Mobile phase— Prepare a degassed and filtered mixture of water and methanol (50:50).

Standard preparation— Dissolve an accurately weighed quantity of USP Hydrocortisone Acetate RSin a mixture of Mobile phaseand alcohol (80:20)to obtain a solution having a known concentration of about 0.06mg per mL.

Assay preparation— Transfer an accurately measured volume of Ophthalmic Suspension,equivalent to about 30mg of hydrocortisone acetate,to a separator containing 25mLof pH9.0alkaline borate buffer (see under Buffer Solutionsin the section Reagents,Indicators,and Solutions).Extract with four 25-mLportions of chloroform,filtering each chloroform extract through a thin layer of chloroform-washed anhydrous sodium sulfate into a 250-mLvolumetric flask.Rinse the sodium sulfate with chloroform,collecting the filtrate in the volumetric flask,dilute with chloroform to volume,and mix.Transfer 25.0mLof the resulting solution to a 50-mLconical flask,and evaporate slowly with the aid of mild heat until about 5mLremains.Add about 15mLof alcohol,and evaporate slowly until about 5mLremains.Transfer this solution to a 50-mLvolumetric flask,dilute with a mixture of Mobile phaseand alcohol (80:20)to volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the column efficiency determined from the analyte peak is not less than 235theoretical plates,the tailing factor for the analyte peak is not more than 1.7,and the relative standard deviation of replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 25µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C23H32O6in each mLof the Ophthalmic Suspension taken by the formula: in which Cis the concentration,in mg per mL,of USP Hydrocortisone Acetate RSin the Standard preparation;Vis the volume,in mL,of Ophthalmic Suspension taken;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.