- British Pharmacopoeia Volume III
- Formulated Preparations: Specific Monographs
Gentamicin and Hydrocortisone Acetate Ear Drops |
Aminoglycoside antibacterial (Gentamicin Sulphate); corticosteroid (Hydrocortisone Acetate).
Gentamicin and Hydrocortisone Acetate Ear Drops contain Gentamicin Sulphate and Hydrocortisone Acetate suspended in a suitable vehicle.
The ear drops comply with the requirements stated under Ear Preparations and with the following requirements.
95.0 to 105.0% of the stated amount.
A. Carry out the method for thin-layer chromatography, Appendix III A, using the following solutions.
(1) Filter a volume of the ear drops diluted in water, if necessary, to produce a solution containing the equivalent of 0.3% w/v of gentamicin and use the filtrate.
(2) 0.5% w/v of gentamicin sulphate BPCRS in water.
(a) Use a silica gel precoated plate (Merck silica gel 60 plates are suitable).
(b) Use as the mobile phase the lower layer obtained by shaking together equal volumes of 13.5m ammonia , dichloromethane and methanol and allowing to separate.
(c) Develop to 15 cm.
(d) Application volume of 10 µl for each solution.
(e) After removal of the plate, allow it to dry in air, spray with ninhydrin solution R1 and heat at 105° for 2 minutes.
The three principal spots in the chromatogram obtained with solution (1) are similar in position, colour and size to those in the chromatogram obtained with solution (2).
B. Comply with the test for Composition of gentamicin sulphate.
C. Filter 20 ml of the well-mixed ear drops (Whatman No. 1 filter paper is suitable), wash the residue with the minimum amount of water, dry at 105° and dissolve 50 mg of the residue obtained in 50 ml of ethanol (96%) . Add 2 ml of sulphuric acid to 2 ml of the resulting solution; an intense yellow colour is produced which exhibits a green fluorescence which is particularly intense when viewed under ultraviolet light (365 nm) . Add the solution to 10 ml of water and mix; the fluorescence under ultraviolet light (365 nm) does not disappear.
D. In the Assay for hydrocortisone acetate, the chromatogram obtained with solution (1) shows a peak with the same retention time as the principal peak in the chromatogram obtained with solution (2).
pH, 6.0 to 7.0, Appendix V L.
Carry out the method for liquid chromatography , Appendix III D, using the following solutions.
(1) Add 5 ml of methanol to 10 ml of a 0.065% w/v solution of gentamicin sulphate BPCRS , swirl and add 4 ml of phthalaldehyde reagent , mix, add sufficient methanol to produce 25 ml, heat in a water bath at 60° for 15 minutes and cool. If the solution is not used immediately, cool to 0° and use within 4 hours.
(2) Prepare solution (2) in the same manner as solution (1) but using 10 ml of a solution prepared by diluting a suitable volume of the ear drops with water to contain the equivalent of 0.045% w/v of gentamicin.
chromatographic conditions
(a) Use a stainless steel column (12.5 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatography (5 µm) (Hypersil ODS is suitable).
(b) Use isocratic elution and the mobile phase described below.
(c) Use a flow rate of 1.5 ml per minute.
(d) Use ambient column temperature.
(e) Use a detection wavelength of 330 nm.
(f) Inject 5 µl of each solution.
0.025m sodium heptanesulphonate monohydrate in a mixture of 5 volumes of glacial acetic acid , 25 volumes of water and 70 volumes of methanol .
When the chromatograms are recorded under the prescribed conditions the retention time of component C2 is 10 to 20 minutes. The retention times relative to component C2 are: about 0.13 (reagent); about 0.27 (component C1); about 0.65 (component C1a); about 0.85 (component C2a).
The test is not valid unless, in the chromatogram obtained with solution (1), the resolution factor between the peaks due to components C2a and C2 is at least 1.3.
Using the chromatogram obtained with solution (2) calculate the percentage content of components C1, C1a, C2 and C2a in the ear drops by normalisation . The proportions are within the following limits:
C1, 25.0 to 50.0%;
C1a, 10.0 to 35.0%;
C2 plus C2a, 25.0 to 55.0%.
Dilute a volume of the ear drops containing the equivalent of 15 mg of gentamicin to 50 ml with sterile phosphate buffer pH 8.0 and dilute 10 ml of the resulting solution to 50 ml with the same solvent. 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.
Calculate the content of gentamicin in the ear drops, taking each 1000 IU found to be equivalent to 1 mg of gentamicin. The upper fiducial limit of error is not less than 90.0% and the lower fiducial limit of error is not more than 120.0% of the stated content.
Carry out the method for liquid chromatography , Appendix III D, using the following solutions.
(1) Shake a quantity of the ear drops containing 0.1 g of Hydrocortisone Acetate with 25 ml of methanol , add sufficient methanol to produce 100 ml and dilute 10 ml of this solution to 50 ml with mobile phase.
(2) Dilute 10 ml of a 0.1% w/v solution of hydrocortisone acetate BPCRS in methanol to 50 ml with the mobile phase.
(a) Use a stainless steel column (12.5 cm × 4.6 mm) packed with octadecylsilyl silica gel for chromatography (5 µm) (Spherisorb ODS 1 is suitable).
(b) Use isocratic elution and the mobile phase described below.
(c) Use a flow rate of 2 ml per minute.
(d) Use ambient column temperature.
(e) Use a detection wavelength of 256 nm.
(f) Inject 20 µl of each solution.
A mixture of 400 volumes of water and 600 volumes of methanol.
Calculate the content of C23H32O6 in the ear drops using the declared content of C23H32O6 in hydrocortisone acetate BPCRS .
Gentamicin and Hydrocortisone Acetate Ear Drops should not be allowed to freeze.
The quantity of Gentamicin Sulphate is stated in terms of the equivalent amount of gentamicin.