EUROPEAN PHARMACOPOEIA 5.

Colestyramine

the solution at the absorption maximum at 420 nm, using as

compensation liquid a solution prepared in the same manner
but using 5.0 ml of water R instead of the test solution.
Repeat the operation using 5.0 ml of the reference solution.
The absorbance obtained with the test solution is not greater
than that obtained with the reference solution.
Impurity A. Liquid chromatography (2.2.29).
Test solution. Shake 5.0 g with 10 ml of acetone R for
30 min. Centrifuge and use the supernatant liquid.
Reference solution (a). Dissolve 5 mg of styrene R in
D. N-[(7S,12aS)-3-(-D-glucopyranosyloxy)-1,2,10-trimethoxy- acetone R and dilute to 100.0 ml with the same solvent.
9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide
Dilute 1.0 ml to 100.0 ml with acetone R.
(colchicoside).
Reference solution (b). Dissolve 0.35 ml of styrene R in
acetone R and dilute to 100.0 ml with the same solvent.
01/2005:1775 Dilute 1.0 ml to 100.0 ml with acetone R.
Reference solution (c). Dissolve 0.35 ml of toluene R in
COLESTYRAMINE
acetone R and dilute to 100.0 ml with the same solvent.
Reference solution (d). Mix 1.0 ml of reference solution (b)
Colestyraminum
and 1.0 ml of reference solution (c) with acetone R and dilute
to 100.0 ml with the same solvent.
DEFINITION
Column :
Strongly basic anion-exchange resin in chloride form,
size : l = 0.30 m, = 3.9 mm,
consisting of styrene-divinylbenzene copolymer with
stationary phase : octadecylsilyl silica gel for
quaternary ammonium groups.
chromatography R (10 m) with a specific surface area of
Nominal exchange capacity : 1.8 g to 2.2 g of sodium
330 m2/g and a pore size of 12.5 nm.
glycocholate per gram (dried substance).
Mobile phase : acetonitrile R, water R (50:50 V/V).
CHARACTERS
Flow rate : 2.0 ml/min.
Appearance : white or almost white, fine powder,
Detection : spectrophotometer at 254 nm.
hygroscopic.
Injection : 20 l of test solution, reference solutions (a)
Solubility : insoluble in water, in methylene chloride and in and (d).
ethanol (96 per cent).
System suitability : reference solution (d) :
IDENTIFICATION
resolution : minimum 1.5 between the peaks due to
impurity A and toluene.
A. Infrared absorption spectrophotometry (2.2.24).
Limit
:
Comparison : colestyramine CRS.
impurity A : not more than the area of the principal peak
B. It complies with the test for chlorides (see Tests).
in the chromatogram obtained with reference solution (a)
TESTS
(1 ppm).
pH (2.2.3) : 4.0 to 6.0.
Chloride : 13.0 per cent to 17.0 per cent (dried substance).
Suspend 0.100 g in 10 ml of water R and allow to stand for To 0.2 g add 100 ml of water R and 50 mg of potassium
10 min.
nitrate R. Add, with stirring, 2 ml of nitric acid R and
titrate with 0.1 M silver nitrate, determining the end-point
Dialysable quaternary amines : maximum 500 ppm,
potentiometrically (2.2.20).
expressed as benzyltrimethylammonium chloride.
1 ml of 0.1 M silver nitrate is equivalent to 3.55 mg of Cl.
Test solution. Place a 25 cm piece of cellulose dialysis
tubing having a molecular weight cut-off of 12 000-14 000
Heavy metals (2.4.8) : maximum 20 ppm.
and an inflated diameter of 3-6 cm (flat width of 5-9 cm)
1.0 g complies with limit test F. Prepare the reference
in water R to hydrate until pliable, appropriately sealing
solution using 2 ml of lead standard solution (10 ppm Pb) R.
one end. Introduce 2.0 g of the substance to be examined
Loss
on drying (2.2.32) : maximum 12 per cent, determined
into the tube and add 10 ml of water R. Seal the tube and
on
1.000
g by drying in an oven at 70 C over diphosphorus
completely immerse it in 100 ml of water R in a suitable
pentoxide
R at a pressure not exceeding 7 kPa for 16 h.
vessel and stir the liquid for 16 h to effect dialysis. Use the
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
dialysate as test solution.
on 1.0 g.
Reference solution. Prepare the reference solution in a
similar manner but using 10 ml of a freshly prepared 0.1 g/l ASSAY
solution of benzyltrimethylammonium chloride R instead
Exchange capacity. Liquid chromatography (2.2.29).
of the substance to be examined.
Transfer 5.0 ml of the test solution to a separating funnel and Solution A. Dissolve 1.500 g of sodium glycocholate R
add 5 ml of a 3.8 g/l solution of disodium tetraborate R, 1 ml in a solution containing 4 g/l of potassium dihydrogen
of a solution containing 1.5 g/l of bromothymol blue R and phosphate R and 12 g/l of dipotassium hydrogen
4.05 g/l of sodium carbonate R and 10 ml of chloroform R. phosphate R and dilute to 100.0 ml with the same solution.
Shake the mixture vigourously for 1 min, allow the phases
Test solution. Add 20.0 ml of solution A to a quantity of the
to separate and transfer the clear organic layer to a 25 ml
substance to be examined equivalent to about 0.100 g of the
volumetric flask. Repeat the extraction with a further 10 ml dried substance. Shake mechanically for 2 h and centrifuge
of chloroform R, combine the organic layers and dilute to
for 15 min. Dilute 5.0 ml of the supernatant liquid to 50.0 ml
25 ml with chloroform R. Measure the absorbance (2.2.25) of with water R.
General Notices (1) apply to all monographs and other texts

