Gentamicin and Prednisolone Acetate 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

Gentamicin and Prednisolone Acetate Ophthalmic Ointment contains the equivalent of NLT 90.0% and NMT 120.0% of the labeled amount of gentamicin, and NLT 90.0% and NMT 110.0% of the labeled amount of prednisolone acetate (C₂₃H₃₀O₆).

2 IDENTIFICATION

A. THIN-LAYER CHROMATOGRAPHY

Standard solution: 1 mg/mL of USP Gentamicin Sulfate RS in water

Sample solution: Nominally 1 mg/mL of gentamicin from Ophthalmic Ointment prepared as follows. Shake a quantity of Ophthalmic Ointment, containing nominally 5 mg of gentamicin, with a mixture of 200 mL of chloroform and 5 mL of water. Allow to separate, and filter the aqueous layer.

Chromatographic system

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

Adsorbent: 0.25-mm layer of chromatographic silica gel

Application volume: 20 µL

Developing solvent system: Mix chloroform, methanol, and ammonium hydroxide (20:13:10), allow to separate, and use the lower layer.

Analysis

Samples: Standard solution and Sample solution

Apply the Standard solution and Sample solution to the plate. Place the plate in a chromatographic chamber, and develop the chromatogram in the Developing solvent system until the solvent front has moved three-fourths of the length of the plate. Remove the plate from the chamber, air-dry, and expose the plate to vapors of iodine in a detection jar containing iodine crystals.

Acceptance criteria: The intensities and R_F values of the three principal spots from the Sample solution correspond to those from the Standard solution.

B. The retention time of the prednisolone acetate peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay for Prednisolone Acetate.

3 ASSAY

3.1 GENTAMICIN

(See Antibiotics-Microbial Assays (81).)

Sample solution: Shake a portion of Ophthalmic Ointment containing nominally 1 mg of gentamicin with about 50 mL of ether in a separator, and extract with four 20-mL portions of Buffer B.3 (see the chapter). Combine the buffer extracts, and dilute with Buffer B.3 to a suitable volume to obtain a Test Dilution having a gentamicin concentration that is nominally equivalent to the median level of the standard.

Analysis: Proceed as directed in the chapter.

Acceptance criteria: 90.0%-120.0%

3.2 PREDNISOLONE ACETATE

Mobile phase: Acetonitrile and water (40:60)

Internal standard solution: 2.7 mg/mL of Fluorometholone acetate in methanol

Standard stock solution: 0.38 mg/mL of USP Prednisolone Acetate RS in methanol

Standard solution: 0.06 mg/mL of USP Prednisolone Acetate RS in methanol prepared as follows. Transfer 8.0 mL of Standard stock solution to a 50-mL volumetric flask, add 25 mL of n-hexane, and shake. Add 2.0 mL of Internal standard solution, dilute with methanol to volume, and shake vigorously for 30 s. Allow the layers to separate, remove the upper n-hexane layer by aspiration, and discard the aspirate. Dilute the solution in the volumetric flask with methanol to volume. Centrifuge a portion of this solution, and use the clear supernatant.

Sample solution: Nominally 0.06 mg/mL of prednisolone acetate from Ophthalmic Ointment in methanol prepared as follows. Transfer a portion of Ophthalmic Ointment, containing nominally 3 mg of prednisolone acetate, to a 50-mL volumetric flask, add 25 mL of n-hexane, and shake. Add 2.0 mL of Internal standard solution, dilute with methanol to volume, and shake vigorously for 30 s. Allow the layers to separate, remove the upper n-hexane layer by aspiration, and discard the aspirate. Dilute the solution in the volumetric flask with methanol to volume. Centrifuge a portion of this solution, and use the clear supernatant.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 3.9-mm × 30-cm; 10-µm packing L1

Flow rate: 2 mL/min

Injection volume: 30 µL

System suitability

Sample: Standard solution

Suitability requirements

Resolution: NLT 2.0 between the prednisolone acetate and fluorometholone acetate peaks

Tailing factor: NMT 1.5

Relative standard deviation: NMT 2.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of prednisolone acetate (C₂₃H₃₀O₆) in the portion of Ophthalmic Ointment taken:

Result = (R_U/R_S) x (C_S/C_U) x 100

R_U = peak area response ratio of prednisolone acetate to fluorometholone acetate from the Sample solution

R_S = peak area response ratio of prednisolone acetate to fluorometholone acetate from the Standard solution

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

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

Acceptance criteria: 90.0%-110.0%

4 SPECIFIC TESTS

4.1 STERILITY TESTS (71)

Meets the requirements

4.2 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

5.1 PACKAGING AND STORAGE

Preserve in collapsible ophthalmic ointment tubes. Store at controlled room temperature.

5.2 USP REFERENCE STANDARDS (11)

USP Gentamicin Sulfate RS

USP Prednisolone Acetate RS