Mobile phase— Prepare a suitable degassed and filtered solution of water and acetonitrile (about 75:25),such that the retention time of hydrocortisone is about 10minutes.

Standard preparation— Dissolve an accurately weighed quantity of USP Hydrocortisone RSin methanol to obtain a solution having a known concentration of about 500µg per mL.Quantitatively dilute 1volume of this solution with 9volumes of dilute methanol (1in 2).The Standard preparationhas a final known concentration of about 50µg per mL.[NOTE—If methanol is used in the final dilution of the Assay preparation,similarly use methanol instead of aqueous methanol in the final dilution of the Standard preparation.]

Assay preparation— Transfer an accurately weighed quantity of Cream,equivalent to about 10mg of hydrocortisone,to a 150-mLbeaker.Add 40mLof methanol,and heat on a steam bath while stirring to melt and disperse the cream.Cool to room temperature,and filter through glass wool into a 100-mLvolumetric flask.Repeat the extraction with two 20-mLportions of methanol,combining the filtrates in the 100-mLvolumetric flask.Add methanol to volume,and mix.Quantitatively dilute 1volume of this solution with an equal volume of water,and filter through a 5µm membrane filter.If precipitation occurs on dilution with water,and the solution is still cloudy after filtration,dilute the initial test solution with methanol instead of water.Filter this solution through a 5µm membrane filter.

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.Adjust operating parameters such that the peak obtained from the Standard preparationis about 0.6full-scale.Chromatograph five replicate injections of the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation is not more than 3.0%.

Procedure— Separately inject equal volumes (about 10to 25µL)of the Standard preparationand the Assay preparationinto the chromatograph by means of a sampling valve,record the chromatograms,and measure the responses for the major peaks at equivalent retention times.Calculate the quantity,in mg,of C21H30O5in the portion of Cream taken by the formula: in which Cis the concentration,in µg per mL,of USP Hydrocortisone RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.