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Sulfasalazine

EUROPEAN PHARMACOPOEIA 5.0

Reference solution (b). Dilute 1.25 ml of test solution (a)


to 50 ml with a mixture of 2 volumes of concentrated
ammonia R and 48 volumes of methanol R.
Reference solution (c). Dissolve 20 mg of the substance to
be examined and 20 mg of sulfamerazine CRS in 3 ml of
a mixture of 2 volumes of concentrated ammonia R and
48 volumes of methanol R and dilute to 5 ml with the same
mixture of solvents.
Apply to the plate 5 l of each solution. Develop over a
path corresponding to two-thirds of the plate height using a
mixture of 3 volumes of dilute ammonia R1, 5 volumes of
water R, 40 volumes of nitromethane R and 50 volumes of
dioxan R. Dry the plate at 100 C to 105 C and examine in
ultraviolet light at 254 nm. Any spot in the chromatogram
obtained with test solution (b), 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 obtained with reference
solution (c) shows two clearly separated principal spots.
Heavy metals (2.4.8). 12 ml of solution S complies with limit
test A for heavy metals (20 ppm). Prepare the standard using
lead standard solution (1 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.
ASSAY
Carry out the determination of primary aromatic
amino-nitrogen (2.5.8), using 0.140 g and determining the
end-point electrometrically.
1 ml of 0.1 M sodium nitrite is equivalent to 17.22 mg of
C6H8N2O2S.
STORAGE
Store protected from light.
01/2005:0863

SULFASALAZINE

IDENTIFICATION
Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
sulfasalazine CRS. Examine the substances prepared as
discs.
TESTS
Related substances. Examine by liquid chromatography
(2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be
examined in dilute ammonia R3 and dilute to 25.0 ml with
the same solvent.
Reference solution (a). Dilute 1.0 ml of the test solution to
100.0 ml with dilute ammonia R3.
Reference solution (b). Dissolve 1.0 mg of sulfasalazine
derivative for resolution CRS in 10.0 ml of reference
solution (a). Dilute 1.0 ml of this solution to 10.0 ml with
reference solution (a).
The chromatographic procedure may be carried out using :
a stainless steel column 0.25 m long and 4.6 mm in
internal diameter, packed with octadecylsilyl silica gel
for chromatography R (5 m),
as mobile phase at a flow rate of 1 ml/min :
Mobile phase A. In a 1000 ml volumetric flask dissolve
1.13 g of sodium dihydrogen phosphate R and 2.5 g of
sodium acetate R in 900 ml of water R. Adjust to pH 4.8
with glacial acetic acid R and adjust the volume to
1000 ml with water R,
Mobile phase B. Mix 1 volume of mobile phase A with
4 volumes of methanol R,
Time
(min)

Mobile phase A
(per cent V/V)

Mobile phase B
(per cent V/V)

Comment

0 - 15

60 45

40 55

linear gradient

15 - 25

45

55

isocratic

25 - 60

45 0

55 100

linear gradient

60 - 65

100

isocratic

65 - 67

0 60

100 40

switch to initial
composition

67 - 77

60

40

re-equilibration

as detector a spectrophotometer set at 320 nm.


Inject 20 l of reference solution (a). Adjust the sensitivity
of the detector so that the height of the principal peak in
the chromatogram obtained is at least 50 per cent of the full
scale of the recorder. Inject 20 l of reference solution (b).
The test is not valid unless the resolution between the peaks
corresponding to sulfasalazine and sulfasalazine derivative
for resolutionis at least 3.0.
Inject 20 l of the test solution and 20 l of reference
solution (a). When the chromatograms are recorded in the
prescribed conditions, the approximate retention times
C18H14N4O5S
Mr 398.4 relative to sulfasalazine are the following : impurity A = 2.00 ;
impurity B = 1.85 ; impurity C = 0.80 ; impurity D = 1.90 ;
impurity E = 1.63 ; impurity F = 0.85 ; impurity G = 1.39 ;
DEFINITION
impurity H = 0.16 ; impurity I = 0.28.
Sulfasalazine contains not less than 97.0 per cent and not
In the chromatogram obtained with the test solution : the
more than the equivalent of 101.5 per cent of 2-hydroxy-5area of any peak, apart from the principal peak, is not greater
[2-[4-(pyridin-2-ylsulphamoyl)phenyl]diazenyl]benzoic acid,
than the area of the principal peak in the chromatogram
calculated with reference to the dried substance.
obtained with reference solution (a) (1 per cent) ; the sum of
CHARACTERS
the areas of all peaks, apart from the principal peak is not
A bright yellow or brownish-yellow, fine powder, practically greater than four times the area of the principal peak in the
insoluble in water, very slightly soluble in alcohol, practically chromatogram obtained with reference solution (a) (4 per
insoluble in methylene chloride. It dissolves in dilute
cent). Disregard any peak with a retention time shorter than
solutions of alkali hydroxides.
6 min (corresponding to salicylic acid and sulfapyridine) and

