Mobile phase— Mix approximately equal volumes of acetonitrile and water,adjusting the ratio of solvents as necessary to achieve acceptable chromatography.

Internal standard solution— Dissolve Butylparaben in acetonitrile to obtain a solution having a concentration of 6µg per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Halcinonide RSin Internal standard solutionto obtain a solution having a known concentration of about 0.04mg per mL.Mix 5.0mLof this solution with 5.0mLof the Mobile phase.Each mLof the Standard preparationhas a known concentration of about 0.02mg of USP Halcinonide RS.

Assay preparation— Transfer an accurately weighed quantity of Ointment,equivalent to about 1mg of halcinonide,to a glass-stoppered,50-mLcentrifuge tube,and add 25.0mLof Internal standard solutionand 5.0mLof hexane.Place in a water bath at 58±2for 3minutes,then mix in a vortex mixer for about 1minute until the specimen is well dispersed.Repeat the above-specified heating and mixing step one more time.Cool in an ice-methanol bath for 15minutes or until the two phases separate,centrifuging if necessary.Transfer 5.0mLof the lower layer into a 15-mLcentrifuge tube,add 5.0mLof Mobile phase,and mix.

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 L1.The flow rate is about 2mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed under Procedure:the resolution,R,between the analyte and internal standard peaks is not less than 2.0,and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 0.6for butylparaben and 1.0for halcinonide.Calculate the quantity,in mg,of C24H32ClFO5in the portion of Ointment taken by the formula: in which Cis the concentration,in mg per mL,of USP Halcinonide RSin the Standard preparation,and RUand RSare the ratios of the peak responses of halcinonide to internal standard obtained from the Assay preparationand the Standard preparation,respectively.