A: Infrared Absorption á197Mñ.

B: Ultraviolet Absorption á197Uñ—

Test solution: 10mg per mL,in acetone.

Mobile phase— Prepare a filtered and degassed mixture of water,acetonitrile,and methanol (700:285:15).Add 3.0mLof glacial acetic acid per Liter of this solution.Mix thoroughly.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Diluting solution— Prepare a mixture of water,acetonitrile,tetrahydrofuran,and glacial acetic acid (500:250:250:1).Mix thoroughly.

Standard solution— Dissolve an accurately weighed quantity of USP Hydrocortisone Hemisuccinate RSin Diluting solution,and dilute quantitatively,and stepwise if necessary,with Diluting solutionto obtain a solution having a known concentration of about 6.6µg per mL.

Test solution— Transfer about 6.6mg of Hydrocortisone Hemisuccinate,accurately weighed,to a 10-mLvolumetric flask,dissolve in and dilute with Diluting solutionto volume,and mix.[NOTE—Samples should be maintained at 5or colder during analysis.]

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 packing L1.The flow rate is about 0.8mLper minute.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the column efficiency is not less than 5000theoretical plates;and the relative standard deviation for replicate injections is not more than 5.0%.

Procedure— Inject a volume (about 15µL)of the Test solutioninto the chromatograph,record the chromatogram,and measure the peak responses.Calculate the percentage of each impurity in the portion of Hydrocortisone Hemisuccinate taken by the formula: in which riis the peak response for each impurity;and rsis the sum of the responses of all the peaks:not more than 1.0%of any individual impurity is found;and not more than 2.0%of total impurities is found.Disregard any peak representing less than 0.05%.

Internal standard solution— Prepare a solution of USP Fluorometholone RSin tetrahydrofuran containing about 3mg per mL.

Mobile phase— Prepare a filtered mixture of butyl chloride,water-saturated butyl chloride,tetrahydrofuran,methanol,and glacial acetic acid (95:95:14:7:6).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Transfer an accurately weighed amount of USP Hydrocortisone Hemisuccinate RSto a suitable container to obtain a solution containing 0.6mg per mL.Add an accurately measured volume of Internal standard solutionso that the Standard preparationcontains 10%Internal standard solution.Dilute with chloroform containing 3%glacial acetic acid to volume.

Assay preparation— Transfer about 30mg of Hydrocortisone Hemisuccinate,accurately weighed,to a 50-mLvolumetric flask,add 5.0mLof Internal standard solution,and dilute with chloroform containing 3%glacial acetic acid to volume.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm ×30-cm column that contains packing L3.The flow rate is about 1.0mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the resolution,R,between hydrocortisone hemisuccinate and the internal standard is not less than 2.0;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 6µL)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 C25H34O8in the portion of Hydrocortisone Hemisuccinate taken by the formula: in which Cis the concentration,in mg per mL,of USP Hydrocortisone Hemisuccinate RSin the Standard preparation;and RUand RSare the peak area ratios of hydrocortisone hemisuccinate to the internal standard obtained from the Assay preparationand the Standard preparation,respectively.