Sulfasalazinum

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See the information section on general monographs (cover pages)

Sulfasalazine

EUROPEAN PHARMACOPOEIA 5.0

any peak with an area less than 0.05 times the area of the
principal peak in the chromatogram obtained with reference
solution (a).
Salicylic acid and sulfapyridine. Examine by liquid
chromatography (2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be
examined in dilute ammonia R3 and dilute to 25.0 ml with
the same solvent.
Reference solution (a). Dissolve 5.0 mg of salicylic acid R
and 5.0 mg and sulfapyridine CRS in dilute ammonia R3
and dilute to 10.0 ml with the same solvent.
Reference solution (b). Dilute 2.0 ml of reference solution (a)
to 100.0 ml with dilute ammonia R3.
The chromatographic procedure may be carried out using :
a stainless steel column 0.25 m long and 4.6 mm in
internal diameter, packed with octadecylsilyl silica gel
for chromatography R (5 m),
as mobile phase at a flow rate of 1 ml/min a mixture of
70 volumes of mobile phase A (described in the test for
related substances) and 30 volumes of mobile phase B
(described in the test for related substances),
as detector a spectrophotometer set at 300 nm.
Inject 20 l of reference solution (b). Adjust the sensitivity
of the system so that the height of the principal peaks
in the chromatogram obtained is at least 50 per cent of
the full scale of the recorder. When the chromatogram is
recorded in the prescribed conditions, the retention times
are : salicylic acid about 6 min and sulfapyridine about 7 min.
The test is not valid unless the resolution between the peaks
corresponding to salicylic acid and sulfapyridine is at least 2.
Inject 20 l of the test solution and 20 l of reference
solution (b). Continue the chromatography for 10 min. In
the chromatogram obtained with the test solution the area
of the peak corresponding to salicylic acid is not greater
than 0.5 times the area of the first peak in the chromatogram
obtained with reference solution (b) (0.5 per cent) and the
peak corresponding to sulfapyridine is not greater than
0.5 times the area of the second peak in the chromatogram
obtained with reference solution (b) (0.5 per cent). Disregard
any peak with an area less than 0.05 times the area of the
principal peak in the chromatogram obtained with reference
solution (b).
Chlorides (2.4.4.). To 1.25 g add 50 ml of distilled water R.
Heat at about 70 C for 5 min. Cool and filter. To 20 ml of
the filtrate add 1 ml of nitric acid R, allow to stand for 5 min
and filter to obtain a clear solution. 15 ml of the filtrate
complies with the limit test for chlorides (140 ppm).
Sulphates (2.4.13). To 20 ml of the filtrate prepared for the
test for chlorides add 1 ml of dilute hydrochloric acid R,
allow to stand for 5 min and filter. 15 ml of the filtrate
complies with the limit test for sulphates (400 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 1.0 per cent,
determined on 1.000 g by drying in an oven at 100 C to
105 C for 2 h.
Sulphated ash (2.4.14). Not more than 0.5 per cent,
determined on 1.0 g.
ASSAY
Dissolve 0.150 g in 0.1 M sodium hydroxide and dilute
to 100.0 ml with the same solvent. Transfer 5.0 ml of this
solution to a 1000 ml volumetric flask containing about
750 ml of water R. Add 20.0 ml of 0.1 M acetic acid and
General Notices (1) apply to all monographs and other texts

dilute to 1000.0 ml with water R. Prepare a standard solution


at the same time and in the same manner using 0.150 g of
sulfasalazine CRS. Measure the absorbance (2.2.25) of the
two solutions at the maximum at 359 nm.
Calculate the content of C18H14N4O5S from the absorbances
measured and the concentration of the solutions.
STORAGE
Store protected from light.
IMPURITIES

A. 4,4-[(4-hydroxy-1,3-phenylene)bis(diazenediyl)]bis[N(pyridin-2-yl)benzenesulphonamide],

B. 2-hydroxy-3,5-bis[2-[4-(pyridin-2-ylsulphamoyl)phenyl]diazenyl]benzoic acid,

C. 2-hydroxy-5-[2-[4-(2-iminopyridin-1(2H)yl)phenyl]diazenyl]benzoic acid,

D. 4-[2-(2-hydroxyphenyl)diazenyl]-N-(pyridin-2yl)benzenesulphonamide,

E. 2-hydroxy-4-(pyridin-2-ylsulphamoyl)-5-[2-[4-(pyridin-2ylsulphamoyl)phenyl]diazenyl]biphenyl-3-carboxylic acid,

F. 2-hydroxy-3-[2-[4-(pyridin-2-ylsulphamoyl)phenyl]diazenyl]benzoic acid,
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Sulfathiazole

