability : reference solution (b) :
— peak-to-valley ratio : minimum 1.2 between the peaks due
TESTS
to linoleyl alcohol and oleyl alcohol.
Appearance. The substance to be examined is clear (2.2.1)
Limits
:
and not more intensely coloured than reference solution B6
—
palmityl
alcohol : maximum 8.0 per cent,
(2.2.2, Method II).
— stearyl alcohol: maximum 5.0 per cent,
Refractive index (2.2.6) : 1.458 to 1.460, determined
— oleyl alcohol (sum of oleyl and elaidyl alcohols) :
at 25 °C.

minimum 80.0 per cent,
Cloud point : maximum 10 °C.
—
linoleyl
alcohol : maximum 3.0 per cent,
Introduce about 60 g into a cylindrical flat-bottomed
— linolenyl alcohol : maximum 0.5 per cent,
container, 30-33.5 mm internal diameter and 115-125 mm
high. Heat to 30 °C, cool, and immerse the container in iced — arachidyl alcohol : maximum 0.3 per cent.
water with the surfaces of the water and the sample at the
same level. Insert a thermometer and, using it as a stirring
rod begin stirring rapidly and steadily when the temperature
01/2005:1878
falls below 20 °C. Keep the thermometer immersed
throughout the test, remove and examine the container at
OLIVE LEAF
regular intervals. The cloud point is the temperature at
which the immersed portion of the thermometer, positioned
Oleae folium
vertically in the centre of the container, is no longer visible
when viewed horizontally through the container and sample.
DEFINITION
Acid value (2.5.1) : maximum 1.0, determined on 5.0 g.
Dried leaf of Olea europaea L.
Hydroxyl value (2.5.3, Method A) : 205 to 215.
Content : minimum 5.0 per cent of oleuropein (C25H32O13 ;
Mr 540.5) (dried drug).
Saponification value (2.5.6) : maximum 2.0.
2134

See the information section on general monographs (cover pages)


Olive oil, refined

EUROPEAN PHARMACOPOEIA 5.0

CHARACTERS
Macroscopic and microscopic characters described under
identification tests A and B.
IDENTIFICATION
A. The leaf is simple, thick and coriaceous, lanceolate to
obovate, 30 mm to 50 mm long and 10 mm to 15 mm
wide, with a mucronate apex and tapering at the base
to a short petiole ; the margins are entire and reflexed
abaxially. The upper surface is greyish-green, smooth and
shiny, the lower surface paler and pubescent, particularly
along the midrib and main lateral veins.
B. Reduce to a powder (355). The powder is yellowish-green.
Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic
characters: fragments of the epidermis in surface view
with small, thick-walled polygonal cells and, in the
lower epidermis only, small anomocytic stomata (2.8.3) ;
fragments of the lamina in sectional view showing a thick
cuticle, a palisade composed of 3 layers of cells and a
small-celled spongy parenchyma ; numerous sclereids,
very thick-walled and mostly fibre-like with blunt or,
occasionally, forked ends, isolated or associated with
the parenchyma of the mesophyll ; abundant, very large

peltate trichomes, with a central unicellular stalk from
which radiate some 10 to 30 thin-walled cells which
become free from the adjoining cells at the margin of the
shield, given an uneven, jagged appearance.
C. Thin-layer chromatography (2.2.27).
Test solution. To 1.0 g of the powdered drug (355) add
10 ml of methanol R. Boil under a reflux condenser for
15 min. Cool and filter.
Reference solution. Dissolve 10 mg of oleuropein R and
1 mg of rutin R in 1 ml of methanol R.
Plate : TLC silica gel plate R.
Mobile phase : water R, methanol R, methylene
chloride R (1.5:15:85 V/V/V).
Application : 10 µl as bands.
Development : over a path of 10 cm.
Drying : in air.
Detection : spray with vanillin reagent R and heat at
100-105 °C for 5 min ; examine in daylight.
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

_______
Oleuropein : a brownish-green
zone
_______

A dark violet-blue zone (solvent
front)
A dark violet-blue zone
_______

Time
(min)

Mobile phase A
(per cent V/V)

Mobile phase B
(per cent V/V)

0-5

85 → 40

15 → 60

5 - 12

40 → 20

60 → 80

12 - 15

20 → 85

80 → 15

Flow rate : 1 ml/min.
Detection : spectrophotometer at 254 nm.
Injection : 20 µl.
Retention time : oleuropein = about 9 min.
Calculate the percentage content of oleuropein from the
expression.

