(Official January 1, 2007)

Mobile phase— Prepare a suitable solution of about 500 volumes of methanol, 500 volumes of water, and 1 volume of glacial acetic acid, such that the retention time of hydrocortisone is between 6 and 10 minutes.

Standard preparation— Dissolve a suitable quantity of USP Hydrocortisone RS, accurately weighed, in a mixture of methanol and water (1:1) to obtain a solution having a known concentration of about 0.15 mg per mL.

Assay preparation— Transfer an accurately measured volume of Otic Solution, equivalent to about 15 mg of hydrocortisone, to a 100-mL volumetric flask, dilute with a mixture of methanol and water (1:1) to volume, and mix.

Chromatographic system (see Chromatography 621)—The chromatograph is equipped with a 254-nm detector and a 4-mm × 30-cm column that contains packing L1. The flow rate is about 2 mL per minute. 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 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph by means of a suitable microsyringe or sampling valve, adjusting the specimen size and other operating parameters such that the peak obtained from the Standard preparation is about 0.6 full-scale. Record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C21H30O5 in each mL of the Otic Solution taken by the formula: in which C is the concentration, in mg per mL, of USP Hydrocortisone RS in the Standard preparation, V is the volume, in mL, of the portion of Otic Solution taken, and HU and HS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.