Tobramycin for Injection

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 for Injection contains an amount of Tobramycin Sulfate equivalent to NLT 90.0% and NMT 115.0% of the labeled amount of tobramycin (C₁₈H₃₇N₅O₉).

2 IDENTIFICATION

2.1 A. THIN-LAYER CHROMATOGRAPHY

Diluent: Butyl alcohol and pyridine (100:1)

Standard solution: 6 mg/mL of USP Tobramycin RS

Sample solution: 6 mg/ml. in water, from Tobramycin for Injection diluted with water

Solution A: Standard solution and Sample solution (1:1)

Chromatographic system

(See Chromatography (621), Thin-Layer Chromatography.)

Adsorbent: 0.25-mm layer of chromatographic silica gel mixture

Application volume: 3 µL

Developing solvent system: Methanol, chloroform, and ammonium hydroxide (60:25:30)

Spray reagent: 10 mg/mL of ninhydrin in Diluent

Analysis

Samples: Standard solution, Sample solution, and Solution A

Apply the Standard solution, the Sample solution, and Solution A to the plate. Place the plate in a suitable chromatographic chamber, and develop the chromatogram in the Developing solvent system 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 110° for 15 min. Immediately locate the spots on the plate by spraying it with Spray reagent.

Acceptance criteria: Tobramycin appears as a pink spot, and the R_F values of the spots of the Sample solution and of Solution A, respectively, correspond to those of the Standard solution.

2.2 B.

The retention time of the major peak of Derivatized sample solution 1 or Derivatized sample solution 2 corresponds to that of the Derivatized standard solution, obtained as directed in the Assay.

2.3 C. IDENTIFICATION TESTS GENERAL, Sulfate (191)

Meets the requirements

3 ASSAY

PROCEDURE

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

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

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

Solution C: 3 mg/mL of tris(hydroxymethyl)aminomethane prepared as follows. Transfer 40 mL of Solution B to a 200-mL volumetric flask.

Add Dimethyl sulfoxide while 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. Transfer 55 mg of USP Tobramycin RS to a 50-mL volumetric flask. Add 1 mL of 1 N sulfuric acid and enough water to dissolve it, and dilute with water to volume.

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

Sample solution 1 (where it is represented as being in a single-dose container): Nominally 0.2 mg/mL of tobramycin prepared as follows. Constitute a container of Tobramycin for Injection in a volume of water corresponding to the volume of diluent specified in the labeling. Withdraw all of the withdrawable contents using a suitable hypodermic needle and syringe, and dilute with water.

Sample solution 2 (where the label states the quantity of tobramycin in a given volume of constituted solution): Nominally 0.2 mg/mL of tobramycin prepared as follows. Constitute a container of Tobramycin for Injection in a volume of water equivalent to the volume of diluent specified in the labeling. Dilute a suitable aliquot of the constituted solution with water to an appropriate volume.

Derivatized standard solution, Derivatized sample solution 1 or Derivatized sample solution 2, and Blank solution: Proceed as follows.

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 Sample solution 1 or Sample solution 2, and 4.0 mL of water. To each flask add 10 mL of Solution A and 10 mL of Solution C, 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 1 or Derivatized sample solution 2, and the Blank solution, respectively.

System suitability stock solution: 0.24 mg/mL of p-naphtholbenzein in acetonitrile. Prepare freshly.

System suitability solution: Transfer 2 mL of the System suitability stock solution to a 10-mL volumetric flask, dilute with the 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 - 1 cm; packing L1

Flow rate: 1.2 mL/min

Injection volume: 20 µL

System suitability

Samples: Derivatized standard solution and System suitability solution

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

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, Derivatized sample solution 1 or Derivatized sample solution 2, and Blank solution

Use the Blank solution to identify the solvent and reagent peaks.

Calculate the percentage of the labeled amount of tobramycin (C₁₈H₃₇N₅O₉) in the portion of Tobramycin for Injection taken:

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

r_U = peak area of tobramycin from Derivatized sample solution 1 or Derivatized sample solution 2

r_S = peak area of tobramycin 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 Sample solution 1 or Sample solution 2 (mg/mL)

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

F = conversion factor, 0.001 mg/µg

Acceptance criteria: 90.0%-115.0%

4 PERFORMANCE TESTS

UNIFORMITY OF DOSAGE UNITS (905): Meets the requirements

5 IMPURITIES

RESIDUE ON IGNITION (281)

Analysis: Moisten the charred residue with 2 mL of nitric acid and 5 drops of sulfuric acid.

Acceptance criteria: NMT 1.0%

6 SPECIFIC TESTS

INJECTIONS AND IMPLANTED DRUG PRODUCTS (1), Specific Tests. Completeness and clarity of solutions: At the time of use, it meets the requirements.

BACTERIAL ENDOTOXINS TEST (85): It contains NMT 2.00 USP Endotoxin Units/mg of tobramycin.

STERILITY TESTS (71): It meets the requirements in Test for Sterility of the Product to Be Examined. Membrane Filtration, 6 g being used if it is not packaged for dispensing.

pH (791)

Sample solution: 40 mg/mL or where packaged for dispensing, use the solution constituted as directed in the labeling.

Acceptance criteria: 6.0-8.0

WATER DETERMINATION, Method (921): NMT 2.0%

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

OTHER REQUIREMENTS: It meets the requirements in Labeling (7), Labels and Labeling for Injectable Products.

7 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve as described in Packaging and Storage Requirements (659), Injection Packaging, Packaging for constitution.

USP REFERENCE STANDARDS (11)

USP Tobramycin RS