Reagent blank— Dilute 200 mL of 22 N sulfuric acid with 100 mL of dehydrated alcohol.

Phenylhydrazine reagent— Dissolve 43.33 mg of phenylhydrazine hydrochloride in 100 mL of Reagent blank.

Standard preparation— Dissolve a suitable quantity of USP Hydrocortisone Acetate RS, accurately weighed, in chloroform, and dilute quantitatively and stepwise with chloroform to obtain a solution having a known concentration of about 10 µg per mL.

Assay preparation— Transfer 5.0 mL of freshly mixed Otic Suspension to a 125-mL separator. Extract with three 20-mL portions of chloroform, filtering each chloroform extract through a pledget of cotton previously saturated with chloroform, collect the filtrates in a 100-mL volumetric flask, dilute with chloroform to volume, and mix. Pipet 10 mL of this solution into a 100-mL volumetric flask, dilute with chloroform to volume, and mix. Pipet 20 mL of this solution into a 100-mL volumetric flask, dilute with chloroform to volume, and mix.

Procedure— Pipet 50 mL each of the Standard and the Assay preparation into separate 125-mL separators, add 2 mL of 0.1 N sodium hydroxide to each separator, shake, and allow the layers to separate. Filter both chloroform layers through glass wool, and collect the filtrates in separate beakers. Pipet two 20-mL portions of each chloroform filtrate into separate 125-mL separators. Add 25.0 mL of Phenylhydrazine reagent to one separator each of the filtrates from the Standard preparation and the Assay preparation, respectively, and add 25.0 mL of Reagent blank to the remaining two separators. Shake all four separators well, allow the layers to separate, and discard the chloroform layers. Drain the aqueous layers into separate centrifuge tubes, and centrifuge for 2 minutes. Pipet 10 mL of each solution into separate glass-stoppered test tubes. Place the tubes in a water bath maintained at a temperature of 60 for 30 minutes, then cool the solution to room temperature. Concomitantly determine the absorbances of the solutions at the wavelength of maximum absorbance at about 410 nm, with a suitable spectrophotometer, using water to set the instrument. Calculate the quantity, in mg, of hydrocortisone acetate (C23H32O6) in each mL of the Otic Suspension taken by the formula: in which C is the concentration, in µg per mL, of USP Hydrocortisone Acetate RS in the Standard preparation; AU and AS are the absorbances of the solutions from the Assay preparation and the Standard preparation treated with Phenylhydrazine reagent, respectively; and AUB and ASB are the absorbances of the solution from the Assay preparation and the Standard preparation treated with the Reagent blank, respectively.