Detailed Product Description

Definition

Gentamicin Injection is a sterile solution of Gentamicin Sulphate in Water for Injections.

The injection complies with the requirements stated under Parenteral Preparations and with the following requirements.

Identification

A.    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 as the mobile phase the lower layer obtained by shaking together equal volumes of 13.5M ammonia, chloroform and methanol  and allowing to separate. Apply separately to the plate (1) a volume of the injection containing the equivalent of 30 µg of gentamicin and (2) 0.1 mg of gentamicin sulphate EPCRS dissolved in a volume of water  equal to the volume of the injection used. 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) correspond to the three principal spots in the chromatogram obtained with solution (2).

B.    In the test for Composition of gentamicin sulphate, the retention times of the four principal peaks in the chromatogram obtained with solution (2) correspond to those of the four principal peaks in the chromatogram obtained with solution (1).

Acidity

pH, 3.0 to 5.5, Appendix V L.

Composition of gentamicin sulphate

Carry out the method for liquid chromatography, Appendix III D, using the following solutions. For solution (1) add 5 ml of methanol  to 10 ml of a 0.065% w/v solution of gentamicin sulphate EPCRS, 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. 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 injection with water  to contain the equivalent of 0.045% of gentamicin.

The chromatographic procedure may be carried out using (a) a stainless steel column (10 to 12.5 cm × 4.6 to 5 mm) packed with stationary phase C (5 µm) (Hypersil ODS is suitable), (b) 0.025M sodium heptanesulphonate monohydrate in a mixture of 70 volumes of methanol , 25 volumes of water  and 5 volumes of glacial acetic acid as the mobile phase with a flow rate of 1.5 ml per minute and (c) a detection wavelength of 330 nm. When the chromatograms are recorded under the operating conditions described above, in the chromatogram obtained with solution (1) the retention time of component C2 is 10 to 20 minutes and the peaks are well separated with relative retention times of about 0.13 (reagent), 0.27 (component C1), 0.65 (component C1a), 0.85 (component C2a) and 1.00 (component C2).

Adjust the sensitivity and the volume of solution (1) injected so that the height of the peak due to component C1 is about 75% of full-scale deflection. Plot a horizontal baseline on the chromatogram from the horizontal portion of the curve immediately before the reagent peak. Measure the peak height above this baseline for each component. Repeat the procedure with solution (2). The test is not valid unless the resolution factor  between the peaks due to components C2a and C2 is at least 1.3.

From the peak heights in the chromatogram obtained with solution (1) and the proportions of the components declared for gentamicin sulphate EPCRS, calculate the response factors for components C1, C1a, C2a and C2. From these response factors and the peak heights in the chromatogram obtained with solution (2), calculate the proportions of components C1, C1a, C2a and C2 in the injection. 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%.

Bacterial endotoxins

Carry out the test for bacterial endotoxins, Appendix XIV C. Dilute the injection, if necessary, with water BET to give a solution containing the equivalent of 10 mg of gentamicin per ml (solution A). The endotoxin limit concentration of solution A is 16.7 IU per ml. Carry out the test using the maximum valid dilution of solution A calculated from the declared sensitivity of the lysate used in the test.

Assay

Carry out the biological 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 injection, taking each 1000 IU found to be equivalent to 1 mg of gentamicin. 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 content.

Labelling

The strength is stated in terms of the equivalent amount of gentamicin in a suitable dose-volume.