EUROPEAN PHARMACOPOEIA 5.
0 Phenylbutazone
Specific optical rotation (2.2.7). Dissolve 0.50 g in water R
and dilute to 25.0 ml with the same solvent. The specific
optical rotation is − 33.0 to − 35.5, calculated with reference
to the dried substance.
Ninhydrin-positive substances. Examine by thin-layer
chromatography (2.2.27), using a TLC silica gel plate R.
Test solution (a). Dissolve 0.10 g of the substance to be
examined in a mixture of equal volumes of glacial acetic
acid R and water R and dilute to 10 ml with the same
mixture of solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 50 ml
with a mixture of equal volumes of glacial acetic acid R
and water R.
Reference solution (a). Dissolve 10 mg of phenylalanine CRS
in a mixture of equal volumes of glacial acetic acid R
and water R and dilute to 50 ml with the same mixture of
solvents.
Reference solution (b). Dilute 5 ml of test solution (b) to
20 ml with a mixture of equal volumes of glacial acetic
acid R and water R.
Reference solution (c). Dissolve 10 mg of phenylalanine CRS
and 10 mg of tyrosine CRS in a mixture of equal volumes of C19H20N2O2
glacial acetic acid R and water R and dilute to 25 ml with
the same mixture of solvents.
Apply separately to the plate 5 µl of each solution. Develop Content : 99.0 per cent to 101.0 per cent (dried substance).
over a path of 15 cm using a mixture of 20 volumes of glacial
acetic acid R, 20 volumes of water R and 60 volumes of
butanol R. Allow the plate to dry in air, spray with ninhydrin Appearance : white or almost white, crystalline powder.
solution R and heat at 100 °C to 105 °C for 15 min. Any spot Solubility : practically insoluble in water, sparingly soluble
in the chromatogram obtained with test solution (a), apart
from the principal spot, is not more intense than the spot
in the chromatogram obtained with reference solution (b)
(0.5 per cent). The test is not valid unless the chromatogram First identification : A, C.
obtained with reference solution (c) shows two clearly
separated spots.
Chlorides (2.4.4). Dissolve 0.25 g in 3 ml of dilute nitric
acid R and dilute to 15 ml with water R. The solution
complies with the limit test for chlorides, without any further
addition of nitric acid (200 ppm).
Sulphates (2.4.13). Dissolve 0.5 g in a mixture of 5 volumes
of dilute hydrochloric acid R and 25 volumes of distilled
water R and dilute to 15 ml with the same mixture of
solvents. The solution complies with the limit test for
sulphates (300 ppm).
Ammonium (2.4.1). 50 mg complies with limit test B for
ammonium (200 ppm). Prepare the standard using 0.1 ml of
ammonium standard solution (100 ppm NH4) R.
Iron (2.4.9). In a separating funnel, dissolve 1.0 g in 10 ml
of dilute hydrochloric acid R. Shake with three quantities,
each of 10 ml, of methyl isobutyl ketone R1, shaking for
3 min each time. To the combined organic layers add 10 ml
of water R and shake for 3 min. The aqueous layer complies
with the limit test for iron (10 ppm).
Heavy metals (2.4.8). 2.0 g complies with limit test D for
heavy metals (10 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.
General Notices (1) apply to all monographs and other texts
ASSAY
Dissolve 0.100 g in 3 ml of anhydrous formic acid R.
Add 30 ml of anhydrous acetic acid R. Titrate with 0.1 M
perchloric acid using 0.1 ml of naphtholbenzein solution R
as indicator, until the colour changes from yellow to green.
1 ml of 0.1 M perchloric acid is equivalent to 16.52 mg of
C9H11NO2.
STORAGE
Store protected from light.
01/2005:0422
PHENYLBUTAZONE
Phenylbutazonum
Mr 308.4
DEFINITION
4-Butyl-1,2-diphenylpyrazolidine-3,5-dione.
CHARACTERS
in alcohol. It dissolves in alkaline solutions.
IDENTIFICATION
Second identification : A, B, D.
A. Melting point (2.2.14) : 104 °C to 107 °C.
B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 25 mg of the substance to be
examined in a mixture of equal volumes of ethanol R and
methylene chloride R and dilute to 25 ml with the same
mixture of solvents.
Reference solution. Dissolve 25 mg of
phenylbutazone CRS in a mixture of equal
volumes of ethanol R and methylene chloride R and
dilute to 25 ml with the same mixture of solvents.
Plate : TLC silica gel GF254 plate R.
Mobile phase : acetone R, methylene chloride R
(20:80 V/V).
Application : 5 µl.
Development : over a path of 10 cm.
Drying : in air.
Detection : examine in ultraviolet light at 254 nm.
Results : 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
the reference solution.
C. Infrared absorption spectrophotometry (2.2.24).
Comparison : phenylbutazone CRS.
