Edition: BP 2025 (Ph. Eur. 11.6 update)
General Notices
Action and use
Aminoglycoside antibacterial.
DEFINITION
Gentamicin Ointment is a dispersion of Gentamicin Sulfate in microfine powder in White Soft Paraffin or other suitable anhydrous greasy basis.
The ointment complies with the requirements stated under Topical Semi-solid Preparations and with the following requirements.
IDENTIFICATION
A. Carry out the method for thin-layer chromatography, Appendix III A, using the following solutions.
(1) Disperse a quantity of the ointment containing the equivalent of 7.5 mg of gentamicin in 20 mL of chloroform, extract with 10 mL of water and use the aqueous layer.
(2) A solution of gentamicin sulfate BPCRS in water containing the equivalent of 0.075% w/v of gentamicin.
CHROMATOGRAPHIC CONDITIONS
(a) Use a silica gel precoated plate (Merck silica gel 60 plates are suitable).
(b) Use the mobile phase as described below.
(c) Apply 20 μL of each solution.
(d) Develop the plate to 15 cm.
(e) After removal of the plate, allow it to dry in air, spray with ninhydrin solution R1 and heat at 105° for 2 minutes.
MOBILE PHASE
The lower layer obtained by shaking together equal volumes of 13.5M ammonia, chloroform and methanol and allowing to separate.
CONFIRMATION
The three principal spots in the chromatogram obtained with solution (1) correspond to the three principal spots in the chromatogram obtained with solution (2).
B. In the test for Composition of gentamicin sulfate, the retention times of the four principal peaks in the chromatogram obtained with solution (1) correspond to those of the four principal peaks in the chromatogram obtained with solution (2).
TESTS
Composition of gentamicin sulfate
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
(1) Disperse a quantity of the ointment containing the equivalent of 20 mg of gentamicin in 10 mL of chloroform, add 20 mL of a 0.25% w/v solution of sodium tetraborate, shake vigorously, centrifuge and separate the aqueous layer. Filter and dilute to 50 mL with water. To 10 mL add 5 mL of methanol, swirl, 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 to be used immediately, cool to 0° and use within 4 hours
(2) Prepare in the same manner as solution (1) but using 10 mL of a 0.065% w/v solution of gentamicin sulfate BPCRS place of the preparation being examined and beginning at the words ‘To 10 mL add …’.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (12.5 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatograph (5 μm) (Hypersil ODS and Kromasil C18 are suitable).
(b) Use isocratic elution and the mobile phase described below.
(c) Use a flow rate of 1.5 mL per minute.
(d) Use an ambient column temperature.
(e) Use a detection wavelength of 330 nm.
(f) Inject 20 μL of each solution.
MOBILE PHASE
0.025M sodium heptanesulfonate monohydrate in a mixture of 5 volumes of glacial acetic acid, 25 volumes of water and 7 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).
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (2), the resolution factor between the peaks due t components C2a and C2 is at least 1.3.
LIMITS
Using the chromatogram obtained with solution (1) calculate the percentage content of components C1, C1a, C2 and C2a in the ointment 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%.
ASSAY
Dissolve as completely as possible a quantity of the ointment containing the equivalent of 4 mg of gentamicin in 50 mL of chloroform, extract with three 20-mL quantities of phosphate buffer pH 8.0 and dilute the combined extracts to 100 mL wit phosphate buffer pH 8.0. Dilute 10 mL of the resulting solution to 50 mL with phosphate buffer pH 8.0. 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 ointment, taking each 1000 IU found to be equivalent to 1 mg of gentamicin. Thupper 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.
LABELLING
The quantity of active ingredient is stated in terms of the equivalent amount of gentamicin.