A1

=

area of the peak due to oleuropein in the
chromatogram obtained with the test solution,

A2

=

area of the peak due to oleuropein in the
chromatogram obtained with the reference
solution,

m1

=

mass of the drug to be examined, in grams,

m2

=

mass of oleuropein R in the reference solution,
in grams,

p

=

percentage content of oleuropein in oleuropein R.
01/2005:1456

OLIVE OIL, REFINED

A brownish-green zone
(oleuropein)
_______

Rutin : a brownish-yellow zone
Reference solution

ASSAY
Liquid chromatography (2.2.29).
Test solution. In a flask, place 1.000 g of the powdered drug
(355) and add 50 ml of methanol R. Heat in a water-bath at
60 °C for 30 min with shaking. Allow to cool and filter into
a 100 ml volumetric flask. Rinse the flask and the filter with
methanol R and dilute to 100.0 ml with the same solvent.

Dilute 2.0 ml of the solution to 20.0 ml with water R.
Reference solution. Dissolve 5.0 mg of oleuropein R in
5.0 ml of methanol R. Dilute 1.0 ml of the solution to 25.0 ml
with water R.
Column :
— size : l = 0.15 m, Ø = 3.9 mm,
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 µm),
— temperature : 25 °C.
Mobile phase :
— mobile phase A : dilute 1.0 ml of glacial acetic acid R to
100 ml with water R,
— mobile phase B : methanol R,

Test solution

TESTS
Foreign matter (2.8.2) : maximum 2 per cent.
Loss on drying (2.2.32) : maximum 10.0 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 9.0 per cent.
General Notices (1) apply to all monographs and other texts

Olivae oleum raffinatum
DEFINITION
Refined olive oil is the fatty oil obtained by refining of crude
olive oil, obtained by cold expression or other suitable
mechanical means from the ripe drupes of Olea europaea L.
A suitable antioxidant may be added.

CHARACTERS
A clear, colourless or greenish-yellow, transparent liquid,
practically insoluble in alcohol, miscible with light petroleum
(50 °C to 70 °C).
2135


Olive oil, virgin

EUROPEAN PHARMACOPOEIA 5.0

When cooled, it begins to become cloudy at 10 °C and
becomes a butter-like mass at about 0 °C. It has a relative
density of about 0.913.

— oleic acid (equivalent chain length on polyethyleneglycol
adipate 18.3) : 56.0 per cent to 85.0 per cent,
— linoleic acid (equivalent chain length on
polyethyleneglycol adipate 18.9) : 3.5 per cent to
IDENTIFICATION
20.0 per cent,
A. It complies with the test for absorbance (see Tests).
— linolenic acid (equivalent chain length on
B. Carry out the test for identification of fatty oils by
polyethyleneglycol adipate 19.7) : not more than 1.2 per
thin-layer chromatography (2.3.2). The chromatogram
cent,
obtained shows spots corresponding to those in the
— arachidic acid : not more than 0.7 per cent,
typical chromatogram for olive oil. For certain types of

refined olive oil, the difference in the size of spots E and — eicosenoic acid (equivalent chain length on
polyethyleneglycol adipate 20.3) : not more than 0.4 per
F is less pronounced than in the typical chromatogram.
cent,
TESTS
— behenic acid : not more than 0.2 per cent,
Acid value (2.5.1). Not more than 0.5, determined on 10.0 g. — lignoceric acid : not more than 0.2 per cent.
Peroxide value (2.5.5, Method A). Not more than 10.0. If
Sterols (2.4.23). The sterol fraction of the oil has the
intended for use in the manufacture of parenteral dosage
following composition :
forms, not more than 5.0.
— sum of contents of β-sitosterol, ∆5,23-stigmastadienol,
Unsaponifiable matter. Not more than 1.5 per cent. Place
clerosterol, sitostanol, ∆5-avenasterol and
5.0 g (m g) in a 150 ml flask fitted with a reflux condenser.
∆5,24-stigmastadienol: not less than 93.0 per cent,
Add 50 ml of 2 M alcoholic potassium hydroxide R and heat — cholesterol : not more than 0.5 per cent,
on a water-bath for 1 h, shaking frequently. Add 50 ml of
— ∆7-stigmasterol : not more than 0.5 per cent,
water R through the top of the condenser, shake, allow to
—
campesterol : not more than 4.0 per cent,
cool and transfer the contents of the flask to a separating
and the content of stigmasterol is not more than that of
funnel. Rinse the flask with several portions to a total
campesterol.
of 50 ml of light petroleum R1 and add the rinsings to
the separating funnel. Shake vigorously for 1 min. Allow
Sesame oil. In a ground-glass-stoppered cylinder shake

