Neomycin Eye Drops

Aminoglycoside antibacterial.

Neomycin Eye Drops are a sterile solution of Neomycin Sulfate in Purified Water.

The eye drops comply with the requirements stated under Eye Preparations and with the following requirements.

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

(1) A volume of the eye drops containing 3.5 IU.

(2) 0.5% w/v of neomycin sulfate BPCRS in water.

(3) Equal volumes of solutions (1) and (2).

(a) Use as the coating silica gel (Merck silica gel 60 plates are suitable).

(b) Use the mobile phase as described below.

(c) Apply all of solution (1) and 1 µL of each of solutions (2) and (3).

(d) Develop the plate to 15 cm.

(e) After removal of the plate, allow it to dry in air, spray with a 1% w/v solution of ninhydrin in butan-1-ol and heat at 105° for 2 minutes.

20 volumes of chloroform, 40 volumes of 13.5m ammonia and 60 volumes of methanol.

The principal red spot in the chromatogram obtained with solution (1) corresponds to that in the chromatogram obtained with solution (2) and the principal red spot in the chromatogram obtained with solution (3) appears as a single spot.

B. Yield the reactions characteristic of sulfates, Appendix VI.

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

(1) A volume of the eye drops containing 3.5 IU.

(2) The same volume of water containing 0.1 µg of neamine EPCRS.

(a) Use as the coating silica gel H.

(b) Use the mobile phase as described below.

(c) Apply all of solutions (1) and (2).

(d) Develop the plate to 15 cm.

(e) After removal of the plate, dry it in a current of warm air, heat at 110° for 10 minutes and spray the hot plate with a solution prepared immediately before use by diluting sodium hypochlorite solution with water to contain 0.5% w/v of available chlorine. Dry in a current of cold air until a sprayed area of the plate below the line of application gives not more than a very faint blue colour with one drop of a 0.5% w/v solution of potassium iodide in starch mucilage; avoid prolonged exposure to cold air. Spray the plate with a 0.5% w/v solution of potassium iodide in starch mucilage.

A freshly prepared 3.85% w/v solution of ammonium acetate.

Any spot corresponding to neamine in the chromatogram obtained with solution (1) is not more intense than the spot in the chromatogram obtained with solution (2).

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

(1) Dilute the eye drops with 0.02m sodium tetraborate to contain 700 IU per mL. To 0.5 mL of this solution add 1.5 mL of a freshly prepared 2% w/v solution of 1-fluoro-2,4-dinitrobenzene in methanol, heat in a water bath at 60° for 1 hour and cool; dilute the solution to 25 mL with the mobile phase, allow to stand and use the clear lower layer.

(2) Add 1.5 mL of a freshly prepared 2% w/v solution of 1-fluoro-2,4-dinitrobenzene in methanol to 0.5 mL of a 0.10% w/v solution of neomycin sulfate BPCRS in 0.02m sodium tetraborate, heat in a water bath at 60° for 1 hour and cool; dilute the solution to 25 mL with the mobile phase, allow to stand and use the clear lower layer.

(a) Use a stainless steel column (20 cm × 4.6 mm) packed with silica gel for chromatography (5 µm) (Nucleosil 100-5 is suitable).

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

(c) Use a flow rate of 1.6 mL per minute.

(d) Use an ambient column temperature.

(e) Use a detection wavelength of 350 nm.

(f) Inject 10 µL of each solution.

(g) Record the chromatograms for 1.4 times the retention time of the peak due to neomycin B.

Mix 97 mL of tetrahydrofuran, 1.0 mL of water and 0.5 mL of glacial acetic acid with sufficient of a 2.0% v/v solution of absolute ethanol in ethanol-free chloroform to produce 250 mL. Pass the mobile phase through the column for several hours before injecting the solutions.

The test is not valid unless:

the chromatogram obtained with solution (2) shows a principal peak due to neomycin B and a major secondary peak due to neomycin C with a retention time relative to neomycin B of about 0.6;

the column efficiency, determined using the peak due to neomycin B in the chromatogram obtained with solution (2), should be not less than 13,000 theoretical plates per metre.

In the chromatogram obtained with solution (1) the area of the peak corresponding to neomycin C is not less than 3% and not more than 15% of the sum of the areas of the peaks corresponding to neomycin B and neomycin C.

Dilute a quantity containing 3500 IU to 50 mL with sterile phosphate buffer pH 8.0; dilute 10 mL of the resulting solution to 100 mL with the same solvent 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 90.0% and the lower fiducial limit of error is not more than 115.0% of the stated number of IU per mL.

Neomycin Eye Drops should be protected from light.

The strength is stated as the number of IU (Units) per mL.