Identification—
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.
Assay for gentamicin—
Proceed with Ophthalmic Ointment as directed for gentamicin under
Antibiotics—Microbial Assays 
81
, using an accurately weighed quantity of Ophthalmic Ointment, equivalent to about 1 mg of gentamicin, shaken with about 50 mL of ether in a separator, and extracted with four 20-mL portions of
Buffer No. 3. Combine the aqueous extracts, and dilute quantitatively and stepwise with
Buffer No. 3 to obtain a
Test Dilution having a concentration of gentamicin assumed to be equal to the median dose level of the Standard.
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—
Calculate the quantity, in mg, of prednisolone acetate (C
23H
30O
6) in the portion of Ophthalmic Ointment taken by the formula:
50C(RU / RS),
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.
