Tobramycin, chemical structure, molecular formula, Reference Standards
»Tobramycin has a potency of not less than 900µg of C18H37N5O9per mg,calculated on the anhydrous basis.
Packaging and storage
Preserve in tight containers.
Where it is intended for use in preparing injectable or ophthalmic dosage forms,the label states that it is sterile or must be subjected to further processing during the preparation of injectable or ophthalmic dosage forms.
USP Reference standards á11ñ
USP Endotoxin RS.USP Tobramycin RS.
Prepare a solution of it in water containing 6mg per mL.Apply 3µLof this test solution,3µLof a Standard solution of USP Tobramycin RScontaining 6mg per mL,and 3µLof a mixture of equal volumes of the two solutions to a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Place the plate in a suitable chromatographic chamber,and develop the chromatogram in a solvent system consisting of a mixture of methanol,ammonium hydroxide,and chloroform (60:30:25)until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the chamber,allow the solvent to evaporate,and heat the plate at 110for 15minutes.Immediately locate the spots on the plate by spraying with a 1in 100solution of ninhydrin in a mixture of butyl alcohol and pyridine (100:1):tobramycin appears as a pink spot,and the spots obtained from the test solution and from the mixture of test solution and Standard solution,respectively,correspond in distance from the origin to that obtained from the Standard solution.
The chromatogram of the Derivatized assay preparationobtained as directed in the Assayexhibits a major peak for tobramycin,the retention time of which corresponds to that exhibited in the chromatogram of the Derivatized standard preparationobtained as directed in the Assay.
between 9and 11,in a solution (1in 10).
not more than 8.0%.
Residue on ignition á281ñ:
not more than 1.0%,the charred residue being moistened with 2mLof nitric acid and 5drops of sulfuric acid.
Heavy metals,Method IIá231ñ:
Diluted sodium hypochlorite solution
Dilute 20mLof sodium hypochlorite solution with water to obtain 100mLof solution.
Starch-potassium iodide reagent
Dissolve 1.1g of potassium iodide in 60mLof water,boil for 15minutes,and slowly add a suspension of 1.5g of soluble starch in 10mLof water.Add 25mLof water,and boil for 10minutes.Allow to cool,dilute with water to 100mL,and mix.
Transfer 50mg of Tobramycin to a 10-mLvolumetric flask,add 7mLof water to dissolve it,and adjust with 1Nsulfuric acid to a pHof 5.5±0.4.Dilute with water to volume,and mix to obtain the test solution.Prepare a standard solution by diluting the test solution quantitatively with water to obtain a solution containing 0.05mg per mL.Separately apply 1µLof these solutions to a thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel,and allow to dry.Develop the chromatogram in a saturated chromatographic chamber containing a mixture of sodium chloride solution (29.2in 100),alcohol,and water (50:30:20)until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the chromatographic chamber,evaporate the solvent in a current of hot air,then heat it at 110for 10minutes.Lightly spray the hot plate with Diluted 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 color with a drop of Starch-potassium iodide reagent.Then spray the plate with Starch-potassium iodide reagent:bluish-purple spots are immediately visible.Other than the principal tobramycin spot,no spot observed in the chromatogram obtained from the test solution is more intense than the principal spot obtained from the standard solution (1.0%).
Where the label states that Tobramycin is sterile,it meets the requirements for Sterilityand Bacterial endotoxinsunder Tobramycin for Injection.Where the label states that Tobramycin must be subjected to further processing during the preparation of injectable dosage forms,it meets the requirements for Bacterial endotoxinsunder Tobramycin for Injection.Where it is intended for use in preparing ophthalmic dosage forms,it is exempt from the requirements for Bacterial endotoxins.
Dissolve 2.0g of tris(hydroxymethyl)aminomethane in about 800mLof water.To this solution add 20mLof 1Nsulfuric acid,dilute with acetonitrile to obtain 2000mLof solution,and mix.Allow to cool,and pass through a filter of 0.2-µm or finer porosity.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).
