Colistin Sulfate

Colistin Sulphate

(Ph. Eur. monograph 0320)

        1264-72-8

Antibacterial.

Colistin Tablets

Ph Eur

A mixture of the sulfates of polypeptides produced by certain strains of Bacillus polymyxa var. colistinus or obtained by any other means.

Minimum 19 000 IU/mg (dried substance).

White or almost white, hygroscopic powder.

Freely soluble in water, practically insoluble in acetone and in ethanol (96 per cent).

First identification   B, E.

Second identification   A, C, D, E.

A. Thin-layer chromatography (2.2.27).

Test solution  Dissolve 5 mg of the substance to be examined in 1 mL of a mixture of equal volumes of hydrochloric acid R and water R. Heat at 135 °C in a sealed tube for 5 h. Evaporate to dryness on a water-bath and continue the heating until moistened blue litmus paper R does not turn red. Dissolve the residue in 0.5 mL of water R.

Reference solution (a)  Dissolve 20 mg of leucine R in water R and dilute to 10 mL with the same solvent.

Reference solution (b)  Dissolve 20 mg of threonine R in water R and dilute to 10 mL with the same solvent.

Reference solution (c)  Dissolve 20 mg of phenylalanine R in water R and dilute to 10 mL with the same solvent.

Reference solution (d)  Dissolve 20 mg of serine R in water R and dilute to 10 mL with the same solvent.

Plate  TLC silica gel G plate R.

Carry out the following procedures protected from light.

Mobile phase  water R, phenol R (25:75 V/V).

Application  5 µL as bands of 10 mm, then place the plate in the chromatographic tank so that it is not in contact with the mobile phase, and allow it to become impregnated with the vapour of the mobile phase for at least 12 h.

Development  Over half of the plate.

Drying  At 105 °C.

Detection  Spray with ninhydrin solution R1 and heat at 110 °C for 5 min.

Results  The chromatogram obtained with the test solution shows zones corresponding to those in the chromatograms obtained with reference solutions (a) and (b), but shows no zones corresponding to those in the chromatograms obtained with reference solutions (c) and (d); the chromatogram obtained with the test solution also shows a zone with a very low R_F value (2,4-diaminobutyric acid).

B. Examine the chromatograms obtained in the test for composition.

Results  The peaks due to polymyxin E1 and polymyxin E2 in the chromatogram obtained with the test solution are similar in retention time to the corresponding peaks in the chromatogram obtained with reference solution (a).

C. 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 a 10 g/L solution of copper sulfate R. A violet colour is produced.

D. Dissolve about 50 mg in 1 mL of 1 M hydrochloric acid and add 0.5 mL of 0.01 M iodine. The solution remains coloured.

E. It gives reaction (a) of sulfates (2.3.1).

4.0 to 6.0.

Dissolve 0.1 g in carbon dioxide-free water R and dilute to 10 mL with the same solvent.

-63 to -73 (dried substance).

Dissolve 1.25 g in water R and dilute to 25.0 mL with the same solvent.

Liquid chromatography (2.2.29).

Test solution  Dissolve 25.0 mg of the substance to be examined in 40 mL of water R and dilute to 50.0 mL with acetonitrile R1.

Reference solution (a)  Dissolve 25.0 mg of colistin sulfate CRS in 40 mL of water R and dilute to 50.0 mL with acetonitrile R1.

Reference solution (b)  Dilute 1.0 mL of reference solution (a) to 100.0 mL with a mixture of 20 volumes of acetonitrile R1 and 80 volumes of water R.

• — size: l = 0.15 m, Ø = 4.6 mm;

• — stationary phase: end-capped octadecylsilyl silica gel for chromatography R (3.5 µm);

• — temperature: 30 °C.

Mobile phase  Mix 22 volumes of acetonitrile R1 and 78 volumes of a solution prepared as follows: dissolve 4.46 g of anhydrous sodium sulfate R in 900 mL of water R, adjust to pH 2.4 with dilute phosphoric acid R and dilute to 1000 mL with water R.

Flow rate  1.0 mL/min.

Detection  Spectrophotometer at 215 nm.

Injection  20 µL of the test solution and reference solution (a).

Run time  1.5 times the retention time of polymyxin E1.

Identification of peaks  Use the chromatogram supplied with colistin sulfate CRS to identify the peaks due to polymyxins E1, E2, E3, E1-I and E1-7MOA.

Relative retention  With reference to polymyxin E1 (retention time = about 16 min): polymyxin E2 = about 0.45; polymyxin E3 = about 0.5; polymyxin E1-I = about 0.8; polymyxin E1-7MOA = about 1.1.

System suitability  Reference solution (a):

• — resolution: minimum 8.0 between the peaks due to polymyxin E2 and polymyxin E1; minimum 6.0 between the peaks due to polymyxin E2 and polymyxin E1-I; minimum 2.5 between the peaks due to polymyxin E1-I and polymyxin E1; minimum 1.5 between the peaks due to polymyxin E1 and polymyxin E1-7MOA.

Calculate the percentage content of polymyxin E3, of polymyxin E1-I, of polymyxin E1-7MOA, and of the sum of polymyxins E1, E2, E3, E1-I and E1-7MOA, using the following expression:

C E i	=	percentage content of polymyxin Ei;
A E i	=	area of the peak due to polymyxin Ei in the chromatogram obtained with the test solution;
m 1	=	mass of the substance to be examined (dried substance) used to prepare the test solution, in milligrams;
B E i	=	area of the peak due to polymyxin Ei in the chromatogram obtained with reference solution (a);
m2	=	mass of colistin sulfate CRS used to prepare reference solution (a), in milligrams;
D E i	=	assigned percentage content of polymyxin Ei in colistin sulfate CRS.

• — polymyxin E3: maximum 10.0 per cent (dried substance);

• — polymyxin E1-I: maximum 10.0 per cent (dried substance);

• — polymyxin E1-7MOA: maximum 10.0 per cent (dried substance);

• — sum of polymyxins E1, E2, E3, E1-I and E1-7MOA: minimum 77.0 per cent (dried substance).

Liquid chromatography (2.2.29) as described in the test for composition with the following modifications. Use the normalisation procedure.

Injection  Test solution and reference solution (b).

• — any impurity: maximum 4.0 per cent;

• — total: maximum 23.0 per cent;

• — disregard limit: the area of the peak due to polymyxin E1 in the chromatogram obtained with reference solution (b); disregard the peaks due to polymyxins E2, E3, E1-I, E1 and E1-7MOA.

16.0 per cent to 18.0 per cent (dried substance).

Dissolve 0.250 g in 100 mL of water R and adjust to pH 11 with concentrated ammonia R. Add 10.0 mL of 0.1 M barium chloride and about 0.5 mg of phthalein purple R. Titrate with 0.1 M sodium edetate, adding 50 mL of ethanol (96 per cent) R when the colour of the solution begins to change and continuing the titration until the violet-blue colour disappears.

1 mL of 0.1 M barium chloride is equivalent to 9.606 mg of SO₄.

Maximum 3.5 per cent, determined on 1.000 g by drying at 60 °C over diphosphorus pentoxide R at a pressure not exceeding 0.67 kPa for 3 h.

Maximum 1.0 per cent, determined on 1.0 g.

Carry out the microbiological assay of antibiotics (2.7.2).

In an airtight container, protected from light.

Ph Eur