Alcohol mixture— Prepare a mixture of dehydrated alcohol,isopropyl alcohol,and methanol (90:5:5).

Mobile phase— Prepare a filtered and degassed mixture of Alcohol mixture and water (55:45).Make adjustments if necessary (seeSystem SuitabilityunderChromatography á621ñ).

Diluted alcohol mixture— Prepare a mixture of Alcohol mixture and water (7:3).

Resolution solution— Dissolve an accurately weighed quantity of USP Methyltestosterone RSinMobile phase,and dilute quantitatively,and stepwise if necessary,withMobile phase to obtain a solution having a known concentration of about 0.4mg per mL.Separately and similarly prepare a solution inMobile phase having a known concentration of about 0.4mg of USP Progesterone RSper mL.Transfer 2.0mLof each solution to a 10-mLvolumetric flask,dilute withMobile phase to volume,and mix.

Standard preparation 1— Dissolve an accurately weighed quantity of USP Progesterone RSin n-propyl alcohol,and dilute quantitatively,and stepwise if necessary,with n-propyl alcohol to obtain a solution having a known concentration of about 0.25mg per mL.Mix 5.0mLof this solution with 10.0mLofDiluted alcohol mixture.

Standard preparation 2— Dissolve an accurately weighed quantity of USP Progesterone RSin n-propyl alcohol,and dilute quantitatively,and stepwise if necessary,with n-propyl alcohol to obtain a solution having a known concentration of about 3mg per mL.Transfer 3.0mLof this solution to a 100-mLvolumetric flask,dilute withDiluted alcohol mixture to volume,and mix.

Assay preparation 1— Transfer 1Suppository,containing not more than 100mg of progesterone,to a 100-mLvolumetric flask,dissolve in about 90mLof n-propyl alcohol,heat at 45for 4minutes,sonicate for 10minutes,cool,dilute with n-propyl alcohol to volume,and mix.Dilute quantitatively,and stepwise if necessary,with n-propyl alcohol,sonicating if necessary,to obtain a solution containing about 0.25mg of progesterone per mL.Transfer 5.0mLof this solution to a 50-mLcentrifuge tube,add 10.0mLof Diluted alcohol mixture,mix,sonicate for 1minute,and centrifuge for 10minutes at 2000rpm.Pass the supernatant through a filter having a 0.45-µm or finer porosity,discarding the first 4mLof the filtrate.

Assay preparation 2— Transfer 1Suppository,containing more than 100mg of progesterone,to a 200-mLvolumetric flask,dissolve in about 180mLof n-propyl alcohol,heat at 45for 8minutes,sonicate for 5minutes,cool,dilute with n-propyl alcohol to volume,and mix.Dilute quantitatively,and stepwise if necessary,sonicating each dilution for 1minute,to obtain a solution containing about 0.09mg of progesterone per mL.Transfer about 15mLof this solution to a 50-mLcentrifuge tube,and centrifuge for 10minutes at 2000rpm.Pass the supernatant through a filter having a 0.45-µm or finer porosity,discarding the first 4mLof the filtrate.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 245-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The column temperature is maintained at 40.The flow rate is about 1.0mLper minute.Chromatograph theResolution solution,and record the peak responses as directed forProcedure:the relative retention times are about 0.8for methyltestosterone and 1.0for progesterone;the resolution,R,between methyltestosterone and progesterone is not less than 2.0;the tailing factor is not more than 2.0for the progesterone peak;and the relative standard deviation for replicate injections is not more than 2.0%for progesterone.

Procedure— Separately inject equal volumes (about 10µL)ofStandard preparation 1andAssay preparation 1,or ofStandard preparation 2andAssay preparation 2,into the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in mg,of progesterone (C21H30O2)in the Suppository taken by the formula: in whichCis the concentration,in mg per mL,of USP Progesterone RSin theStandard preparation;Dis the dilution factor for obtaining theAssay preparation;andrUandrSare the peak responses obtained from theAssay preparation and theStandard preparation,respectively.

Progesterone (micronized)	25mg to 600mg
Silica Gel	25mg
Polyethylene Glycol Base,a sufficient quantity to make one suppository

Alcohol mixture,Mobile phase,Resolution solution,and Chromatographic system— Proceed as directed in theCompliance assay for suppositories compounded in fatty acid base.

Standard preparation— Dissolve an accurately weighed quantity of USP Progesterone RSinMobile phase,and dilute quantitatively,and stepwise if necessary,withMobile phase,to obtain a solution having a known concentration of about 0.1mg per mL.

Assay preparation— Dissolve 1Suppository in 200mLofMobile phase,and dilute quantitatively,and stepwise if necessary,withMobile phase to obtain a solution containing about 0.1mg of progesterone per mL.Pass a 10-mLportion of the mixture through a filter having a 0.45-µm or finer porosity,discarding the first 4mLof the filtrate.

Procedure— Separately inject equal volumes (about 10µL)of theStandard preparation and theAssay preparation into the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in mg,of progesterone (C21H30O2)in the Suppository taken by the formula: in whichCis the concentration,in mg per mL,of USP Progesterone RSin theStandard preparation;Dis the dilution factor for obtaining theAssay preparation;andrUandrSare the peak responses obtained from theAssay preparation and theStandard preparation,respectively.