,4-Dinitrofluorobenzene reagentPrepare a solution of 2,4-dinitrofluorobenzene in alcohol containing 10mg per mL.This solution may be used for 5days if refrigerated when not in use.
(hydroxymethyl)aminomethane reagentPrepare a stock solution of tris(hydroxymethyl)aminomethane in water containing 15mg per mL.This stock solution may be used for 1month if refrigerated when not in use.Transfer 40mLof this stock solution to a 200-mLvolumetric flask,add dimethyl sulfoxide with mixing,dilute with dimethyl sulfoxide to volume,and mix.Use this reagent within 4hours.[NOTEIf kept immersed in an ice-water bath below 10,the reagent may be used for up to 8hours.]
Transfer about 55mg of USP Tobramycin RS,accurately weighed,to a 50-mLvolumetric flask,add 1mLof 1Nsulfuric acid and enough water to dissolve it,dilute with water to volume,and mix.Transfer 10.0mLof this solution to a second 50-mLvolumetric flask,dilute with water to volume,and mix.This solution contains about 0.22mg of USP Tobramycin RSper mL.
Transfer about 55mg of Tobramycin,accurately weighed,to a 50-mLvolumetric flask,add 1mLof 1Nsulfuric acid and enough water to dissolve it,dilute with water to volume,and mix.Transfer 10.0mLof this solution to a second 50-mLvolumetric flask,dilute with water to volume,and mix.
[NOTEHeat all solutions at the same temperature and for the same duration of time as indicated.Move all flasks to and from the 60constant temperature bath at the same time.]To separate 50-mLvolumetric flasks transfer 4.0mLof the Standard preparation,4.0mLof the Assay preparation,and 4.0mLof water.To each flask add 10mLof 2,4-Dinitrofluorobenzene reagentand 10mLof Tris(hydroxymethyl)aminomethane reagent,shake,and insert the stopper.Place the flasks in a constant temperature bath at 60±2,and heat for 50±5minutes.Remove the flasks from the bath,and allow to stand for 10minutes.Add acetonitrile to about 2mLbelow the 50-mLmark,allow to cool to room temperature,then dilute with acetonitrile to volume,and mix.The solutions thus obtained are the Derivatized standard preparation,the Derivatized assay preparation,and the Blank preparation,respectively.
Prepare a fresh solution of p-naphtholbenzein in acetonitrile containing about 0.24mg per mL.Transfer 2mLof this solution to a 10-mLvolumetric flask,dilute with Derivatized standard preparationto volume,and use promptly.
(see Chromatography á621ñ)The liquid chromatograph is equipped with a 365-nm detector and a 3.9-mm ×30-cm column containing packing L1.The flow rate is about 1.2mLper minute.Chromatograph the Blank preparation,and record the responses as directed for Procedure.Identify the solvent and reagent peaks.Chromatograph the Resolution solution,and record the responses as directed for Procedure:the relative retention times are about 0.6for p-naphtholbenzein and 1.0for tobramycin,and the resolution,R,between the two peaks is not less than 4.0.Chromatograph the Derivatized standard preparation,and record the responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 2.0%.
Separately inject equal volumes (about 20µL)of the Derivatized standard preparationand the Derivatized assay preparationinto the chromatograph,record the chromatograms,and measure the area responses for the major peaks.Calculate the quantity,in µg,of C18H37N5O9in each mg of the Tobramycin taken by the formula:
in which Cis the concentration,in mg per mL,of USP Tobramycin RSin the Standard preparation;Eis the tobramycin equivalent,in µg per mg,of USP Tobramycin RS;Wis the weight,in mg,of the portion of Tobramycin taken;and rUand rSare the tobramycin peak area responses obtained from the Derivatized assay preparationand the Derivatized standard preparation,respectively.
Staff Liaison:William W.Wright,Ph.D.,Scientific Fellow
Expert Committee:(PA7)Pharmaceutical Analysis 7
信息录入：awin 责任编辑：awin 【发表评论