A:Infrared Absorption á197Kñ.

B:Ultraviolet Absorption á197Uñ—

C: To about 50mg contained in a test tube add 2mLof alcohol and 2mLof dilute sulfuric acid (1in 3.5),and boil gently for about 1minute:the odor of ethyl acetate is perceptible.

Test solution: 10mg per mL,in dioxane.

Mobile phase— Prepare a filtered and degassed mixture of isooctane,butyl chloride and methanol (49:49:2).Make adjustments if necessary (seeSystem Suitability underChromatography á621ñ).

Test solution— Transfer about 10mg of Prednisolone Acetate,accurately weighed,to a suitable container,dissolve in 10mLof chloroform,and mix.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 6.0-mm ×4.0-cm column that contains packing L3.The flow rate is about 3mLper minute.Chromatograph theTest solution,and record the peak responses as directed forProcedure:the column efficiency is not less than 800theoretical plates;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Inject a volume (about 10µL)of theTest solution into the chromatograph,record the chromatogram,and measure all the peak responses.Calculate the percentage of each impurity in the portion of Prednisolone Acetate taken by the formula: in whichriis the peak response for each impurity;andrsis the sum of the responses for all the peaks:not more than 1.0%of any individual impurity is found;and not more than 2.0%of total impurities is found.

Mobile phase— Prepare a solution containing a mixture ofn-butyl chloride,water-saturatedn-butyl chloride,tetrahydrofuran,methanol,and glacial acetic acid (95:95:14:7:6).Make adjustments if necessary (seeSystem Suitability underChromatography á621ñ).

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 final concentration of about 1mg of betamethasone per mL.

Standard preparation— Transfer about 10mg of USP Prednisolone Acetate RS,accurately weighed,to a 100-mLvolumetric flask;add 20.0mLofInternal standard solution;and dissolve,using sonication if necessary.Dilute with water-saturated chloroform to volume,and mix.Dilute 5.0mLof the solution so obtained with water-saturated chloroform to 20.0mLto obtain a solution having a known concentration of about 25µg of USP Prednisolone Acetate RSper mL.

Assay preparation— Transfer about 10mg of Prednisolone Acetate,accurately weighed,to a 100-mLvolumetric flask;add 20.0mLofInternal standard solution;and dissolve,using sonication if necessary.Dilute with water-saturated chloroform to volume,and mix.Dilute 5.0mLof the solution so obtained with water-saturated chloroform to 20.0mL.

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 theStandard preparation,and record the peak responses as directed forProcedure:the relative retention times are about 1.6for betamethasone and 1.0for prednisolone acetate;the resolution,R,between prednisolone acetate and betamethasone 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 theStandard preparation and theAssay preparation into the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C23H30O6in the portion of Prednisolone Acetate taken by the formula: in whichCis the concentration,in µg per mL,of USP Prednisolone Acetate RSin theStandard preparation;andRUandRSare the peak response ratios obtained from theAssay preparation and theStandard preparation,respectively.