Colistin Tablets

Antibacterial.

Colistin Tablets contain Colistin Sulfate.

The tablets comply with the requirements stated under Tablets and with the following requirements

To a quantity of the powdered tablets containing 200,000 IU add 10 mL of water, shake and filter. Use the filtrate for the following tests.

A. Carry out the method for thin-layer chromatography, Appendix III A, protected from light using the following solutions.

(1) Add 0.5 mL of hydrochloric acid to 0.5 mL of the filtrate, heat in a sealed tube at 135° for 5 hours, evaporate to dryness on a water bath, continue to heat until any residual hydrogen chloride has been removed, dissolve the residue in 0.5 mL of water and centrifuge, if necessary.

(2) 0.25% w/v of l-leucine in water.

(3) 0.25% w/v of l-threonine in water.

(4) 0.25% w/v of l-phenylalanine in water.

(5) 0.25% w/v of l-serine in water.

(a) Use as the coating silica gel.

(b) Use the mobile phase described below.

(c) Apply 5 µL of each solution, as 10-mm bands. Place the plate in the tank so that it is not in contact with the mobile phase and expose it to the vapour of the mobile phase.

(d) After exposure of the plate to the mobile phase vapour for at least 12 hours, develop to 12 cm.

(e) Remove the plate, heat it at 100° to 105°, spray with ninhydrin solution R1 and heat at 110° for 5 minutes.

25 volumes of water and 75 volumes of phenol.

The bands in the chromatogram obtained with solution (1) correspond to those in the chromatograms obtained with solutions (2) and (3) and do not correspond to those in the chromatograms obtained with solutions (4) and (5). The chromatogram obtained with solution (1) also shows a band with a very low Rf value (2,4-diaminobutanoic acid).

B. Heat 0.5 mL of the filtrate with 0.5 mL of chromotropic acid–sulfuric acid solution at 100° for 30 minutes. No purple colour is produced (distinction from colistin sulfomethate).

C. The filtrate yields reaction A characteristic of sulfates, Appendix VI.

Carry out the method for liquid chromatography, Appendix III D, using the following solutions.

(1) Shake a quantity of the powdered tablets containing 500,000 IU with 40 mL of water for 20 minutes, add sufficient acetonitrile to produce 50 mL and filter through a Whatman GF/C filter and then though a 0.45-µm nylon filter.

(2) Dissolve 25 mg of colistin sulfate EPCRS in 40 mL of water and add sufficient acetonitrile to produce 50 mL.

(a) Use a stainless steel column (15 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatography (3.5 µm) (SunFire C18 and Symmetry C18 are suitable).

(b) Use isocratic elution and the mobile phase described below.

(c) Use a flow rate of 1 mL per minute.

(d) Use a column temperature of 30º.

(e) Use a detection wavelength of 215 nm.

(f) Inject 20 µL of each solution.

(g) For solution (1) allow the chromatography to proceed for 1.5 times the retention time of polymyxin E1.

22 volumes of acetonitrile and 78 volumes of a solution prepared by dissolving 4.46 g of anhydrous sodium sulfate in 900 mL of water, adjusting the pH to 2.4 with dilute orthophosphoric acid and adding sufficient water to produce 1000 mL.

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

When the chromatograms are recorded under the prescribed conditions the retention time of polymyxin E1 is about 16 minutes. The retention times relative to polymyxin E1 are: polymyxin E2, about 0.45; polymyxin E3, about 0.5; polymyxin E1-I, about 0.8; polymyxin E1-7MOA, about 1.1.

The test is not valid unless, in the chromatogram obtained with solution (2):

the resolution factor between the peaks due to polymyxins E2 and E1 is at least 8.0;

the resolution factor between the peaks due to polymyxins E2 and E1-I is at least 6.0;

the resolution factor between the peaks due to polymyxins E1-I and E1 is at least 2.5;

the resolution factor between the peaks due to polymyxins E1 and E1-7MOA is at least 1.5.

Calculate the content of polymyxin E3, polymyxin E1-I and polymyxin E1-7MOA and the sum of the contents of polymyxins E1, E2, E3, E1-I and E1-7MOA using the declared contents of polymyxins E1, E2, E3, E1-I and E1-7MOA in colistin sulfate EPCRS.

The content of polymyxin E1-I is not more than 10.0%;

the content of polymyxin E1-7MOA is not more than 10.0%;

the content of polymyxin E3 is not more than 10.0%;

the sum of the contents of polymyxin E1, polymyxin E2, polymyxin E3, polymyxin E1-I and polymyxin E1-7MOA is not less than 77.0%.

Carry out the method for liquid chromatography, Appendix III D, using the following solutions.

(1) Shake a quantity of the powdered tablets containing 500,000 IU with 40 mL of water for 20 minutes, add sufficient acetonitrile to produce 50 mL and filter through a Whatman GF/C filter and then though a 0.45-µm nylon filter.

(2) Dissolve 25 mg of colistin sulfate EPCRS in 40 mL of water and add sufficient acetonitrile to produce 50 mL. Dilute 1 volume of the resulting solution to 100 volumes with a mixture of 20 volumes of acetonitrile and 80 volumes of water.

The chromatographic conditions described under Composition may be used.

In the chromatogram obtained with solution (1):

the area of any secondary peak is not greater than 4.0% by normalisation;

the sum of the areas of all secondary peaks is not greater than 23.0% by normalisation.

Disregard any peak with an area less than the area of the peak due to polymyxin E1 in the chromatogram obtained with solution (2) (1%) and any peaks due to polymyxins E1, E2, E3, E1-I and E1-7MOA.

Weigh and powder 20 tablets. Dissolve a suitable quantity of the powder in phosphate buffer pH 6.0 and carry out the microbiological assay of antibiotics, Appendix XIV A. The precision of the assay is such that the fiducial limits of error are not less than 95% and not more than 105% of the estimated potency. The upper fiducial limit of error is not less than 97.0% and the lower fiducial limit of error is not more than 110.0% of the stated number of IU.

Colistin Tablets should be protected from light.

The strength is stated as the number of IU (units) in each tablet.