EUROPEAN PHARMACOPOEIA 5.0

D. Dissolve about 10 mg in a mixture of 10 ml of water R


and 2 ml of 0.1 M sodium hydroxide and add 0.5 ml of
copper sulphate solution R. A greyish-blue or purple
precipitate is formed.
E. Dissolve about 5 mg in 10 ml of 1 M hydrochloric acid.
Dilute 1 ml of the solution to 10 ml with water R. The
solution, without further addition of acid, gives the
reaction of primary aromatic amines (2.3.1).
TESTS
Appearance of solution. Dissolve 1.0 g in 10 ml of 1 M
sodium hydroxide. The solution is clear (2.2.1) and not
H. salicylic acid,
more intensely coloured than reference solution GY4 (2.2.2,
Method II).
Acidity. To 1.0 g add 50 ml of carbon dioxide-free water R.
Heat to 70 C for 5 min. Cool rapidly to 20 C and filter.
To 25 ml of the filtrate add 0.1 ml of bromothymol blue
solution R1. Not more than 0.1 ml of 0.1 M sodium
hydroxide is required to change the colour of the indicator.
Related substances. Examine by thin-layer chromatography
(2.2.27), using silica gel H R as the coating substance.
Test solution (a). Dissolve 0.10 g of the substance to
I. 2-hydroxy-5-[2-(4-sulphophenyl)diazenyl]benzoic acid.
be examined in a mixture of 1 volume of concentrated
01/2005:0742 ammonia R and 9 volumes of alcohol R and dilute to 10 ml
with the same mixture of solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 5 ml with
SULFATHIAZOLE
a mixture of 1 volume of concentrated ammonia R and
9 volumes of alcohol R.
Sulfathiazolum
Reference solution (a). Dissolve 20 mg of sulfathiazole CRS
in a mixture of 1 volume of concentrated ammonia R and
9 volumes of alcohol R and dilute to 10 ml with the same
mixture of solvents.
Reference solution (b). Dissolve 50 mg of sulfanilamide R
in a mixture of 1 volume of concentrated ammonia R and
9 volumes of alcohol R and dilute to 100 ml with the same
C 9 H 9 N3 O 2 S2
Mr 255.3
mixture of solvents. Dilute 1 ml of this solution to 10 ml with
the same mixture of solvents.
DEFINITION
Apply to the plate 10 l of each solution. Develop over a
Sulfathiazole contains not less than 99.0 per cent
path of 15 cm using a mixture of 18 volumes of ammonia R
and not more than the equivalent of 101.0 per cent of
and 90 volumes of butanol R. Dry the plate at 100 C
4-amino-N-(thiazol-2-yl)benzenesulphonamide, calculated
to 105 C for 10 min and spray with a 1 g/l solution of
with reference to the dried substance.
dimethylaminobenzaldehyde R in alcohol R containing
CHARACTERS
1 per cent V/V of hydrochloric acid R. Any spot in the
A white or slightly yellowish, crystalline powder, practically chromatogram obtained with test solution (a), apart from
insoluble in water, slightly soluble in alcohol, practically
the principal spot, is not more intense than the spot in the
insoluble in methylene chloride. It dissolves in dilute
chromatogram obtained with reference solution (b) (0.5 per
solutions of alkali hydroxides and in dilute mineral acids.
cent).
Heavy metals (2.4.8). 1.0 g complies with limit test C for
IDENTIFICATION
heavy metals (20 ppm). Prepare the standard using 2 ml of
First identification : A, B.
lead standard solution (10 ppm Pb) R.
Second identification : A, C, D, E.
Loss on drying (2.2.32). Not more than 0.5 per cent,
A. Melting point (2.2.14) : 200 C to 203 C. Melting may
determined on 1.000 g by drying in an oven at 100 C to
occur at about 175 C, followed by solidification and a
105 C.
second melting between 200 C and 203 C.
Sulphated ash (2.4.14). Not more than 0.1 per cent,
B. Examine by infrared absorption spectrophotometry
determined
on 1.0 g.
(2.2.24), comparing with the spectrum obtained with
sulfathiazole CRS. Examine the substances prepared as
ASSAY
discs. If the spectra obtained show differences, dissolve
the substance to be examined and the reference substance Carry out the determination of primary aromatic aminonitrogen (2.5.8), using 0.200 g, determining the end-point
separately in alcohol R, evaporate to dryness in vacuo
electrometrically.
and record the spectra again using the residues.
1 ml of 0.1 M sodium nitrite is equivalent to 25.53 mg of
C. Examine the chromatograms obtained in the test
C9 H 9 N 3 O 2 S 2 .
for related substances. The principal spot in the
chromatogram obtained with test solution (b) is similar
STORAGE
in position, colour and size to the principal spot in the
chromatogram obtained with reference solution (a).
Store protected from light.
G. 5-[2-[4,5-bis(pyridin-2-ylsulphamoyl)biphenyl-2yl]diazenyl]-2-hydroxybenzoic acid,

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See the information section on general monographs (cover pages)