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EUROPEAN PHARMACOPOEIA 5.0 Colestyramine

D. N-[(7S,12aS)-3-(-D-glucopyranosyloxy)-1,2,10-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[ a]heptalen-7-yl]acetamide(colchicoside).

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COLESTYRAMINE

Colestyraminum

DEFINITION

Strongly basic anion-exchange resin in chloride form,consisting of styrene-divinylbenzene copolymer withquaternary ammonium groups.

Nominal exchange capacity: 1.8 g to 2.2 g of sodiumglycocholate per gram (dried substance).

CHARACTERS

Appearance : white or almost white, fine powder,hygroscopic.

Solubility: insoluble in water, in methylene chloride and inethanol (96 per cent).

IDENTIFICATION

A. Infrared absorption spectrophotometry (2.2.24).Comparison: colestyramine CRS.

B. It complies with the test for chlorides (see Tests).

TESTS

pH(2.2.3): 4.0 to 6.0.

Suspend 0.100 g in 10 ml ofwater Rand allow to stand for10 min.

Dialysable quaternary amines: maximum 500 ppm,expressed as benzyltrimethylammonium chloride.

Test solution. Place a 25 cm piece of cellulose dialysistubing having a molecular weight cut-off of 12 000-14 000and an inflated diameter of 3-6 cm (flat width of 5-9 cm)

in water Rto hydrate until pliable, appropriately sealingone end. Introduce 2.0 g of the substance to be examinedinto the tube and add 10 ml ofwater R. Seal the tube andcompletely immerse it in 100 ml ofwater Rin a suitablevessel and stir the liquid for 16 h to effect dialysis. Use thedialysate as test solution.

Reference solution. Prepare the reference solution in asimilar manner but using 10 ml of a freshly prepared 0.1 g/lsolution ofbenzyltrimethylammonium chloride R insteadof the substance to be examined.

Transfer 5.0 ml of the test solution to a separating funnel andadd 5 ml of a 3.8 g/l solution ofdisodium tetraborate R, 1 mlof a solution containing 1.5 g/l ofbromothymol blue R and4.05 g/l ofsodium carbonate R and 10 ml ofchloroform R.

Shake the mixture vigourously for 1 min, allow the phasesto separate and transfer the clear organic layer to a 25 mlvolumetric flask. Repeat the extraction with a further 10 mlofchloroform R, combine the organic layers and dilute to25 ml withchloroform R. Measure the absorbance (2.2.25) of

the solution at the absorption maximum at 420 nm, using ascompensation liquid a solution prepared in the same mannerbut using 5.0 ml ofwater Rinstead of the test solution.

Repeat the operation using 5.0 ml of the reference solution.

The absorbance obtained with the test solution is not greaterthan that obtained with the reference solution.

Impurity A. Liquid chromatography (2.2.29).

Test solution. Shake 5.0 g with 10 ml ofacetone Rfor30 min. Centrifuge and use the supernatant liquid.

Reference solution (a). Dissolve 5 mg ofstyrene Rinacetone Rand dilute to 100.0 ml with the same solvent.Dilute 1.0 ml to 100.0 ml withacetone R.

Reference solution (b). Dissolve 0.35 ml ofstyrene Rinacetone Rand dilute to 100.0 ml with the same solvent.Dilute 1.0 ml to 100.0 ml withacetone R.

Reference solution (c). Dissolve 0.35 ml oftoluene R inacetone Rand dilute to 100.0 ml with the same solvent.

Reference solution (d). Mix 1.0 ml of reference solution (b)and 1.0 ml of reference solution (c) withacetone Rand diluteto 100.0 ml with the same solvent.

Column : size : l= 0.30 m, = 3.9 mm,

stationary phase : octadecylsilyl silica gel forchromatography R(10 m) with a specific surface area of330 m2/g and a pore size of 12.5 nm.

Mobile phase : acetonitrile R,water R(50:50V/V).

