Tobramycin and Dexamethasone 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 and Dexamethasone Ophthalmic Ointment contains NLT 90.0% and NMT 120.0% of the labeled amount of tobramycin (C₁₈H₃₇N₅O₉), and NLT 90.0% and NMT 110.0% of the labeled amount of dexamethasone (C₂₂H₂₉FO₅).

2 IDENTIFICATION

2.1 A. THIN-LAYER CHROMATOGRAPHY

Solution A: 100 mg/ml of sodium sulfate in water

Diluent: Butyl alcohol and pyridine (100:1)

Standard solution: 6 mg/mL of USP Tobramycin RS in water

Sample solution: To 1 g of Ophthalmic Ointment in a test tube add 2 mL of chloroform, and shake to dissolve. Add 0.5 mL of Solution A, shake vigorously, and centrifuge. Use the clear supernatant aqueous liquid. If, after centrifuging, an oily film remains on top of the supernatant aqueous liquid, transfer the supernatant aqueous liquid to a second test tube, and wash it with 2 mL of chloroform.

Solution B: 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 B

Apply the Standard solution, the Sample solution, and Solution B 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, values of the spots of the Sample solution and of Solution B, respectively, correspond to those of the Standard solution.

2.2 B.

The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay for Dexamethasone.

3 ASSAY

3.1 TOBRAMYCIN

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

r_U = peak area of tobramycin 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 (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%-120.0%

3.2 DEXAMETHASONE

Mobile phase: Methanol and water (55:45)

Diluent: Methanol and water (75:25)

System suitability stock solution: 1 mg/mL of anhydrous Chlorobutanol and 0.2 mg/mL of USP Dexamethasone RS in Diluent

System suitability solution: 0.3 mg/mL of anhydrous chlorobutanol and 0.06 mg/mL of USP Dexamethasone RS in Diluent prepared as follows. Transfer 15.0 mL of the System suitability stock solution to a separator containing about 50 mL of n-hexane, and shake. Allow the layers to separate, and drain the lower phase into a 50-mL volumetric flask. Repeat the extraction with two 15-mL portions of Diluent, combining the lower phase from each extraction in the same 50-mL volumetric flask. Dilute with Diluent to volume.

Standard stock solution: 0.2 mg/mL of USP Dexamethasone RS in Diluent

Standard solution: 0.06 mg/mL of USP Dexamethasone RS in Diluent prepared as follows. Transfer 15.0 mL of the Standard stock solution to a separator containing about 50 ml of n-hexane, and shake. Allow the layers to separate, and drain the lower phase into a 50-mL volumetric flask. Repeat the extraction with two 15-mL portions of Diluent, combining the lower phase from each extraction in the same 50-mL volumetric flask. Dilute with Diluent to volume.

Sample solution: Transfer a portion of Ophthalmic Ointment containing nominally 3 mg of dexamethasone to a separator containing 50 mL of n-hexane, and shake. Add 15 mL of Diluent, and shake. Allow the layers to separate, and drain the lower phase into a 50-mL, volumetric flask. Repeat the extraction with two 15-mL portions of Diluent, combining the lower phase from each extraction in the same 50-mL volumetric flask. Dilute with Diluent to volume, mix, and centrifuge. Use the clear solution.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 206 nm

Column: 8.0-mm x 10-cm; packing L1

Flow rate: 3 mL/min

Injection volume: 100 µL

System suitability

Samples: System suitability solution and Standard solution

[NOTE-The relative retention times for chlorobutanol and dexamethasone are about 0.7 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 1.8 between chlorobutanol and dexamethasone, System suitability solution

Tailing factor: NMT 2, Standard solution

Column efficiency: NLT 350 theoretical plates, Standard solution

Relative standard deviation: NMT 2.0%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of dexamethasone (C₂₂H₂₉FO₅) in the portion of Ophthalmic Ointment taken:

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

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution s

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

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

P = potency of dexamethasone in USP Dexamethasone RS (mg/mg)

Acceptance criteria: 90.0%~110.0%

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 Dexamethasone RS

USP Tobramycin RS