Sulfuric acid reagent— Prepare a solution of sulfuric acid,dehydrated alcohol,and water solution (4:3:3).

Modified phenylhydrazine-sulfuric acid TS— Dissolve 65mg of phenylhydrazine hydrochloride in 100mLof Sulfuric acid reagent.

Standard preparation— Dissolve in dehydrated alcohol a suitable quantity,accurately weighed,of USP Prednisolone RS,and dilute quantitatively and stepwise with dehydrated alcohol to obtain a solution having a known concentration of about 0.1mg per mL.

Assay preparation— To an accurately weighed portion of Cream,equivalent to about 20mg of prednisolone,add 25mLof dehydrated alcohol.Warm on a steam bath to disperse the assay specimen,then chill to congeal it.Filter through paper,previously washed with dehydrated alcohol,into a 200-mLvolumetric flask.Repeat twice more,starting with “add 25mLof dehydrated alcohol.”Dilute with dehydrated alcohol to volume.(Retain about 5mLof this dilution for the Identificationtest.)

Procedure— Pipet 2mLof Assay preparationinto each of two 50-mLconical flasks (identify as Assay preparationand Assay preparation blank).Pipet 2mLof Standard preparationinto each of two 50-mLconical flasks (identify as Standard preparationand Standard preparation blank).Pipet 2mLof dehydrated alcohol into a 50-mLconical flask (identify as Reagent blank).Add 20.0mLof Sulfuric acid reagentto the Assay preparation blankand to the Standard preparation blank.Add 20.0mLof Modified phenylhydrazine-sulfuric acid to the Assay preparation,to the Standard preparation,and to the Reagent blank,respectively.Maintain the flasks in a water bath at 60for about 45minutes,then chill in a water-ice bath.Filter each through a fine-porosity,sintered-glass funnel,and identify the filtrate flasks correspondingly.Concomitantly determine the absorbances of the solutions at the wavelength of maximum absorbance at about 410nm,with a suitable spectrophotometer,against dehydrated alcohol.Calculate the quantity,in mg,of C21H28O5in the portion of Cream taken by the formula: in which Cis the concentration,in mg per mL,of USP Prednisolone RSin the Standard preparation,AUand ASare the absorbances of the solutions from the Assay preparationand the Standard preparation,respectively,AUBand ASBare the absorbances of the solutions from the Assay preparation blankand the Standard preparation blank,respectively,and ARis the absorbance of the Reagent blank.