A: Infrared Absorption á197Mñ.

B: Ultraviolet Absorption á197Uñ—

Solution: 10µg per mL.

Medium: methanol.

Test solution: 5mg per mL,in dioxane.

Solution A— Prepare a filtered and degassed mixture of water and acetonitrile (80:20).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Solution B— Prepare a filtered and degassed mixture of acetonitrile and water (70:30).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Mobile phase— Use variable mixtures of Solution Aand Solution Bas 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 RSin Diluting solution,and dilute quantitatively,and stepwise if necessary,with Diluting solutionto obtain a solution having a known concentration of about 5µg per mL.

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

Chromatographic system (see Chromatography á621ñ)—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 1mLper 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 10µL)of the Standard solutionand the Test solutioninto 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 riis the peak response for each impurity;and rSis 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.

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 Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Hydrocortisone Acetate RSin Mobile phase,and dilute quantitatively,and stepwise if necessary,with Mobile phaseto obtain a solution having a known concentration of about 0.10mg per mL.

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

Chromatographic system (see Chromatography á621ñ)—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 1.0mLper 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 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C23H32O6in the portion of Hydrocortisone Acetate taken by the formula: in which Cis the concentration,in mg per mL,of USP Hydrocortisone Acetate RSin the Standard preparation;and rUand rSare the hydrocortisone acetate peak responses obtained from the Assay preparationand the Standard preparation,respectively.