Citrate buffer— Prepare a 0.0033Msolution of citric acid in water,adjust with 1Nsodium hydroxide to a pHof 6.2,and mix.

Diluting solution: a mixture of methanol and water (1:1).

Mobile phase— Prepare a filtered and degassed mixture of Citrate bufferand methanol (31:19).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

System suitability solution— Dissolve suitable quantities of prednisolone and hydrocortisone in a mixture of methanol and water (1:1)to obtain a solution containing about 100µg per mLand 90µg per mL,respectively.

Standard preparation— Dissolve an accurately weighed quantity of USP Prednisolone RSin Diluting solution,and dilute quantitatively,and stepwise if necessary,with Diluting solutionto obtain a solution having a known concentration of about 0.1mg per mL.

Assay preparation— Transfer an accurately measured volume of Oral Solution,equivalent to about 5.0mg of prednisolone,to a 50-mLvolumetric flask,dissolve in Diluting solution,shake by mechanical means for 15minutes,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 packing L10.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 0.8for hydrocortisone and 1.0for prednisolone;the resolution,R,between hydrocortisone and prednisolone is not less than 2.5;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 prednisolone (C21H28O5)in the volume of Oral Solution taken by the formula: in which Cis the concentration,in mg per mL,of USP Prednisolone RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.