A: Infrared Absorption á197Mñ.

B: Ultraviolet Absorption á197Uñ—

Test solution: 10mg per mL,in dioxane.

Mobile phase— Prepare a filtered and degassed mixture of butyl chloride,tetrahydrofuran,methanol,glacial acetic acid,and water (890:56:28:24:0.4),and sonicate to dissolve.Make adjustments if necessary (see System SuitabilityunderChromatography á621ñ).

Diluting solution— Prepare a solution of butyl chloride,tetrahydrofuran,methanol,and glacial acetic acid (81.5:10:8:0.5).

Standard solution— Prepare a solution in Diluting solutioncontaining 40µg of USP Hydrocortisone RSper mL.Sonicate for about 5minutes.

Test solution— Transfer 20mg of Hydrocortisone to a 10-mLvolumetric flask,dissolve in Diluting solution,add Diluting solutionto volume to obtain a solution having a known concentration of about 2.0mg per mL,and sonicate for about 5minutes.

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 L3.The flow rate is about 1.5mLper minute.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the tailing factor is not more than 2.0;and the relative standard deviation for replicate injections is not more than 5%.

Procedure— Separately inject equal volumes (about 5µL)of the Standard solution,the Diluting solution,and the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for all the peaks,ignoring artifact peaks.Calculate the percentage of impurities in the portion of Hydrocortisone taken by the formula: in which Cis the concentration,in mg per mL,of USP Hydrocortisone RSin the Standard solution;Wis the weight,in mg,of Hydrocortisone taken;riis the response of each individual impurity peak in the Test solution;and rSis the response of the major peak obtained from the Standard solution:not more than 0.5%of any individual impurity and not more than 2.0%of total impurities is found.

Mobile phase— Prepare a filtered and degassed mixture of water,acetonitrile,and methanol (50:25:25).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Diluent— Prepare a mixture of methanol and water (1:1).

Internal standard solution— Prepare a solution of propylparaben in methanol having a concentration of about 1mg per mL.

Standard stock solution— Dissolve an accurately weighed quantity of USP Hydrocortisone RSin methanol to obtain a solution having a known concentration of about 1mg per mL.

Standard preparation— Transfer 2.0mLof Standard stock solutionand 2.0mLof Internal standard solutionto a 50-mLvolumetric flask,dilute with Diluentto volume,and mix.

Assay preparation— Transfer about 50mg of Hydrocortisone,accurately weighed,to a 50-mLvolumetric flask,dissolve in and dilute with methanol to volume,and mix.Transfer 2.0mLof this solution and 2.0mLof Internal standard solutionto a 50-mLvolumetric flask,dilute with Diluentto 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 5-µm packing L1.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 1.8for propylparaben and 1.0for hydrocortisone;the resolution,R,between the hydrocortisone and propylparaben peaks is not less than 9.0;the column efficiency is not less than 3000theoretical plates for hydrocortisone;the tailing factor is not more than 1.2;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 C21H30O5in the portion of Hydrocortisone taken by the formula: in which Cis the concentration,in mg per mL,of USP Hydrocortisone RSin the Standard preparation;and RUand RSare the ratios of the peak response of hydrocortisone to that of propylparaben obtained from the Assay preparationand the Standard preparation,respectively.