Mobile phase— Prepare a filtered and degassed mixture of water, acetonitrile, and methanol (58:21:21). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Hydrocortisone RS in alcohol and dilute quantitatively, and stepwise if necessary, with alcohol to obtain a solution having a known concentration of about 0.1 mg per mL. Quantitatively dilute a known volume of the final solution with an equal volume of water to obtain the Standard preparation.

Assay preparation— In a tared, 100-mL volumetric flask, weigh 100 mL of Lotion that previously has been shaken to ensure homogeneity, allow to stand until the entrapped air rises, and finally invert carefully just prior to transfer to the volumetric flask. Transfer an accurately weighed quantity of Lotion, freshly mixed but free from air bubbles, equivalent to about 10 mg of hydrocortisone, to a 40-mL beaker, and add about 30 mL of alcohol. Warm gently until the Lotion is dispersed, and cool to room temperature. Quantitatively transfer the mixture, filter through a pledget of cotton previously moistened with alcohol to a 100-mL volumetric flask, rinse the beaker with two 20-mL portions of alcohol, and collect the washings in the same volumetric flask. Dilute with alcohol to volume, and mix. Quantitatively dilute a known volume of this solution with an equal volume of water to obtain the Assay preparation.

System suitability preparation— Dissolve about 5 mg of propylparaben in 100 mL of alcohol. Dilute 1 mL of this solution with the Standard preparation to 10 mL, and mix.

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. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation and the System suitability preparation, and record the peak responses as directed for Procedure: the column efficiency determined from the analyte peak is not less than 1000 theoretical plates, the tailing factor for the analyte peak is not more than 1.2, the resolution, R, between the propylparaben and hydrocortisone peaks is not less than 2.0, and the relative standard deviation for replicate injections of the Standard preparation is not more than 3.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of hydrocortisone (C21H30O5) in the portion of Lotion taken by the formula: in which C is the concentration, in mg per mL, of USP Hydrocortisone RS in the Standard preparation; and rU and the rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively. From the observed weight of 100 mL of the Lotion, calculate the quantity of C21H30O5 in each 100 mL.