D. To 0.1 g add 1 ml of glacial acetic acid R and 2 ml
of hydrochloric acid R and heat the mixture under a
reflux condenser for 30 min. Cool, add 10 ml of water R
and filter. To the filtrate add 3 ml of a 7 g/l solution of
2229
Phenylbutazone
sodium nitrite R. A yellow colour is produced. To 1 ml of System suitability : reference solution (b) :
the solution add a solution of 10 mg of β-naphthol R in
5 ml of sodium carbonate solution R. A brownish-red to
violet-red precipitate is formed.
TESTS
Solution S. Dissolve 1.0 g with shaking in 20 ml of dilute
sodium hydroxide solution R and maintain the solution at
25 °C for 3 h.
Appearance of solution. Solution S is clear (2.2.1).
Acidity or alkalinity. Heat to boiling 1.0 g in 50 ml of
water R, cool with shaking in a closed flask and filter.
To 25 ml of the filtrate add 0.5 ml of phenolphthalein
solution R. The solution is colourless. Not more than 0.5 ml
of 0.01 M sodium hydroxide is required to change the colour
of the indicator. Add 0.6 ml of 0.01 M hydrochloric acid and
0.1 ml of methyl red solution R ; the solution is red or orange. — total : not more than 5 times the area of the principal peak
Absorbance (2.2.25) : maximum 0.20 for solution S at
420 nm in a 4 cm cell.
Related substances. Liquid chromatography (2.2.29).
Prepare the solutions immediately before use.
Test solution. Dissolve 100.0 mg of the substance to be
examined in acetonitrile R and dilute to 10.0 ml with the
same solvent.
Reference solution (a). Dilute 1.0 ml of the test solution to
100.0 ml with acetonitrile R. Dilute 1.0 ml to 10.0 ml with
acetonitrile R.
Reference solution (b). Dissolve 5 mg of phenylbutazone
impurity B CRS and 5 mg of 1,2-diphenylhydrazine R in
acetonitrile R, add 0.5 ml of the test solution and dilute
to 50 ml with acetonitrile R. Dilute 2.5 ml to 10 ml with
acetonitrile R.
Reference solution (c). Dissolve 1.0 mg of benzidine R in
acetonitrile R and dilute to 100.0 ml with the same solvent.
Dilute 1.0 ml to 100.0 ml with acetonitrile R. Dilute 5.0 ml
to 10.0 ml with acetonitrile R.
Column :
— size : l = 0.125 m, Ø = 4.0 mm,
— stationary phase: octadecylsilyl silica gel for
chromatography R (5 µm),
— temperature : 30 °C.
Mobile phase :
— mobile phase A : dissolve 1.36 g of sodium acetate R in
water R, adjust to pH 5.2 with a 52.5 g/l solution of citric Dissolve 0.250 g in 25 ml of acetone R and add 0.5 ml of
acid R and dilute to 1000 ml with water R,
— mobile phase B : acetonitrile R,
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 10 70 30
10 - 20 70 → 40 30 → 60
20 - 35 40 60
35 - 40 40 → 70 60 → 30
Flow rate : 1.5 ml/min.
Detection : spectrophotometer at 240 nm.
Injection : 20 µl ; inject the test solution and reference
solutions (a) and (b).
Relative retentions with reference to phenylbutazone
(retention time = about 13 min) : impurity E = about 0.2 ;
impurity A = about 0.5 ; impurity B = about 1.2 ;
impurity C = about 1.3 ; impurity D = about 1.7.
2230 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 5.0
— resolution : minimum 2.0 between the peaks due to
phenylbutazone and to impurity B.
Limits :
— correction factor : for the calculation of content, multiply
the peak area of impurity C by 0.55,
— impurities A, B : for each impurity, not more than 2.5 times
the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.25 per cent),
— impurity C : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.20 per cent),
— any other impurity : not more than the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.1 per cent),
in the chromatogram obtained with reference solution (a)
(0.5 per cent),
— disregard limit : 0.25 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.025 per cent) ; disregard any peak due to impurity E.
Impurity E. Liquid chromatography (2.2.29) as described
in the test for related substances with the following
modifications.
Detection : spectrophotometer at 280 nm.
Injection : test solution and reference solution (c).
System suitability : reference solution (c) :
— signal-to-noise ratio : minimum 10 for the principal peak.
Limit :
— impurity E : not more than the area of the principal peak
in the chromatogram obtained with reference solution (c)
(5 ppm).
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with limit test C. Prepare the standard using
2 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.2 per cent, determined
on 1.000 g by drying in vacuo at 80 °C for 4 h.
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
bromothymol blue solution R1. Titrate with 0.1 M sodium
hydroxide until a blue colour is obtained which persists for
15 s. Carry out a blank titration.
