Mobile phase— Prepare a filtered and degassed solution containing butyl chloride,water-saturated butyl chloride,tetrahydrofuran,methanol,and glacial acetic acid (475:475:70:35:30).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Hydrocortisone Acetate RSin water-saturated chloroform to obtain a solution having a known concentration of about 0.10mg per mL.

Assay preparation— Transfer an accurately weighed quantity of Lotion,equivalent to about 2.5mg of hydrocortisone acetate,to a closable container.Add 25.0mLof water-saturated chloroform and about 10glass beads.Securely close the container,and shake vigorously for approximately 15minutes.Centrifuge,and use the clear,lower chloroform layer.

Procedure— Introduce equal volumes of the Assay preparationand the Standard preparationinto a high-pressure liquid chromatograph fitted with a 254-nm detector.Typically the apparatus is fitted with a 4-mm ×30-cm column containing packing L3and operated at room temperature.Six replicate injections of the Standard preparationshow a relative standard deviation of not more than 2.0%.Calculate the quantity,in mg,of hydrocortisone acetate (C23H32O6)in the portion of Lotion taken by the formula: in which Cis the concentration,in mg per mL,of USP Hydrocortisone Acetate RSin the Standard preparation;and rUand rSare the hydrocortisone acetate peak responses obtained from the Assay preparationand the Standard preparation,respectively.