Tobramycin Inhalation Solution »Tobramycin Inhalation Solution is a sterile,nonpyrogenic,preservative-free solution of Tobramycin in Water for Injection containing Sodium Chloride.It is prepared with the aid of Sulfuric Acid or Sodium Hydroxide and contains,in each mL,not less than 90.0percent and not more than 110.0percent of the labeled amount of tobramycin (C18H37N5O9). Packaging and storage— Preserve in low-density,polyethylene,single-use ampules stored in light-resistant foil over-wrapped packaging,in a refrigerator. USP Reference standards á11ñ USP Endotoxin RS.USP Tobramycin RS. Absorbance— The absorbance of the Inhalation Solution determined at 410nm in a 1-cm cell is not more than 0.24. Bacterial endotoxins á85ñ It contains not more than 60USP Endotoxin Units per mL. Sterility á71ñ It meets the requirements when tested as directed forMembrane FiltrationunderTest for Sterility of the Product to be Examined. Uniformity of dosage units á905ñ: meets the requirements. pHá791ñ: between 5.5and 6.5. Particulate matter á788ñ: meets the requirements for small-volume injections. Osmolarity á785ñ: the osmolality is between 135and 200mOsmol per kg. Chromatographic purity— Solution A— Prepare a filtered and degassed mixture of water,acetonitrile,and phosphoric acid (95:5:0.08). Solution B— Prepare a filtered and degassed mixture of acetonitrile,water,and phosphoric acid (75:25:0.08). Mobile phase— Use variable mixtures ofSolution AandSolution Bas directed forChromatographic system.Make adjustments if necessary (seeSystem Suitability underChromatography á621ñ). Blank solution— Use water. 2,4-Dinitrofluorobenzene reagentand Tris(hydroxymethyl)aminomethane reagent— Proceed as directed in theAssay underTobramycin. System suitability stock solution— Dissolve an accurately weighed quantity of USP Tobramycin RSin water,and adjust with 1Nsulfuric acid to a pHof 6.0.Dilute with water to obtain a solution having a known concentration of about 1.1mg per mL. System suitability solution 1— Dilute theSystem suitability stock solution quantitatively,and stepwise if necessary,with water to obtain a solution having a known concentration of about 0.22mg per mL. System suitability solution 2— Heat a portion of theSystem suitability stock solution in a suitable sealed glass container at 100for 8to 9hours.Cool to room temperature,and dilute with water to obtain a solution having a known concentration of about 0.22mg per mL. Standard solution— Prepare a solution of about 55mg of USP Tobramycin RS,accurately weighed,in a 50-mLvolumetric flask.Dissolve in water,add 1.0mLof 1.0Nsulfuric acid,dilute with water to volume,and mix.Dilute quantitatively,and stepwise if necessary,with water to obtain a solution having a concentration of 1.10µg of tobramycin per mL. Test solution— Transfer an accurately measured volume of Inhalation Solution,equivalent to about 240mg of tobramycin,to a 50-mLvolumetric flask,dilute with water to volume,and mix.Dilute quantitatively,and stepwise if necessary,with water to obtain a solution having a concentration of 192µg of tobramycin per mL. Derivatization procedure— [NOTE—Heat all solutions at the same temperature and for the same duration as indicated.Move all flasks to and from the 60constant-temperature bath at the same time.]To separate 50-mLflasks transfer 15.0mLofSystem suitability solution 1,15.0mLofSystem suitability solution 2,15.0mLofStandard solution,15.0mLofTest solution,and 15.0mLofBlank solution.To each flask,add 10mLof2,4-Dinitrofluorobenzene reagent and 10mLofTris(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,dilute with acetonitrile to volume,and mix.Allow the solutions to stand for 16hours.The solutions thus obtained areDerivatized system suitability solution 1,Derivatized system suitability solution 2,theDerivatized standard solution,theDerivatized test solution,and theDerivatized blank solution. Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 365-nm detector and a 4.6-mm ×25-cm column that contains packing L11.The flow rate is about 1.2mLper minute.The chromatograph is programmed as follows. Time
(minutes) Solution A