1359

Colistimethate sodium

EUROPEAN PHARMACOPOEIA 5.0

Reference solution (a). Dilute 4.0 ml of solution A to

IDENTIFICATION
100.0 ml with water R.
A. Examine by thin-layer chromatography (2.2.27), using
silica gel G R as the coating substance.
Reference solution (b). Dissolve 60 mg of sodium
glycocholate R and 30 mg of sodium taurodeoxycholate R
Test solution. Dissolve 5 mg of the substance to be
in water R and dilute to 100 ml with the same solvent. Dilute
examined in 1 ml of a mixture of equal volumes of
1 ml of the solution to 10 ml with water R.
hydrochloric acid R and water R. Heat at 135 C in a
sealed tube for 5 h. Evaporate to dryness on a water-bath
Column :
and continue the heating until the hydrochloric acid has
size : l = 0.25 m, = 4.6 mm,
evaporated. Dissolve the residue in 0.5 ml of water R.
stationary phase: octadecylsilyl silica gel for
Reference
solution (a). Dissolve 20 mg of leucine R in
chromatography R (5 m).
water R and dilute to 10 ml with the same solvent.
Mobile phase : mix 35 volumes of acetonitrile R and
Reference solution (b). Dissolve 20 mg of threonine R in
65 volumes of a 10.9 g/l solution of potassium dihydrogen
water R and dilute to 10 ml with the same solvent.
phosphate R adjusted to pH 3.0 with phosphoric acid R.
Reference solution (c). Dissolve 20 mg of phenylalanine R
Flow rate : 1.5 ml/min.
in water R and dilute to 10 ml with the same solvent.
Detection : spectrophotometer at 214 nm.
Reference solution (d). Dissolve 20 mg of serine R in
Injection : 50 l.
water R and dilute to 10 ml with the same solvent.
Run time : twice the retention time of glycocholate.
Carry out the following procedures protected from light.
System suitability : reference solution (b) :
Apply to the plate as 10 mm bands 5 l of each solution.
resolution : minimum 1.5 between the peaks due to
Place the plate in the chromatographic tank so that it
glycocholate and taurodeoxycholate.
is not in contact with the mobile phase consisting of a
mixture of 25 volumes of water R and 75 volumes of
Calculate the nominal exchange capacity using the following
phenol R. Leave the plate to become impregnated with
expression :
the vapour of the solvent for at least 12 h. Develop over a
path of 12 cm using the same mobile phase. Dry the plate
at 100-105 C and spray with ninhydrin solution R1.
Heat at 110 C for 5 min. The chromatogram obtained
with the test solution shows zones corresponding to
A1 = area of the peak due to glycocholate in
those in the chromatograms obtained with reference
the chromatogram obtained with reference
solutions (a) and (b), but shows no zones corresponding
solution (a),
to those in the chromatograms obtained with reference
A2 = area of the peak due to glycocholate in the
solutions (c) and (d). The chromatogram obtained with
chromatogram obtained with the test solution,
the test solution also shows a zone with a very low Rf
m1 = mass, in milligrams, of sodium glycocholate R
value (2,4-diaminobutyric acid).
used in the preparation of solution A,
B. Dissolve about 5 mg in 3 ml of water R. Add 3 ml of dilute
sodium hydroxide solution R. Shake and add 0.5 ml of
m2 = mass, in milligrams, of the dried substance to
a 10 g/l solution of copper sulphate R. A violet colour
be examined used in the preparation of the test
is produced.
solution,
C.
Dissolve about 50 mg in 1 ml of 1 M hydrochloric
0.73 = correction factor to convert the true exchange
acid
and add 0.5 ml of 0.01 M iodine. The solution is
capacity to the conventionally used nominal
decolourised
and gives reaction (a) of sulphates (2.3.1).
exchange capacity.
D. It gives reaction (b) of sodium (2.3.1).
STORAGE
TESTS
In an airtight container.
Appearance of solution. Dissolve 0.16 g in 10 ml of water R.
The solution is clear (2.2.1).
pH (2.2.3). Dissolve 0.1 g in carbon dioxide-free water R
and dilute to 10 ml with the same solvent. The pH of the
A. styrene.
solution, measured after 30 min, is 6.5 to 8.5.
Specific optical rotation (2.2.7). Dissolve 1.25 g in water R
01/2005:0319 and dilute to 25.0 ml with the same solvent. The specific
optical rotation is 46 to 51, calculated with reference to
the dried substance.
COLISTIMETHATE SODIUM
Free colistin. Dissolve 80 mg in 3 ml of water R. Add
0.1 ml of a 100 g/l solution of silicotungstic acid R ; 10 s to
Colistimethatum natricum
20 s after addition of the reagent, the solution is not more
DEFINITION
opalescent than reference suspension II (2.2.1).
Colistimethate sodium is prepared from colistin by the action Total sulphite. Work in a fume cupboard. Dissolve 0.100 g
of formaldehyde and sodium hydrogen sulphite. The potency in 50 ml of water R and add 5 ml of a 100 g/l solution of
is not less than 11 500 IU/mg, calculated with reference to sodium hydroxide R and 0.3 g of potassium cyanide R.
the dried substance.
Boil gently for 3 min and then cool. Neutralise with 0.5 M
sulphuric acid using 0.2 ml of methyl orange solution R as
CHARACTERS
indicator. Add an excess of 0.5 ml of the acid and 0.2 g of
potassium iodide R. Titrate with 0.05 M iodine using 1 ml of
A white or almost white powder, hygroscopic, very soluble
starch solution R as indicator. The volume of 0.05 M iodine
in water, slightly soluble in alcohol, practically insoluble in
used in the titration is 5.5 ml to 7.0 ml.
acetone.
IMPURITIES
Specified impurities : A.

1360

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