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TOBRAMYCIN

Category Antibacterial.

      Tobramycin contains not less than 900 μg of C18H37N5O9 per mg, calculated on the anhydrous basis.

Description White to off-white powder; hygroscopic.

Solubility Freely soluble in water; very slightly soluble in ethanol; practically insoluble in chloroform and in ether.

Contra-indication; Warning; Precaution; Additional information See under Gentamicin Sulfate, p. 111.

Packaging and storage Tobramycin shall be kept in tightly closed containers and stored at a temperature not exceeding 25º.

Labelling The label on the container states (1) the number of μg of activity per mg; (2) storage condition; (3) parenteral grade.

Identification

      A. The infrared absorption spectrum is concordant with the spectrum obtained from Tobramycin RS (Appendix 2.1) or with the reference spectrum of Tobramycin.

      B. Carry out the test as described in the “Thin-layer Chromatography” (Appendix 3.1), using silica gel G as the coating substance and a mixture of 60 volumes of methanol, 30 volumes of strong ammonia solution and 25 volumes of chloroform as the mobile phase. Apply separately to the plate, 3 μl of each of two solutions containing (A) 6 mg per ml of the test substance and (B) 6 mg per ml of Tobramycin RS and at a third point, apply 3 μl of a mixture of equal volumes of solutions (A) and (B). After removal of the plate, allow to dry in air, and heat at 110º for 15 minutes. Immediately spray with a 1 per cent w/v solution of ninhydrin in a mixture of 100 volumes of 1-butanol and 1 volume of pyridine: the principal pink spot in the chromatogram obtained from solution (A) corresponds to that in the chromatogram obtained from solution (B) and the principal red spot in the third chromatogram appears as a single compact spot.

      C. The retention time of the major peak in the chromatogram of the Derivatized assay preparation corresponds to that of the Derivatized standard preparation, as obtained in the Assay.

pH 9.0 to 11.0, in a 10 per cent w/v solution (Appendix 4.11).

Water Not more than 8.0 per cent w/w (Karl Fischer Method, Appendix 4.12).

Heavy metals Not more than 30 ppm (Method II, Appendix 5.2). Use 0.67 g; for the Standard Preparation, use lead standard solution (1 ppm Pb).

Sulfated ash Not more than 1.0 per cent w/w, the charred residue being moistened with 2 ml of nitric acid and 5 drops of sulfuric acid (Appendix 5.3).

Chromatographic purity Not more than 1.0 per cent Carry out the test as described in the “Thin-layer Chromatography” (Appendix 3.1), using silica gel G as the coating substance and a mixture of 50 volumes of a 29.2 per cent w/v solution of sodium chloride, 30 volumes of ethanol and 20 volumes of water as the mobile phase.

      Dilute sodium hypochlorite solution Dilute 20 ml of sodium hypochlorite TS with water to obtain 100 ml of solution.

      Starch-potassium iodide reagent Dissolve 1.1 g of potassium iodide in 60 ml of water, boil for 15 minutes, and slowly add a suspension of 1.5 g of soluble starch in 10 ml of water. Add 25 ml of water, and boil for 10 minutes. Allow to cool, dilute with water to 100 ml, and mix.

      Test solution Transfer about 50 mg, accurately weighed, of the test substance to a 10-ml volumetric flask, add 7 ml of water to dissolve it, and adjust with 0.5 M sulfuric acid to a pH of 5.5±0.4. Dilute with water to volume and mix.

      Reference solution Dilute 1 ml of Test solution to 10 ml with water.

      Apply separately to the plate 1 μl of each solution. After removal of the plate, evaporate the solvent in a current of hot air, and then heat at 110º for 10 minutes. Lightly spray the hot plate with Dilute sodium hypochlorite solution. Dry the plate in a current of cold air until a sprayed area of the plate below the origin gives at most a faint blue colour with a drop of Starch-potassium iodide reagent. Then spray the plate with Starch-potassium iodide reagent: bluish-purple spots are immediately visible. Any spot obtained from Test solution, other than principal spot, is not more intense than the principal spot obtained from Reference solution.

Assay Carry out the determination as described in the “High-pressure Liquid Chromatography” (Appendix 3.5).

      Mobile phase Dissolve 2.0 g of tris(hydroxymethyl) aminomethane in 800 ml of water. To this solution add 20 ml of 0.5 M sulfuric acid, dilute with acetonitrile to obtain 2000 ml of solution and mix. Allow to cool and filter. Make adjustments if necessary.