to separate and transfer the aqueous layer to a second
10 ml for about 1 min with a mixture of 0.5 ml of a 0.35 per
separating funnel. If an emulsion forms, add small quantities cent V/V solution of furfural R in acetic anhydride R and
of alcohol R or a concentrated solution of potassium
4.5 ml of acetic anhydride R. Filter through a filter paper
hydroxide R. Shake the aqueous layer with 2 quantities,
impregnated with acetic anhydride R. To the filtrate add
each of 50 ml, of light petroleum R1. Combine the light
0.2 ml of sulphuric acid R. No bluish-green colour develops.
petroleum layers in a third separating funnel and wash with
3 quantities, each of 50 ml, of alcohol (50 per cent V/V) R. Water (2.5.32). If intended for use in the manufacture
of parenteral dosage forms, not more than 0.1 per cent,
Transfer the light petroleum layer to a tared 250 ml flask.
determined on 5.0 g by the coulometric method. Use a
Rinse the separating funnel with small quantities of light
mixture of equal volumes of decanol R and anhydrous
petroleum R1 and add to the flask. Evaporate the light
methanol R as solvent.
petroleum on a water-bath and dry the residue at 100 °C
to 105 °C for 15 min, keeping the flask horizontal. Allow
STORAGE
to cool in a desiccator and weigh (a g). Repeat the drying
Store in a well-filled container, protected from light, at a
for successive periods of 15 min until the loss of mass
temperature not exceeding 25 °C. If intended for use in
between 2 successive weighings does not exceed 0.1 per
cent. Dissolve the residue in 20 ml of alcohol R, previously the manufacture of parenteral dosage forms, store under
an inert gas.
neutralised to 0.1 ml of bromophenol blue solution R. If
necessary, titrate with 0.1 M hydrochloric acid (b ml).

LABELLING
Calculate the percentage content of unsaponifiable matter
The label states :
from the expression :
— where applicable, that the substance is suitable for use in
the manufacture of parenteral dosage forms,
— the name and concentration of any added antioxidant,
— the name of the inert gas.
If 0.032b is greater than 5 per cent of a, the test is invalid
and must be repeated.
Alkaline impurities (2.4.19). It complies with the test for
01/2005:0518
alkaline impurities in fatty oils.
Absorbance (2.2.25). Dissolve 1.00 g in cyclohexane R and
OLIVE OIL, VIRGIN
dilute to 100.0 ml with the same solvent. The absorbance
measured at 270 nm is 0.20 to 1.20.
Olivae oleum virginale
Composition of fatty acids (2.4.22, Method A). The fatty acid
DEFINITION
fraction of the oil has the following composition :
Virgin olive oil is the fatty oil obtained by cold expression or
— saturated fatty acids of chain length less than C16 : not
other suitable mechanical means from the ripe drupes of
more than 0.1 per cent,
Olea europaea L.
— palmitic acid : 7.5 per cent to 20.0 per cent,
— palmitoleic acid (equivalent chain length on
polyethyleneglycol adipate 16.3) : not more than 3.5 per
cent,

— stearic acid : 0.5 per cent to 5.0 per cent,
2136

CHARACTERS
A clear, yellow or greenish-yellow, transparent liquid with a
characteristic odour, practically insoluble in alcohol, miscible
with light petroleum (50 °C to 70 °C).

See the information section on general monographs (cover pages)


Olsalazine sodium

EUROPEAN PHARMACOPOEIA 5.0

— linoleic acid (equivalent chain length on
polyethyleneglycol adipate 18.9) : 3.5 per cent to
20.0 per cent,
— linolenic acid (equivalent chain length on
IDENTIFICATION
polyethyleneglycol adipate 19.7) : not more than 1.2 per
cent,
Carry out the test for identification of fatty oils by thin-layer
—
arachidic
acid : not more than 0.7 per cent,
chromatography (2.3.2). The chromatogram obtained shows
spots corresponding to those in the typical chromatogram
— eicosenoic acid (equivalent chain length on
for olive oil. For certain types of olive oil, the difference in

