Test solution: 10mg per mL,in dioxane.

Mobile phase— Prepare a filtered and degassed mixture of water and acetonitrile (55:45).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— Prepare a solution of ethyl benzoate in methanol containing about 2.0mg per mL.

Standard preparation— Immediately prior to use,dissolve an accurately weighed quantity of USP Hydrocortisone Valerate RSin methanol to obtain a solution having a known concentration of about 0.5mg per mL.Pipet 2mLof this solution and 2mLof Internal standard solutioninto a 10-mLvolumetric flask.Dilute with methanol to volume,and mix to obtain a Standard preparationhaving a known concentration of about 100µg of hydrocortisone valerate per mL.

Assay preparation— Transfer about 100mg of Hydrocortisone Valerate,accurately weighed,to a 100-mLvolumetric flask.Dissolve in and dilute with methanol to volume,and mix.Pipet 1mLof this solution and 2mLof Internal standard solutioninto a 10-mLvolumetric flask.Dilute with methanol 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 relative retention times are about 0.8for ethyl benzoate and 1.0for hydrocortisone valerate,the resolution,R,between hydrocortisone valerate and ethyl benzoate 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.Calculate the quantity,in mg,of C26H38O6,in the portion of Hydrocortisone Valerate taken by the formula: in which Cis the concentration,in µg per mL,of USP Hydrocortisone Valerate RSin the Standard preparation;and RUand RSare the peak response ratios of the hydrocortisone valerate peak and the internal standard peak obtained from the Assay preparationand the Standard preparation,respectively.