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 of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

Blank solution— Use water.

2,4-Dinitrofluorobenzene reagent and Tris(hydroxymethyl)aminomethane reagent— Proceed as directed in the Assay under Tobramycin.

System suitability stock solution— Dissolve an accurately weighed quantity of USP Tobramycin RS in water, and adjust with 1 N sulfuric acid to a pH of 6.0. Dilute with water to obtain a solution having a known concentration of about 1.1 mg per mL.

System suitability solution 1— Dilute the System suitability stock solution quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 0.22 mg per mL.

System suitability solution 2— Heat a portion of the System suitability stock solution in a suitable sealed glass container at 100 for 8 to 9 hours. Cool to room temperature, and dilute with water to obtain a solution having a known concentration of about 0.22 mg per mL.

Standard solution— Prepare a solution of about 55 mg of USP Tobramycin RS, accurately weighed, in a 50-mL volumetric flask. Dissolve in water, add 1.0 mL of 1.0 N sulfuric acid, dilute with water to volume, and mix. Dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a concentration of 1.10 µg of tobramycin per mL.

Test solution— Transfer an accurately measured volume of Inhalation Solution, equivalent to about 240 mg of tobramycin, to a 50-mL volumetric flask, dilute with water to volume, and mix. Dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a concentration of 192 µg 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 60 constant-temperature bath at the same time.] To separate 50-mL flasks transfer 15.0 mL of System suitability solution 1, 15.0 mL of System suitability solution 2, 15.0 mL of Standard solution,15.0 mL of Test solution, and 15.0 mL of Blank solution. 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 60 ± 2, 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. Allow the solutions to stand for 16 hours. The solutions thus obtained are Derivatized system suitability solution 1, Derivatized system suitability solution 2, the Derivatized standard solution, the Derivatized test solution, and the Derivatized blank solution.

Chromatographic system (see Chromatography 621)— 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 1.2 mL per minute. The chromatograph is programmed as follows. Chromatograph Derivatized system suitability solution 2, and record the peak responses as directed for Procedure: the capacity factor, k¢, determined from tobramycin is not less than 15.5. Chromatograph Derivatized system suitability solution 1, and use the chromatogram to locate the degradation peaks from comparison to Derivatized system suitability solution 2 (deoxystreptamine kanosaminide and nebramine will increase in response in Derivatized system suitability solution 2 when viewed at a 0–10 mAbs unit or 0–5 mV unit full scale). Record the peak responses as directed for Procedure: the relative retention times are about 0.36 for an impurity, 0.66 for deoxystreptamine kanosaminide, 0.94 for nebramine, 0.96 for kanamycin B, and 1.00 for tobramycin. The resolution, R, between the nebramine and kanamycin peaks is not less than 1.0. The relative standard deviation for replicate injections of the Derivatized standard solution is not more than 2.0%.

Procedure— Separately inject equal volumes (about 45 µL) of Derivatized system suitability solution 1, Derivatized system suitability solution 2, the Derivatized standard solution, the Derivatized test solution, and the Derivatized blank solution, record the chromatograms, and measure the peak responses, disregarding any peak corresponding to those obtained from the Derivatized blank solution, and subtracting the quantities of any such peaks found at the relative retention times of 0.36, 0.66, and 0.94 from those found in the Derivatized test solution. For unknown peak determinations, disregard any peaks found in the chromatogram of the Derivatized test solution that correspond to those in the chromatogram of Derivatized system suitability solution 1. Calculate the percentage of each impurity in relation to the tobramycin content of the Inhalation Solution taken by the formula: in which ri is the peak area of any impurity obtained from the Derivatized test solution; and rS is the peak area for tobramycin obtained from the Derivatized standard solution: not more than 0.25% of the impurity noted at a relative retention time of 0.36 is 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.

(Official January 1, 2007)

Mobile phase, 2,4-Dinitrofluorobenzene reagent, Tris(hydroxymethyl)aminomethane reagent, Standard preparation, Derivatization procedure, Resolution solution, and Chromatographic system— Proceed as directed in the Assay under Tobramycin.

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 192 µg of tobramycin per mL.

Procedure— Proceed as directed in the Assay under Tobramycin. Calculate the quantity, in mg, of tobramycin (C18H37N5O9) in each mL of Inhalation Solution taken by the formula: in which C, E, rU , and rS are as defined therein; L is the labeled quantity, in mg, of tobramycin per mL in the Inhalation Solution taken; and D is the concentration, in µg per mL, of tobramycin in the Assay preparation.