      2,4-Dinitrofluorobenzene reagent Prepare a stock solution of 2,4-dinitrofluorobenzene in ethanol containing 10 mg per ml. This solution may be used for 5 days if refrigerated when not in use. 

      Tris(hydroxymethyl)aminomethane reagent Prepare a stock solution of tris(hydroxymethyl)-aminomethane in water containing 15 mg per ml. This stock solution may be used for 1 month if refrigerated when not in use. Transfer 40 ml of this stock solution to a 200-ml volumetric flask, add sufficient dimethyl sulfoxide with mixing, dilute with the same solvent to volume, and mix. Use this reagent within 4 hours. (Note If kept immersed in an ice-water bath below 10º, the reagent may be used for up to 8 hours.)

      Standard preparation Transfer about 55 mg of Tobramycin RS, accurately weighed, to a 50-ml volumetric flask, add 1 ml of 0.5 M sulfuric acid and sufficient water to dissolve it, dilute with water to volume and mix. Transfer 10.0 ml of this solution to a second 50-ml volumetric flask, dilute with water to volume and mix. This solution contains about 220 μg of tobramycin per ml.

      Assay preparation Transfer about 55 mg of Tobramycin, accurately weighed, to a 50-ml volumetric flask, add 1 ml of 0.5 M sulfuric acid and sufficient water to dissolve it, dilute with water to volume and mix. Transfer 10.0 ml of this solution to a second 50-ml volumetric flask, dilute with water to volume and mix.

      Derivatization procedure (Note Heat all solutions at the same temperature and for the same duration of time as indicated. Move all flasks to and from the 60º constant temperature bath at the same time.) To separate 50-ml volumetric flasks transfer 4.0 ml of Standard preparation, 4.0 ml of Assay preparation, and 4.0 ml of water. To each flask add 10 ml of 2,4-Dinitrofluorobenzene reagent and 10 ml of Tris(hydroxymethyl)aminomethane reagent, shake, and insert the stopper. Place the flasks in a constant temperature bath at 60º±2º and heat for 50±5 minutes. Remove the flasks from the bath, and allow to stand for 10 minutes. Add acetonitrile to about 2 ml below the 50-ml mark, allow to cool to room temperature, dilute with acetonitrile to volume, and mix. The solutions thus obtained are Derivatized standard preparation, Derivatized assay preparation, and Blank preparation, respectively.

      Resolution solution Prepare a fresh solution of p-naphtholbenzein in acetonitrile containing about 240 μg per ml. Transfer 2 ml of this solution to a 10-ml volumetric flask, dilute with Derivatized standard preparation to volume, and use promptly.

      Chromatographic system The chromatographic procedure may be carried out using (a) a stainless steel column (30 cm × 3.9 mm) packed with octadecylsilane chemically bonded to porous silica or ceramic microparticles (5 to 10 μm), (b) Mobile phase at a flow rate of about 1.2 ml per minute and (c) an ultraviolet photometer set at 365 nm.

      To determine the suitability of the chromatographic system, chromatograph Blank preparation and record the peak responses as directed under Procedure. Identify the solvent and reagent peaks. Chromatograph Resolution solution, and record the peak responses as directed under Procedure: the relative retention times are about 1.0 for p-naphtholbenzein and 1.7 for tobramycin and the resolution factor between the two peaks is not less than 4.0. Chromatograph Derivatized standard preparation, and record the peak responses as directed under Procedure: the relative standard deviation for replicate injections is not more than 2.0 per cent.

      Procedure Separately inject equal volumes (about 20 μl) of Derivatized standard preparation and Derivatized assay preparation into the chromatograph, record the chromatograms and measure the responses for the major peaks.

      Calculation Calculate the content of C18H37N5O9 in the Tobramycin taken, using the declared content of C18H37N5O9 in Tobramycin RS.

Other requirements Tobramycin intended for parenteral administration complies with the following additional requirements.

      Bacterial endotoxins When tested as described in the “Test for Bacterial Endotoxins” (Appendix 8.5), it contains not more than 2.00 Endotoxin Units per mg of tobramycin.

Sterilitys Complies with the “Sterility Test” (Method I, Appendix 10.1).

 

 

 

MONOGRAPHS • TOBRAMYCIN
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หมายเหตุ / Note : TP II 2011 PAGE 160 - 162