A: Filter a portion of Ophthalmic Suspension,freshly mixed but free from air bubbles,equivalent to about 60mg of prednisolone acetate,discarding the filtrate.Wash the filter with about 10mLof water,and dry at 105for 3hours:the IRabsorption spectrum of a potassium bromide dispersion of the dried residue on the filter so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of USP Prednisolone Acetate RS.

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 exhibited in the chromatogram of the Standard preparationobtained as directed in the Assay for prednisolone acetate.

Mobile phase— Prepare a solution containing n-butyl chloride,water-saturated n-butyl chloride,tetrahydrofuran,methanol,and glacial acetic acid (95:95:14:7:6).

Internal standard solution— Prepare a solution of betamethasone in tetrahydrofuran containing 10mg per mL.Dilute this solution with water-saturated chloroform,and mix to obtain a solution having a concentration of about 1mg per mL.

Standard preparation— Dissolve about 5mg of USP Prednisolone Acetate RS,accurately weighed,in 10.0mLof Internal standard solution.Use sonication,if necessary,dilute with water-saturated chloroform to 200.0mL,and mix to obtain a solution having a known concentration of about 25µg per mL.

Assay preparation— Transfer an accurately measured volume of Ophthalmic Suspension,freshly mixed and free from air bubbles,equivalent to about 2.5mg of prednisolone acetate,to a suitable container,add 5.0mLof Internal standard solutionand about 100mLof water-saturated chloroform,and shake by mechanical means for about 15minutes.Allow to separate for about 15minutes,and use the clear chloroform layer as the Assay preparation.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4-mm ×30-cm column that contains packing L3.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the resolution,R,between the analyte and internal standard peaks is not less than 3.0,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 1.6for betamethasone and 1.0for prednisolone acetate.Calculate the quantity,in mg,of prednisolone acetate (C23H30O6)in each mLof the Ophthalmic Suspension taken by the formula: in which Cis the concentration,in µg per mL,of USP Prednisolone Acetate RSin the Standard preparation,Vis the volume,in mL,of Ophthalmic Suspension taken,and RUand RSare the peak response ratios of prednisolone acetate to betamethasone obtained from the Assay preparationand the Standard preparation,respectively.