A: Shake a quantity of Ophthalmic Ointment, equivalent to about 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: the filtrate so obtained meets the requirements of the Identification test under Gentamicin Sulfate Injection.

B: The chromatogram of the Assay preparation obtained as directed in the Assay for prednisolone acetate exhibits a major peak for prednisolone acetate, the retention time of which corresponds to that obtained in the chromatogram of the Standard preparation obtained as directed in the Assay for prednisolone acetate.

Mobile phase— Prepare a suitable mixture of water and acetonitrile (60:40), and pass through a suitable filter having a porosity of 1 µm or less. Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Prepare a solution in methanol containing about 2.7 mg of fluorometholone acetate per mL.

Standard preparation— Transfer about 38 mg of USP Prednisolone Acetate RS, accurately weighed, to a 100-mL volumetric flask, dissolve in methanol, dilute with methanol to volume, and mix. Transfer 8.0 mL of this 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 seconds. 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, and mix. Centrifuge a portion of this solution, and use the clear liquid as the Standard preparation. This solution contains about 0.06 mg of USP Prednisolone Acetate RS per mL.

Assay preparation— Transfer an accurately weighed portion of Ophthalmic Ointment, equivalent to about 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 seconds. 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, and mix. Centrifuge a portion of this solution, and use the clear liquid as the Assay preparation.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm × 20-cm column that contains packing L1. The flow rate is about 2 mL per minute. Chromatograph the Standard preparation, and measure the peak responses as directed for Procedure: the tailing factor is not more than 1.5; the resolution, R, between the prednisolone acetate peak and the fluorometholone acetate peak is not less than 2.0; the column efficiency is not less than 2500 theoretical plates; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 30 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of prednisolone acetate (C23H30O6) in the portion of Ophthalmic Ointment taken by the formula: in which C is the concentration, in mg per mL, of USP Prednisolone Acetate RS in the Standard preparation; and RU and RS are the ratios of the response of the prednisolone acetate peak area to that of the fluorometholone acetate peak obtained from the Assay preparation and the Standard preparation, respectively.