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 10 minutes.

Standard preparation— Dissolve an accurately weighed quantity of USP Hydrocortisone RS in methanol to obtain a solution having a known concentration of about 500 µg per mL. Quantitatively dilute 1 volume of this solution with 9 volumes of dilute methanol (1 in 2). The Standard preparation has a final known concentration of about 50 µg 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 10 mg of hydrocortisone, to a 150-mL beaker. Add 40 mL of 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-mL volumetric flask. Repeat the extraction with two 20-mL portions of methanol, combining the filtrates in the 100-mL volumetric flask. Add methanol to volume, and mix. Quantitatively dilute 1 volume of this solution with an equal volume of water, and filter through a 5 µm 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 5 µm membrane filter.

Chromatographic system (see Chromatography 621)—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 preparation is about 0.6 full-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 10 to 25 µL) of the Standard preparation and the Assay preparation into 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 C21H30O5 in the portion of Cream taken by the formula: in which C is the concentration, in µg per mL, of USP Hydrocortisone RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.