A: Infrared Absorption á197Kñ.

B: Ultraviolet Absorption á197Uñ—

Solution: 10µg per mL.

Medium: methanol.Absorptivities at 242nm,calculated on the dried basis,do not differ by more than 3.0%.

Test solution: 10mg per mL,in chloroform.

Solution A— Prepare a filtered and degassed solution of 1g of monobasic potassium phosphate in 1000mLof water,adjust with 45%potassium hydroxide to a pHof 5.5,and mix.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Solution B— Use filtered and degassed acetonitrile.

Mobile phase— Use variable mixtures of Solution Aand Solution Bas directed forChromatographic system.

Solvent— Prepare a mixture of acetonitrile and Solution A(80:20).

Standard solution— Dissolve an accurately weighed quantity of USP Hydrocortisone Butyrate RSin Solventto obtain a solution having a known concentration of about 0.5mg per mL.

Test solution— Transfer about 50mg of Hydrocortisone Butyrate,accurately weighed,to a 50-mLvolumetric flask,dissolve in and dilute with Solventto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×10-cm column that contains 3-µm packing L1.The flow rate is about 2.0mLper minute.The chromatograph is programmed as follows. Chromatograph the Standard solutionand the Test solution,and record the peak responses as directed for Procedure:the resolution,R,between hydrocortisone butyrate and any impurity is not less than 1.0;and the column efficiency is not less than 10,000theoretical plates.

Procedure— Separately inject equal volumes (about 5µL)of theTest solutionand theStandard solutioninto the chromatograph,record the chromatograms,and measure the areas for the major peaks,disregarding any peak having a percentage of 0.05%or less.Calculate the percentage of each impurity in the portion of Hydrocortisone Butyrate taken by the formula: in which CSis the concentration,in mg per mL,of USP Hydrocortisone Butyrate RSin the Standard solution;CUis the concentration,in mg per mL,of Hydrocortisone Butyrate in the Test solution;riis the peak area for each impurity obtained from the Test solution;and rSis the peak area for hydrocortisone butyrate obtained from 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 water,acetonitrile,and glacial acetic acid (124:76:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Solvent— Prepare a mixture of tetrahydrofuran and glacial acetic acid (1000:1).

Diluting solution— Prepare a filtered solution of methanol,water,and glacial acetic acid (500:500:1).

Standard preparation— Dissolve an accurately weighed quantity of USP Hydrocortisone Butyrate RSin Solvent,and dilute quantitatively,and stepwise if necessary,with Solventto obtain a solution having a known concentration of about 0.1mg per mL.Transfer 10.0mLof this solution to a 50-mLvolumetric flask,dilute with Diluting solutionto volume,and mix.

System suitability solution— Dissolve suitable quantities of propyl 4-hydroxybenzoate and USP Hydrocortisone Butyrate RSin Solvent,and dilute quantitatively,and stepwise if necessary,with Solventto obtain a solution having known concentrations of about 0.1mg of each per mL.Transfer 10mLof this solution to a 50-mLvolumetric flask,dilute with Diluting solution to volume,and mix.

Assay preparation— Transfer about 50mg of Hydrocortisone Butyrate,accurately weighed,to a 50-mLvolumetric flask,dissolve in and dilute with Solventto volume,and mix.Transfer 5.0mLof this solution to a 50-mLvolumetric flask,dilute with Solventto volume,and mix.Transfer 10.0mLof this solution to a 50-mLvolumetric flask,dilute with Diluting solutionto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 3.0-mm ×10-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.7for propyl 4-hydroxybenzoate and 1.0for hydrocortisone butyrate;and the resolution,R,between propyl 4-hydroxybenzoate and hydrocortisone butyrate is not less than 4.0.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the column efficiency is not less than 4000theoretical plates;the tailing factor is not more than 1.6;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 5µL)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 C25H36O6in the portion of Hydrocortisone Butyrate taken by the formula: in which Cis the concentration,in mg per mL,of USP Hydrocortisone Butyrate RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.