Test solution— Transfer about 1.5g of Cream to a glass-stoppered,50-mLcentrifuge tube.Add 15mLof a methanol–hydrochloric acid solution prepared by mixing 1volume of dilute hydrochloric acid (1in 120)with 4volumes of methanol.Shake to obtain a homogeneous mixture.Add 30mLof solvent hexane,mix for 10minutes,and centrifuge.Using a suitable syringe,transfer the lower aqueous phase to a second centrifuge tube,add about 20mLof water,and mix.Extract this aqueous mixture with chloroform by shaking,centrifuging,and removing the lower,chloroform phase with a syringe.Evaporate the chloroform on a steam bath with the aid of a stream of nitrogen to dryness,cool,and dissolve the residue in chloroform to obtain a solution containing about 150µg of betamethasone dipropionate per mL.

Standard solution: USP Betamethasone Dipropionate RSin chloroform containing 150µg per mL.

Application volume: 40µL.

Developing solvent system: a mixture of chloroform and acetone (7:1).

Procedure— Proceed as directed in the chapter.

Mobile phase— Prepare as directed in the Assayunder Betamethasone Dipropionate.

Internal standard solution— Prepare a solution of USP Beclomethasone Dipropionate RSin methanol containing acetic acid (1in 1000)having a known concentration of about 0.45mg per mL.

Standard preparation— Prepare a solution of USP Betamethasone Dipropionate RSin methanol containing acetic acid (1in 1000)having a known concentration of about 0.2mg per mL.Transfer 10.0mLof this solution to a suitable vial,and add 5.0mLof Internal standard solution,to obtain a Standard preparationhaving known concentrations of about 0.133mg of betamethasone dipropionate and about 0.15mg of beclomethasone dipropionate per mL.

Assay preparation— Transfer an accurately weighed quantity of Cream,equivalent to about 2mg of betamethasone dipropionate,into a capped 50-mLcentrifuge tube.Add 5.0mLof Internal standard solutionand 10.0mLof methanol containing acetic acid (1in 1000).Heat in a water bath at 60,shaking intermittently,until the assay specimen melts.Remove from the bath,and shake vigorously until the specimen has resolidified.Repeat the heating and shaking.Freeze in an ice-methanol bath for about 15minutes,and centrifuge at 2500rpm for about 5minutes.Transfer a portion of the supernatant to a suitable vial.

Procedure— Proceed as directed for Procedurein the Assayunder Betamethasone Dipropionate.Calculate the quantity,in mg,of betamethasone (C22H29FO5)in the portion of Cream taken by the formula: in which 392.46and 504.60are the molecular weights of betamethasone and betamethasone dipropionate,respectively;Cis the concentration,in mg per mL,of USP Betamethasone Dipropionate RSin the Standard preparation;and RUand RSare the peak height ratios of the betamethasone dipropionate peak and the internal standard peak obtained from the Assay preparationand the Standard preparation,respectively.