Tobramycin Ophthalmic Ointment

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 Ophthalmic Ointment contains NLT 90.0% and NMT 120.0% 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 in water

Sample solution: Vigorously shake by mechanical means a quantity of Ophthalmic Ointment, containing nominally 3 mg of tobramycin with 2 mL of chloroform. Add 1 mL of water, shake vigorously by mechanical means for 1 min, and centrifuge for 15 min. Use the clear upper, aqueous layer.

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 Rf values of the spots of the Sample solution and of Solution A, respectively, correspond to those of the Standard solution.

3 ASSAY

3.1 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-µm 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 into 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: Nominally 0.045 mg/mL of tobramycin from Ophthalmic Ointment in water prepared as follows. Transfer a portion of Ophthalmic Ointment, containing nominally 4.5 mg of tobramycin, to a separator. Add 50 mL of ether, and extract with four 20- to 25-mL portions of water. Combine the water extracts in a 100-mL volumetric flask, and dilute with water to volume.

Derivatized standard solution, Derivatized sample solution, 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, 15.0 mL of the Sample solution, 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, the Derivatized sample solution, 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 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 × 30-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, 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 Ophthalmic Ointment taken:

Result = (rᵤ/rₛ) × (Cₛ/Cᵤ) × P × F × 100

rᵤ = peak area of tobramycin from the Derivatized sample solution

rₛ = peak area of tobramycin from the Derivatized standard solution

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

Cᵤ = 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%–120.0%

4 SPECIFIC TESTS

Sterility Tests 〈71〉: Meets the requirements

Other Requirements: It meets the requirements for Particulate and Foreign Matter and Container Contents in Ophthalmic Products—Quality Tests 〈771〉, Drug Product Quality, Universal Tests, Particulate and Foreign Matter and Container Contents.

5 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in collapsible ophthalmic ointment tubes.

USP Reference Standards 〈11〉

USP Tobramycin RS