(%) Solution B
(%) Elution 0 79 21 equilibration 0–14 79®66 21®34 linear gradient 14–25 66®30 34®70 linear gradient 25–35 30 70 isocratic 35–40 30®20 70®80 linear gradient 40–50 20®5 80®95 linear gradient ChromatographDerivatized system suitability solution 2,and record the peak responses as directed forProcedure:the capacity factor,k¢,determined from tobramycin is not less than 15.5.ChromatographDerivatized system suitability solution 1,and use the chromatogram to locate the degradation peaks from comparison toDerivatized system suitability solution 2(deoxystreptamine kanosaminide and nebramine will increase in response inDerivatized system suitability solution 2when viewed at a 0–10mAbs unit or 0–5mVunit full scale).Record the peak responses as directed forProcedure:the relative retention times are about 0.36for an impurity,0.66for deoxystreptamine kanosaminide,0.94for nebramine,0.96for kanamycin B,and 1.00for tobramycin.The resolution,R,between the nebramine and kanamycin peaks is not less than 1.0.The relative standard deviation for replicate injections of theDerivatized standard solution is not more than 2.0%. Procedure— Separately inject equal volumes (about 45µL)ofDerivatized system suitability solution 1,Derivatized system suitability solution 2,theDerivatized standard solution,theDerivatized test solution,and theDerivatized blank solution,record the chromatograms,and measure the peak responses,disregarding any peak corresponding to those obtained from theDerivatized blank solution,and subtracting the quantities of any such peaks found at the relative retention times of 0.36,0.66,and 0.94from those found in theDerivatized test solution.For unknown peak determinations,disregard any peaks found in the chromatogram of theDerivatized test solution that correspond to those in the chromatogram ofDerivatized system suitability solution 1.Calculate the percentage of each impurity in relation to the tobramycin content of the Inhalation Solution taken by the formula: (110/192)(ri/rS), in which riis the peak area of any impurity obtained from theDerivatized test solution;andrSis the peak area for tobramycin obtained from theDerivatized standard solution:not more than 0.25%of the impurity noted at a relative retention time of 0.36is found;not more than 0.3%of deoxystreptamine kanosaminide is found;not more than 0.4%of nebramine is found;not more than 0.1%of any unknown impurity is found;not more than 0.2%of total unknown impurities is found;and not more than 1.0%of total impurities is found. Content of sodium chloride— Pipet 25mLof Inhalation Solution into a suitable container.Add between 70and 100mLof water.Add 10mLof an acidic gelatin solution,prepared by dissolving 2g of gelatin and 50mLof nitric acid in 1000mLof water.Titrate potentiometrically with 0.1Nsilver nitrate VSusing a suitable silver electrode:not less than 90.0%and not more than 110.0%of the labeled amount of sodium chloride is found. Other requirements— It meets the requirements for theIdentification tests underTobramycin. Assay— Mobile phase,2,4-Dinitrofluorobenzene reagent,Tris(hydroxymethyl)aminomethane reagent,Standard preparation,Derivatization procedure,Resolution solution,and Chromatographic system— Proceed as directed in theAssay underTobramycin. Assay preparation— Transfer an accurately measured volume of Inhalation Solution to a suitable volumetric flask,and quantitatively dilute with water to obtain a solution having a concentration of about 192µg of tobramycin per mL. Procedure— Proceed as directed in theAssay underTobramycin.Calculate the quantity,in mg,of tobramycin (C18H37N5O9)in each mLof Inhalation Solution taken by the formula: (CE)(L/D)(rU/rS), in whichC,E,rU,andrSare as defined therein;Lis the labeled quantity,in mg,of tobramycin per mLin the Inhalation Solution taken;andDis the concentration,in µg per mL,of tobramycin in theAssay preparation. Auxiliary Information— Staff Liaison:William W.Wright,Ph.D.,Scientific Fellow Expert Committee:(PA7)Pharmaceutical Analysis 7 USP28–NF23Page 1940 Pharmacopeial Forum:Volume No.29(2)Page 438 Phone Number:1-301-816-8335