A: Infrared Absorption á197Kñ—If a difference appears,dissolve portions of both the test specimen and the Reference Standard in methanol,evaporate the solutions to dryness,and repeat the tests.

B: Dissolve about 6mg in 2mLof sulfuric acid,and allow to stand for 5minutes:an orange color is produced.Pour the solution into 10mLof water:the color changes first to yellow and then,gradually,to bluish green.

Test solution: 5mg per mL,in dioxane.

Mobile phase— Prepare a filtered and degassed mixture of chloroform and methanol (98:2).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Test solution— Transfer about 25mg of Prednisone,accurately weighed,to a suitable container,dissolve in 20mLof Mobile phase,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 6.0-mm ×4.0-cm column that contains packing L3.The flow rate is about 1mLper minute.Chromatograph the Test solution,and record the peak responses as directed for Procedure:the column efficiency is not less than 2,500theoretical plates;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Inject a volume (about 5µL)of the Test solutioninto the chromatograph,record the chromatogram,and measure the peak responses.Calculate the percentage of each impurity in the portion of Prednisone taken by the formula: in which riis the peak response for each impurity;and rsis the sum of the responses of all peaks:not more than 1.5%of any individual impurity is found,and not more than 2.0%of total impurities is found.

Mobile phase— Prepare a suitable filtered mixture of water,peroxide-free tetrahydrofuran,and methanol (688:250:62)such that at a flow rate of 1.0mLper minute,the retention times of prednisone and acetanilide are about 8and 6minutes,respectively.

Internal standard solution— Prepare a solution of acetanilide in dilute methanol (1in 2)having a concentration of about 110µg per mL.

Standard preparation— Using an accurately weighed quantity of USP Prednisone RS,prepare a solution in dilute methanol (1in 2)having a known concentration of about 0.2mg per mL.Transfer 5.0mLof this solution and 5.0mLof the Internal standard solutionto a 50-mLvolumetric flask.Add dilute methanol (1in 2)to volume,and mix to obtain a Standard preparationhaving a known concentration of about 20µg of USP Prednisone RSper mL.Prepare this solution fresh.

Assay preparation— Using about 50mg of Prednisone,accurately weighed,proceed as directed for Standard preparation,beginning with “prepare a solution in dilute methanol (1in 2).”

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4-mm ×25-cm column that contains packing L1.Chromatograph five replicate injections of the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation is not more than 2.0%;and the resolution factor between prednisone and the internal standard is not less than 3.Adjust the operating parameters so that the peak obtained from the Standard preparationis about one-half full-scale.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph by means of a suitable microsyringe or sampling valve,record the chromatograms,and measure the responses at equivalent retention times.Calculate the quantity,in mg,of C21H26O5in the portion of Prednisone taken by the formula: in which Cis the concentration,in µg per mL,of USP Prednisone RSin the Standard preparation;and RUand RSare the peak response ratios of the prednisone peak to the internal standard peak obtained from the Assay preparationand the Standard preparation,respectively.