A: Infrared Absorption 197M.

B: Ultraviolet Absorption 197U—

Solution: 10 µg per mL.

Medium: methanol.

Test solution: 5 mg per mL, in dioxane.

Solution A— Prepare a filtered and degassed mixture of water and acetonitrile (80:20). Make adjustments if necessary (see System Suitability under Chromatography 621).

Solution B— Prepare a filtered and degassed mixture of acetonitrile and water (70:30). Make adjustments if necessary (see System Suitability under Chromatography 621).

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system.

Diluting solution— Prepare a mixture of acetonitrile, water, and glacial acetic acid (700:300:1).

Standard solution— Dissolve an accurately weighed quantity of USP Hydrocortisone Acetate RS in Diluting solution, and dilute quantitatively, and stepwise if necessary, with Diluting solution to obtain a solution having a known concentration of about 5 µg per mL.

Test solution— Transfer about 10 mg of Hydrocortisone Acetate, accurately weighed, to a 10-mL volumetric flask, dissolve in and dilute with Diluting solution to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 15-cm column that contains 3-µm packing L1. The flow rate is about 1 mL per minute. The chromatograph is programmed as follows: Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 5.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of each impurity in the portion of Hydrocortisone Acetate taken by the formula: in which ri is the peak response for each impurity; and rS is the peak response of the Standard solution: not more than 1.0% of any individual impurity is found, and not more than 2.0% of total impurities is found.

(Official January 1, 2007)

Mobile phase— Prepare a filtered and degassed mixture of 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 Mobile phase, and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having a known concentration of about 0.10 mg per mL.

Assay preparation— Transfer about 10 mg of Hydrocortisone Acetate, accurately weighed, to a 100-mL volumetric flask, add Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4-mm × 30-cm column that contains 10-µm packing L3. The flow rate is about 1.0 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the tailing factor for the hydrocortisone acetate peak is not more than 2.0, and the relative standard deviation for replicate injections 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, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C23H32O6 in the portion of Hydrocortisone Acetate 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.