Tobramycin Inhalation Solution

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

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 milliliter, NLT 90.0% and NMT 110.0% of the labeled amount of tobramycin (C₁₈H₃₇N₅O₉).

2 IDENTIFICATION

A. The retention time of the major peak of the Derivatized sample solution corresponds to that of the Derivatized standard solution, as obtained in the Assay.

3 ASSAY

PROCEDURE

Mobile phase: Dissolve 2.0 g of tris(hydroxymethyl)aminomethane in about 800 mL of water. To this solution add 20 mL of 1 N sulfuric acid, and dilute with acetonitrile to obtain 2000 mL of solution. Allow to cool, and pass through a filter of 0.2-µm or finer pore size.

Blank: Water

2,4-Dinitrofluorobenzene reagent: 10 mg/mL of 2,4-dinitrofluorobenzene in alcohol. This solution may be used for 5 days if refrigerated when not in use.

Tris(hydroxymethyl)aminomethane stock reagent: 15 mg/mL of tris(hydroxymethyl)aminomethane in water. This reagent may be used for 1 month if refrigerated when not in use.

Tris(hydroxymethyl)aminomethane reagent: 3 mg/mL of tris(hydroxymethyl)aminomethane prepared as follows. Transfer 40 mL of Tris(hydroxymethyl)aminomethane stock reagent to a 200-mL volumetric flask, add Dimethyl sulfoxide with mixing, and dilute with dimethyl sulfoxide to volume. Use this reagent within 4 h. If kept immersed in an ice-water bath below 10", the reagent may be used for up to 8 h.

Standard stock solution: 1.1 mg/mL of USP Tobramycin RS prepared as follows. Weigh a suitable amount of USP Tobramycin RS into a suitable volumetric flask. Add 1 N sulfuric acid, using 2% of the final volume, and enough water to dissolve the tobramycin, and dilute with water to volume.

Standard solution: 0.22 mg/mL of USP Tobramycin RS from the Standard stock solution in water

Sample solution: Nominally 0.192 mg/mL of tobramycin from the Inhalation Solution in water

Derivatization procedure: Heat all solutions at the same temperature and for the same duration of time as indicated. Move all flasks to and from the 60° constant temperature bath at the same time.

To separate 50-mL volumetric flasks transfer 4.0 mL of the Standard solution, 4.0 mL of the Sample solution, and 4.0 mL of the Blank. 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 min. Remove the flasks from the bath, and allow to stand for 10 min. Add acetonitrile to about 2 ml. below the 50-ml mark, allow to cool to room temperature, then dilute with acetonitrile to volume. The solutions thus obtained are the Derivatized standard solution, Derivatized sample solution, and Derivatized blank solution, respectively.

System suitability solution: 0.24 mg/mL of p-naphtholbenzein in acetonitrile. Transfer 2 mL of this solution to a 10-ml volumetric flask, dilute with Derivatized standard solution to volume, and use promptly.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 365 nm

Column: 3.9-mm x 30-cm; packing L1

Flow rate: 1.2 mL/min

Injection volume: 20 µL

System suitability

Samples: Derivatized standard solution, Derivatized blank solution, and System suitability solution

[NOTE-The relative retention times for p-naphtholbenzein and tobramycin are about 0.6 and 1.0, respectively.]

Identify the solvent and reagent peaks using the Derivatized blank solution.

Suitability requirements

Resolution: NLT 4.0 between p-naphtholbenzein and tobramycin, System suitability solution

Relative standard deviation: NMT 2.0%, Derivatized standard solution

Analysis

Samples: Derivatized standard solution and Derivatized sample solution

Calculate the percentage of the labeled amount of tobramycin (C₁₈H₃₇N₅O₉) in the portion of Inhalation Solution taken:

                          Result = (r_U/r_S) × (C_S/C_U) × P × F × 100

r_U = peak response from the Derivatized sample solution

r_S = peak response from the Derivatized standard solution

C_S = concentration of USP Tobramycin RS in the Standard solution (mg/mL)

C_U = nominal concentration of tobramycin in the Sample solution (mg/mL)

P = potency of tobramycin in USP Tobramycin RS (µg/mg)

F = conversion factor, 0.001 mg/µg

Acceptance criteria: 90.0%-110.0%

4 OTHER COMPONENTS

CONTENT OF SODIUM CHLORIDE

Solution A: 2 g of gelatin and 50 mL of nitric acid in 1000 mL of water

Sample solution: Pipet 25 mL of Inhalation Solution into a suitable container. Add 70-100 mL of water and 10 mL of Solution A.

