Identification—
A:
Shake a quantity of Ophthalmic Ointment,equivalent to about 5mg of gentamicin,with a mixture of 200mLof chloroform and 5mLof water.Allow to separate,and filter the aqueous layer:the filtrate so obtained meets the requirements of the Identificationtest under Gentamicin Sulfate Injection.
B:
The chromatogram of the
Assay preparationobtained as directed in the
Assay for prednisolone acetateexhibits a major peak for prednisolone acetate,the retention time of which corresponds to that obtained in the chromatogram of the
Standard preparationobtained as directed in the
Assay for prednisolone acetate.
Assay for gentamicin—
Proceed with Ophthalmic Ointment as directed for gentamicin under
Antibiotics—Microbial Assays á81ñ,using an accurately weighed quantity of Ophthalmic Ointment,equivalent to about 1mg of gentamicin,shaken with about 50mLof ether in a separator,and extracted with four 20-mLportions of
Buffer No.3.Combine the aqueous extracts,and dilute quantitatively and stepwise with
Buffer No.3to obtain a
Test Dilutionhaving 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 1µm or less.Make adjustments if necessary (see
System Suitabilityunder
Chromatography á621ñ).
Internal standard solution—
Prepare a solution in methanol containing about 2.7mg of fluorometholone acetate per mL.
Standard preparation—
Transfer about 38mg of
USP Prednisolone Acetate RS,accurately weighed,to a 100-mLvolumetric flask,dissolve in methanol,dilute with methanol to volume,and mix.Transfer 8.0mLof this solution to a 50-mLvolumetric flask,add 25mLof
n-hexane,and shake.Add 2.0mLof
Internal standard solution,dilute with methanol to volume,and shake vigorously for 30seconds.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 0.06mg of
USP Prednisolone Acetate RSper mL.
Assay preparation—
Transfer an accurately weighed portion of Ophthalmic Ointment,equivalent to about 3mg of prednisolone acetate,to a 50-mLvolumetric flask,add 25mLof n-hexane,and shake.Add 2.0mLof Internal standard solution,dilute with methanol to volume,and shake vigorously for 30seconds.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 á621ñ)—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 2mLper 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 2500theoretical 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
Cis the concentration,in mg per mL,of
USP Prednisolone Acetate RSin the
Standard preparation,and
RUand
RSare the ratios of the response of the prednisolone acetate peak area to that of the fluorometholone acetate peak obtained from the
Assay preparationand the
Standard preparation,respectively.