polyethyleneglycol adipate 20.3) : not more than 0.4 per
the size of spots E and F is less pronounced than in the
cent,
typical chromatogram.
— behenic acid : not more than 0.2 per cent,
— lignoceric acid : not more than 0.2 per cent.
TESTS
Sterols (2.4.23). The sterol fraction of the oil has the
Acid value (2.5.1). Not more than 2.0, determined on 5.0 g. following composition :
Peroxide value (2.5.5, Method A). Not more than 20.0.
— sum of contents of β-sitosterol, ∆5,23-stigmastadienol,
clerosterol, sitostanol, ∆5-avenasterol and
Unsaponifiable matter. Not more than 1.5 per cent. Place
∆5,24-stigmastadienol: not less than 93.0 per cent,
5.0 g (m g) in a 150 ml flask fitted with a reflux condenser.
Add 50 ml of 2 M alcoholic potassium hydroxide R and heat — cholesterol : not more than 0.5 per cent,
on a water-bath for 1 h, shaking frequently. Add 50 ml of
— ∆7-stigmasterol : not more than 0.5 per cent,
water R through the top of the condenser, shake, allow to
— campesterol : not more than 4.0 per cent,
cool and transfer the contents of the flask to a separating
and the content of stigmasterol is not more than that of
funnel. Rinse the flask with several portions to a total
campesterol.
of 50 ml of light petroleum R1 and add the rinsings to
Sesame oil. In a ground-glass-stoppered cylinder shake
the separating funnel. Shake vigorously for 1 min. Allow
10 ml for about 1 min with a mixture of 0.5 ml of a 0.35 per
to separate and transfer the aqueous layer to a second
separating funnel. If an emulsion forms, add small quantities cent V/V solution of furfural R in acetic anhydride R and

4.5 ml of acetic anhydride R. Filter through a filter paper
of alcohol R or a concentrated solution of potassium
impregnated with acetic anhydride R. To the filtrate add
hydroxide R. Shake the aqueous layer with 2 quantities,
0.2 ml of sulphuric acid R. No bluish-green colour develops.
each of 50 ml, of light petroleum R1. Combine the light
petroleum layers in a third separating funnel and wash with
3 quantities, each of 50 ml, of alcohol (50 per cent V/V) R. STORAGE
Store in a well-filled container, protected from light, at a
Transfer the light petroleum layer to a tared 250 ml flask.
temperature not exceeding 25 °C.
Rinse the separating funnel with small quantities of light
petroleum R1 and add to the flask. Evaporate the light
01/2005:1457
petroleum on a water-bath and dry the residue at 100 °C
to 105 °C for 15 min, keeping the flask horizontal. Allow
to cool in a desiccator and weigh (a g). Repeat the drying
OLSALAZINE SODIUM
for successive periods of 15 min until the loss of mass
between 2 successive weighings does not exceed 0.1 per
Olsalazinum natricum
cent. Dissolve the residue in 20 ml of alcohol R, previously
neutralised to 0.1 ml of bromophenol blue solution R. If
necessary, titrate with 0.1 M hydrochloric acid (b ml).
Calculate the percentage content of unsaponifiable matter
from the expression :
When cooled, it begins to become cloudy at 10 °C and
becomes a butter-like mass at about 0 °C. It has a relative
density of about 0.913.

C14H8N2Na2O6
If 0.032b is greater than 5 per cent of a, the test is invalid
and must be repeated.
Absorbance (2.2.25). Dissolve 1.00 g in cyclohexane R and
dilute to 100.0 ml with the same solvent. The absorbance
measured at 270 nm is not greater than 0.20. The ratio of the
absorbance at 232 nm to that at 270 nm is greater than 8.
Composition of fatty acids (2.4.22, Method A). The fatty acid
fraction of the oil has the following composition :
— saturated fatty acids of chain length less than C16 : not
more than 0.1 per cent,
— palmitic acid : 7.5 per cent to 20.0 per cent,
— palmitoleic acid (equivalent chain length on
polyethyleneglycol adipate 16.3) : not more than 3.5 per
cent,
— stearic acid : 0.5 per cent to 5.0 per cent,
— oleic acid (equivalent chain length on polyethyleneglycol
adipate 18.3) : 56.0 per cent to 85.0 per cent,
General Notices (1) apply to all monographs and other texts

Mr 346.2

DEFINITION
Olsalazine sodium contains not less than 98.0 per cent and
not more than the equivalent of 102.0 per cent of disodium
3,3′-diazenediylbis(6-hydroxybenzoate), calculated with
reference to the dried and acetate-free substance.
CHARACTERS
A yellow, fine, crystalline powder, sparingly soluble in water,
soluble in dimethyl sulphoxide, very slightly soluble in

methanol.
It shows polymorphism.
IDENTIFICATION
First identification : B, D.
Second identification : A, C, D.
A. Dissolve 40.0 mg in 5 ml of 0.1 M sodium hydroxide
and dilute to 100.0 ml with a 7.8 g/l solution of sodium
dihydrogen phosphate R adjusted to pH 7.2 with strong
sodium hydroxide solution R (buffer solution). Dilute
2137