Flow rate : 2.0 ml/min.

Detection : spectrophotometer at 254 nm.

Injection : 20 l of test solution, reference solutions (a)and (d).

System suitability: reference solution (d):

resolution : minimum 1.5 between the peaks due toimpurity A and toluene.

Limit:

impurity A : not more than the area of the principal peakin the chromatogram obtained with reference solution (a)(1 ppm).

Chloride : 13.0 per cent to 17.0 per cent (dried substance).

To 0.2 g add 100 ml ofwater Rand 50 mg ofpotassiumnitrate R. Add, with stirring, 2 ml ofnitric acid R andtitrate with0.1 M silver nitrate, determining the end-pointpotentiometrically (2.2.20).

1 ml of0.1 M silver nitrateis equivalent to 3.55 mg of Cl.

Heavy metals(2.4.8): maximum 20 ppm.

1.0 g complies with limit test F. Prepare the reference

solution using 2 ml oflead standard solution (10 ppm Pb) R.Loss on drying(2.2.32): maximum 12 per cent, determinedon 1.000 g by drying in an oven at 70 C over diphosphorus

pentoxide R at a pressure not exceeding 7 kPa for 16 h.

Sulphated ash(2.4.14): maximum 0.1 per cent, determinedon 1.0 g.

ASSAY

Exchange capacity. Liquid chromatography (2.2.29).

Solution A. Dissolve 1.500 g ofsodium glycocholate Rin a solution containing 4 g/l ofpotassium dihydrogen

phosphate Rand 12 g/l ofdipotassium hydrogenphosphate Rand dilute to 100.0 ml with the same solution.

Test solution. Add 20.0 ml of solution A to a quantity of thesubstance to be examined equivalent to about 0.100 g of thedried substance. Shake mechanically for 2 h and centrifugefor 15 min. Dilute 5.0 ml of the supernatant liquid to 50.0 mlwithwater R.

General Notices (1) apply to all monographs and other texts 1359

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Colistimethate sodium EUROPEAN PHARMACOPOEIA 5.0

Reference solution (a). Dilute 4.0 ml of solution A to100.0 ml with water R.

Reference solution (b). Dissolve 60 mg ofsodiumglycocholate Rand 30 mg ofsodium taurodeoxycholate Rinwater Rand dilute to 100 ml with the same solvent. Dilute1 ml of the solution to 10 ml with water R.

Column :

size : l= 0.25 m, = 4.6 mm, stationary phase : octadecylsilyl silica gel for

chromatography R(5 m).

Mobile phase : mix 35 volumes ofacetonitrile R and65 volumes of a 10.9 g/l solution ofpotassium dihydrogen

phosphate R adjusted to pH 3.0 with phosphoric acid R.

Flow rate : 1.5 ml/min.

Detection : spectrophotometer at 214 nm.

Injection : 50 l.

Run time : twice the retention time of glycocholate.

System suitability: reference solution (b):

resolution : minimum 1.5 between the peaks due toglycocholate and taurodeoxycholate.

Calculate the nominal exchange capacity using the followingexpression:

A1 = area of the peak due to glycocholate inthe chromatogram obtained with referencesolution (a),

A2 = area of the peak due to glycocholate in thechromatogram obtained with the test solution,

m1 = mass, in milligrams, ofsodium glycocholate Rused in the preparation of solution A,

m2 = mass, in milligrams, of the dried substance tobe examined used in the preparation of the testsolution,

0.73 = correction factor to convert the true exchangecapacity to the conventionally used nominalexchange capacity.

STORAGE

In an airtight container.

IMPURITIES

Specified impurities: A.

A. styrene.

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COLISTIMETHATE SODIUM

Colistimethatum natricum

DEFINITION

Colistimethate sodium is prepared from colistin by the actionof formaldehyde and sodium hydrogen sulphite. The potencyis not less than 11 500 IU/mg, calculated with reference tothe dried substance.

