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 Suitability under Chromatography 621).

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

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

Procedure— Introduce equal volumes of the Assay preparation and the Standard preparation into 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 L3 and operated at room temperature. Six replicate injections of the Standard preparation show 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 C is the concentration, in mg per mL, of USP Hydrocortisone Acetate RS in the Standard preparation; and rU and rS are the hydrocortisone acetate peak responses obtained from the Assay preparation and the Standard preparation, respectively.