1 ml of 0.1 M sodium hydroxide is equivalent to 30.84 mg
of C19H20N2O2.
STORAGE
Protected from light.
IMPURITIES
A. (2RS)-2-[(1,2-diphenyldiazanyl)carbonyl]hexanoic acid,
EUROPEAN PHARMACOPOEIA 5.0 Phenylephrine

D. Dissolve about 10 mg in 1 ml of 1 M hydrochloric acid,

add 0.05 ml of copper sulphate solution R and 1 ml of a
200 g/l solution of sodium hydroxide R. A violet colour
develops. Add 1 ml of ether R and shake. The upper layer
remains colourless.

TESTS
Appearance of solution. Dissolve 1 g in 1 M hydrochloric
B. 4-butyl-4-hydroxy-1,2-diphenylpyrazolidine-3,5-dione, acid and dilute to 10 ml with the same acid. The solution is
clear (2.2.1) and not more intensely coloured than reference
C. C6H5-NH-NH-C6H5 : 1,2-diphenyldiazane, solution Y7 (2.2.2, Method II).
(1,2-diphenylhydrazine), Specific optical rotation (2.2.7). Dissolve 1.250 g in 1 M
hydrochloric acid and dilute to 25.0 ml with the same acid.
D. C6H5-N=N-C6H5 : 1,2-diphenyldiazene, The specific optical rotation is − 53 to − 57, calculated with
reference to the dried substance.
Absorbance. Dissolve 0.50 g in 0.1 M hydrochloric acid
and dilute to 200 ml with the same solvent. Measure the
absorbance (2.2.25) at 315 nm using 0.1 M hydrochloric
acid as the compensation liquid. The absorbance is not more
E. biphenyl-4,4′-diamine (benzidine). than 0.15 (0.4 per cent, calculated as impurity C).
Related substances. Examine by thin-layer chromatography
(2.2.27), using silica gel HF254 R as the coating substance.
Solvent mixture. Prepare a mixture of equal volumes of
01/2005:1035 methylene chloride R and methanolic hydrochloric acid
hydrochloric acid R diluted to 10 volumes with methanol R.
PHENYLEPHRINE Test solution. Dissolve 0.1 g of the substance to be examined
in the solvent mixture and dilute to 5 ml with the same
solvent mixture.
Phenylephrinum Reference solution (a). Dissolve 20 mg of
phenylephrine CRS in the solvent mixture and
dilute to 1 ml with the same solvent mixture.
Reference solution (b). Dilute 0.1 ml of the test solution to
20 ml with the solvent mixture.
Reference solution (c). Dilute 0.1 ml of the test solution to
50 ml with the solvent mixture.
C9H13NO2 Mr 167.2
Apply separately to the plate 10 µl of each solution and
DEFINITION develop over a path of 15 cm using a mixture of 0.5 volumes
of concentrated ammonia R, 25 volumes of methanol R
Phenylephrine contains not less than 99.0 per cent
and 70 volumes of methylene chloride R. Dry the plate in a
and not more than the equivalent of 100.5 per cent of
current of cold air. Examine in ultraviolet light at 254 nm.
(1R)-1-(3-hydroxyphenyl)-2-(methylamino)ethanol, calculated
Spray with a 1 g/l solution of fast red B salt R in a 50 g/l
with reference to the dried substance.
solution of sodium carbonate R and examine in daylight.
Any spot in the chromatogram obtained with the test
CHARACTERS
solution, apart from the principal spot, is not more intense
A white or almost white, crystalline powder, slightly soluble than the spot in the chromatogram obtained with reference
in water, sparingly soluble in methanol, slightly soluble in solution (b) (0.5 per cent) and at most two such spots are
alcohol. It dissolves in dilute mineral acids and in dilute more intense than the spot in the chromatogram obtained
solutions of alkali hydroxides. with reference solution (c) (0.2 per cent).
It melts at about 174 °C. 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
IDENTIFICATION 105 °C.
First identification : A, B. Sulphated ash (2.4.14). Not more than 0.1 per cent,
Second identification : A, C, D. determined on 1.0 g.
A. It complies with the test for specific optical rotation (see ASSAY
Tests).
Dissolve 0.150 g in 60 ml of anhydrous acetic acid R.
B. Examine by infrared absorption spectrophotometry Titrate with 0.1 M perchloric acid determining the end-point
(2.2.24), comparing with the spectrum obtained with potentiometrically (2.2.20).
phenylephrine CRS.
1 ml of 0.1 M perchloric acid is equivalent to 16.72 mg of
C. Examine the chromatograms obtained in the test
C9H13NO2.
for related substances. The principal spot in the
chromatogram obtained with the test solution is similar
in position, colour and size to the principal spot in the STORAGE
chromatogram obtained with reference solution (a). Store in an airtight container, protected from light.

General Notices (1) apply to all monographs and other texts 2231