Analysis: Titrate potentiometrically with 0.1 N silver nitrate VS using a suitable silver electrode.

Acceptance criteria: 90.0%-110.0% of the labeled amount of sodium chloride

5 PERFORMANCE TESTS

UNIFORMITY OF DOSAGE UNITS (905): Meets the requirements

6 IMPURITIES

Change to read:

ORGANIC IMPURITIES

Solution A: Acetonitrile, water, and phosphoric acid (5: 95:0.08)

Solution B: Acetonitrile, water, and phosphoric acid (75: 25:0.08)

Mobile phase: See Table 1.

Blank: Water

2,4-Dinitrofluorobenzene reagent and Tris(hydroxymethyl)aminomethane reagent: Prepare as directed in the Assay.

System suitability stock solution: Dissolve 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 1.1 mg/mL.

System suitability solution 1: 0.22 mg/mL of tobramycin from the System suitability stock solution in water

System suitability solution 2: Heat a portion of the System suitability stock solution in a suitable sealed glass container at 100° for 8-9 h. Cool to room temperature, and dilute with water to obtain a solution containing nominally 0.22 mg/mL of tobramycin.

Standard stock solution: Prepare as directed in the Assay.

Standard solution: 1.10 µg/mL of tobramycin from the Standard stock solution in water

Sample solution: Prepare as directed in the Assay.

Derivatization procedure: 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 7, 15.0 ml of System suitability solution 2, 15.0 mL of Standard solution, 15.0 mL of Sample solution, and 15.0 mL of Blank. 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 min. Remove the flasks from the bath, and allow to stand for 10 min. Add acetonitrile to about 2 mL below the 50-mL mark, allow to cool to room temperature, and dilute with acetonitrile to volume. Allow the solutions to stand for 16 h. The solutions thus obtained are Derivatized system suitability solution 1, Derivatized system suitability solution 2, Derivatized standard solution, Derivatized sample solution, and Derivatized blank solution.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 365 nm

Column: 4.6-mm x 25-cm; packing L11

Flow rate: 1.2 mL/min

Injection volume: 45 µl.

System suitability

Samples: Derivatized system suitability solution 1, Derivatized system suitability solution 2, and Derivatized standard solution

[NOTE-See Table 2 for relative retention times.]

Compare Derivatized system suitability solutions 1 and 2 chromatograms to identify degradation peaks. Deoxystreptamine kanosaminide and nebramine will increase in response in Derivatized system suitability solution 2.

Suitability requirements

Capacity factor (k): NLT 15.5 for tobramycin, Derivatized system suitability solution 2

Resolution: NLT 1.0 between the nebramine and kanamycin peaks, Derivatized system suitability solution 2

Relative standard deviation: NMT 2.0%, Derivatized standard solution

Analysis

Samples: Derivatized system suitability solution 1, Derivatized standard solution, Derivatized sample solution, and Derivatized blank solution Disregard any peak corresponding to those from the Derivatized blank solution, and subtract 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 sample solution. ^▲For unspecified impurity determination, disregard any unidentified peak observed in the Derivatized sample solution that is also observed in Derivatized system suitability solution 1. _{▲(USP 1-Dec-2024)}

Calculate the percentage of each impurity in the portion of Inhalation Solution taken:

                         Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak area of each impurity from the Derivatized sample solution 

r_S = peak area of tobramycin from the Derivatized standard solution 

C_S = concentration of USP Tobramycin RS in the Standard solution (µg/mL)

C_U = nominal concentration of tobramycin in the Sample solution (µg/mL)

Acceptance criteria: See Table 2.

Table 2

7 SPECIFIC TESTS

Change to read:

BACTERIAL ENDOTOXINS TEST (85): Meets the requirements (USP 1-Dec-2024)

STERILITY TESTS (71), Test for Sterility of the Product to Be Examined, Membrane Filtration: Meets the requirements

ABSORBANCE

Sample: Inhalation Solution

Analysis: Determine the absorbance of the Sample at 410 nm in a 1-cm cell.

Acceptance criteria: NMT 0.30

pH (791): 4.5-6.5

PARTICULATE MATTER IN INJECTIONS (788): Meets the requirements for small-volume injections

OSMOLALITY AND OSMOLARITY (785): The osmolality is 135-285 mosmol/kg.

8 ADDITIONAL REQUIREMENTS

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 (11)

USP Tobramycin RS