Hydrocortisone and Neomycin Cream

Corticosteroid + Aminoglycoside antibacterial.

Hydrocortisone and Neomycin Cream contains Hydrocortisone and Neomycin Sulphate in a suitable basis.

The cream complies with the requirements stated under Topical Semi-solid Preparations and with the following requirements.

90.0  to 110.0% of the stated amount.

A.  Carry out the method for thin-layer chromatography , Appendix III A, using silica gel GF ₂₅₄ as the coating substance and a mixture of 90  volumes of dichloromethane and 8  volumes of methanol as the mobile phase. Apply separately to the plate 10 µl of each of the following solutions. For solution (1) add 10  ml of hexane saturated with acetonitrile to a quantity of the preparation being examined containing 5  mg of Hydrocortisone and shake for 2  to 3  minutes. Add 10  ml of acetonitrile saturated with hexane , shake for 10  minutes and allow the layers to separate. Centrifuge, filter the acetonitrile layer if necessary, evaporate 5  ml to dryness and dissolve the residue in 5  ml of a mixture of equal volumes of chloroform and ethanol (96%) . Solution (2) contains 0.05% w/v of hydrocortisone BPCRS in a mixture of equal volumes of chloroform and ethanol (96%) . After removal of the plate, allow it to dry in air and spray with alkaline tetrazolium blue solution . The principal spot in the chromatogram obtained with solution (1) corresponds to that in the chromatogram obtained with solution (2).

B.  In the Assay for hydrocortisone the chromatogram obtained with solution (2) shows a peak with the same retention time as the peak due to hydrocortisone in the chromatogram obtained with solution (1).

C.  Carry out the method for thin-layer chromatography , Appendix III A, using a silica gel precoated plate (Merck silica gel 60 plates are suitable) and a mixture of 60  volumes of methanol , 40  volumes of 13.5m ammonia and 20  volumes of chloroform as the mobile phase. Apply separately to the plate 5 µl of each of the following solutions. For solution (1) disperse a quantity containing 7000  IU of Neomycin Sulphate with 10  ml of chloroform , add 5  ml of water, shake, centrifuge and use the clear, upper layer. Solution (2) contains 0.2% w/v of neomycin sulphate EPCRS in water. 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. The principal red spot in the chromatogram obtained with solution (1) corresponds to that in the chromatogram obtained with solution (2).

Carry out the method for thin-layer chromatography , Appendix III A, using silica gel H as the coating substance and a freshly prepared 3.85% w/v solution of ammonium acetate as the mobile phase. Apply separately to the plate 2 µl of each of the following solutions. For solution (1) dissolve a quantity containing 7000  IU of Neomycin Sulphate in 10  ml of chloroform , shake gently with 5  ml of water, centrifuge and use the aqueous layer. Solution (2) contains 0.004% w/v of neamine EPCRS in water. 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% of available chlorine. Dry in a current of cold air until a sprayed area of the plate below the line of application gives at most a very faint blue colour with a drop of a 0.5% w/v solution of potassium iodide in starch mucilage ; avoid prolonged exposure to the cold air. Spray the plate with a 0.5% w/v solution of potassium iodide in starch mucilage . 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, injecting 10 µl of each of the following solutions. For solution (1) 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 sulphate EPCRS 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. For solution (2) shake a quantity containing 3500  IU of Neomycin Sulphate with 10  ml of chloroform , add 5  ml of 0.02m sodium tetraborate , mix, allow to separate and centrifuge the upper layer. Proceed as for solution (1) but using 0.5  ml of the clear supernatant liquid in place of 0.5  ml of the neomycin sulphate solution.

The chromatographic procedure may be carried out using (a) a stainless steel column (20  cm × 4.6  mm) packed with silica gel for chromatography (5 µm) (Nucleosil 100-5 is suitable), (b) as the mobile phase with a flow rate of 1.6  ml per minute a solution prepared by mixing 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 and (c) a detection wavelength of 350  nm. Pass the mobile phase through the column for several hours before starting the analysis. Record the chromatogram for 1.4  times the retention time of the peak due to neomycin B.

The chromatogram obtained with solution (1) 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 (1), should be at least 13,000 theoretical plates per metre.

In the chromatogram obtained with solution (2) the area of the peak corresponding to neomycin C is 3  to 15% of the sum of the areas of the peaks corresponding to neomycin B and neomycin C.

Carry out the method for liquid chromatography , Appendix III D, using the following solutions. Solution (1) contains 0.010% w/v of hydrocortisone BPCRS and 0.018% w/v of fluoxymesterone BPCRS (internal standard) in chloroform . For solution (2) shake together a quantity of the preparation being examined containing 25  mg of Hydrocortisone, several small glass beads and 25  ml of a 0.036% w/v solution of fluoxymesterone BPCRS in chloroform for 15  minutes, add sufficient chloroform to produce 50  ml, mix and centrifuge. Remove any excipient material present at the interface and use the clear supernatant liquid.

The chromatographic procedure may be carried out using (a) a stainless steel column (30  cm × 3.9  mm) packed with silica gel for chromatography (10 µm) (µPorasil is suitable), (b) a mixture of 425  volumes of butyl chloride , 425  volumes of butyl chloride saturated with water, 70  volumes of tetrahydrofuran , 35  volumes of methanol and 30  volumes of glacial acetic acid as the mobile phase with a flow rate of 1  ml per minute and (c) a detection wavelength of 254  nm.

Calculate the content of C₂₁H₃₀O₅ in the cream using the declared content of C₂₁H₃₀O₅ in hydrocortisone BPCRS .

Stir a quantity containing 4200  IU with 15  ml of chloroform until the emulsion is completely broken. Transfer to a separating funnel with 25  ml of phosphate buffer pH 8.0 and 5  ml of chloroform , shake vigorously, allow to separate and reserve the aqueous phase. Extract the chloroform layer with two 25-ml quantities of phosphate buffer pH 8.0 and discard the chloroform layer. Pass nitrogen through the combined aqueous solutions to remove dissolved chloroform and dilute to 100  ml with sterile phosphate buffer pH 8.0 . Dilute 10  ml of the resulting solution to 50  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 amount.

The strength with respect to Neomycin Sulphate is stated as the number of IU (Units) per g.