Tobramycin and Fluorometholone Acetate Ophthalmic Suspension

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 Fluorometholone Acetate Ophthalmic Suspension is a sterile aqueous suspension of Tobramycin and Fluorometholone Acetate. It contains NLT 90.0% and NMT 120.0% of the labeled amount of tobramycin (C₁₈H₃₇N₅O₉) and NLT 90.0% and NMT 115.0% of the labeled amount of fluorometholone acetate (C₂₄H₃₁FO₅). It may contain suitable buffers, dispersants, tonicity-adjusting agents, and preservatives.

2 IDENTIFICATION

2.1 A.

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

2.2 B. THIN-LAYER CHROMATOGRAPHY

Diluent: Butyl alcohol and pyridine (100:1)

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

Sample solution: Allow the Ophthalmic Suspension to settle, and decant 1 mL of the supernatant into a test tube. Add 0.1 g of sodium sulfate and centrifuge. Use the clear supernatant.

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,

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-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 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 0.09 mg/mL of tobramycin from Ophthalmic Suspension in water

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, 10.0 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 Suspension 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 FLUOROMETHOLONE ACETATE

Mobile phase: Acetonitrile and water (50:50)

System suitability solution: 0.04 mg/mL each of USP Fluorometholone RS and USP Fluorometholone Acetate RS in acetonitrile

Standard solution: 0.04 mg/mL of USP Fluorometholone Acetate RS in acetonitrile

Sample stock solution: Nominally 0.1 mg/mL of fluorometholone acetate in acetonitrile, prepared as follows. Transfer Ophthalmic Suspension, freshly mixed and free from air bubbles and containing nominally 2.5 mg of fluorometholone acetate, to a 25-ml volumetric flask. Dilute with acetonitrile to volume.

Sample solution: Nominally 0.04 mg/mL of fluorometholone acetate in acetonitrile, prepared as follows. Transfer 4.0 mL of Sample stock solution to a 10-mL volumetric flask, and dilute with acetonitrile to volume. Transfer a portion of this solution to a test tube, and centrifuge for 15 min. Use the clear supernatant.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 4-mm x 25-cm, packing L1

Flow rate: 1.5 mL/min

Injection volume: 10 µL

System suitability

Samples: System suitability solution and Standard solution

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

Suitability requirements

Resolution: NLT 2.0 between fluorometholone and fluorometholone acetate, System suitability solution

Capacity factor, k: 1.0-5.0, fluorometholone acetate peak, Standard solution

Column efficiency: NLT 1000 theoretical plates, Standard solution

Tailing factor: NMT 1.35, Standard solution

Relative standard deviation: NMT 2.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of fluorometholone acetate (C₂₄H₃₁FO₅) in the portion of Ophthalmic Suspension taken: 31

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

r_U = peak area of fluorometholone acetate from the Sample solution

r_S = peak area of fluorometholone acetate from the Standard solution

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

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

P = potency of fluorometholone acetate in USP Fluorometholone Acetate RS (mg/mg)

Acceptance criteria: 90.0%-115.0%

4 SPECIFIC TESTS

STERILITY TESTS (71); It meets the requirements in Test for Sterility of the Product to Be Examined. Membrane Filtration.

pH (791): 6.0-7.0

5 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight containers.

USP REFERENCE STANDARDS (11)

USP Fluorometholone RS

USP Fluorometholone Acetate RS

USP Tobramycin RS