CHARACTERS

A white or almost white powder, hygroscopic, very solublein water, slightly soluble in alcohol, practically insoluble inacetone.

IDENTIFICATION

A. Examine by thin-layer chromatography (2.2.27), usingsilica gel G R as the coating substance.

Test solution. Dissolve 5 mg of the substance to beexamined in 1 ml of a mixture of equal volumes ofhydrochloric acid R and water R. Heat at 135 C in asealed tube for 5 h. Evaporate to dryness on a water-bathand continue the heating until the hydrochloric acid hasevaporated. Dissolve the residue in 0.5 ml ofwater R.

Reference solution (a). Dissolve 20 mg ofleucine Rinwater Rand dilute to 10 ml with the same solvent.

Reference solution (b). Dissolve 20 mg ofthreonine Rinwater Rand dilute to 10 ml with the same solvent.

Reference solution (c). Dissolve 20 mg ofphenylalanine Rin water Rand dilute to 10 ml with the same solvent.

Reference solution (d). Dissolve 20 mg ofserine R inwater Rand dilute to 10 ml with the same solvent.

Carry out the following procedures protected from light.

Apply to the plate as 10 mm bands 5 l of each solution.Place the plate in the chromatographic tank so that itis not in contact with the mobile phase consisting of amixture of 25 volumes ofwater Rand 75 volumes of

phenol R. Leave the plate to become impregnated withthe vapour of the solvent for at least 12 h. Develop over apath of 12 cm using the same mobile phase. Dry the plateat 100-105 C and spray with ninhydrin solution R1.Heat at 110 C for 5 min. The chromatogram obtainedwith the test solution shows zones corresponding tothose in the chromatograms obtained with referencesolutions (a) and (b), but shows no zones correspondingto those in the chromatograms obtained with referencesolutions (c) and (d). The chromatogram obtained withthe test solution also shows a zone with a very lowRfvalue (2,4-diaminobutyric acid).

B. Dissolve about 5 mg in 3 ml ofwater R

. Add 3 ml ofdilutesodium hydroxide solution R. Shake and add 0.5 ml of

a 10 g/l solution ofcopper sulphate R. A violet colouris produced.

C. Dissolve about 50 mg in 1 ml of1 M hydrochloricacidand add 0.5 ml of0.01 M iodine. The solution isdecolourised and gives reaction (a) of sulphates (2.3.1).

D. It gives reaction (b) of sodium (2.3.1).

TESTS

Appearance of solution. Dissolve 0.16 g in 10 ml ofwater R.The solution is clear (2.2.1).

pH(2.2.3). Dissolve 0.1 g in carbon dioxide-free water Rand dilute to 10 ml with the same solvent. The pH of the

solution, measured after 30 min, is 6.5 to 8.5.

Specific optical rotation(2.2.7). Dissolve 1.25 g inwater Rand dilute to 25.0 ml with the same solvent. The specificoptical rotation is46 to51, calculated with reference tothe dried substance.

Free colistin. Dissolve 80 mg in 3 ml ofwater R. Add0.1 ml of a 100 g/l solution ofsilicotungstic acid R ; 10 s to20 s after addition of the reagent, the solution is not moreopalescent than reference suspension II (2.2.1).

Total sulphite. Work in a fume cupboard. Dissolve 0.100 gin 50 ml ofwater Rand add 5 ml of a 100 g/l solution of

sodium hydroxide R and 0.3 g ofpotassium cyanide R.Boil gently for 3 min and then cool. Neutralise with0.5 M

sulphuric acidusing 0.2 ml ofmethyl orange solution R asindicator. Add an excess of 0.5 ml of the acid and 0.2 g of

potassium iodide R. Titrate with0.05 M iodineusing 1 ml ofstarch solution Ras indicator. The volume of0.05 M iodineused in the titration is 5.5 ml to 7.0 ml.

1360 See the information section on general monographs (cover pages)