A: The retention times of the two major peaks in the chromatogram of the Assay preparationcorrespond to those of norgestrel and ethinyl estradiol in the Standard preparation,as obtained in the Assay.

B: Finely powder 20Tablets and transfer a portion of the powder,equivalent to about 4mg of levonorgestrel,to a suitable container.Add 250mLof a solvent mixture consisting of isooctane and chloroform (3:1).Sonicate the mixture for 3minutes,and then stir it by mechanical means for 30minutes.Filter the mixture and evaporate the filtrate to dryness in a rotating vacuum evaporator.Dissolve the residue in 3mLof chloroform,and transfer with a pipet to a 60-mLseparator containing 18mLof isooctane.Rinse the evaporator flask with an additional 3-mLportion of chloroform,and add the rinsing to the separator.Add 10mLof 1Nsodium hydroxide,shake vigorously,and allow the layers to separate.Discard the lower aqueous phase,and filter the organic phase through about 3g of anhydrous sodium sulfate on filter paper into a 50-mLbeaker.Rinse the filter with several small portions of the mixture of isooctane and chloroform (3:1),adding the filtered rinsings to the filtrate,and evaporate under nitrogen on a steam bath to dryness.Dissolve the residue in 1to 2mLof hot toluene and transfer to a small glass vial with a pipet.Reduce the volume of the solution to about 0.1mLunder nitrogen with warming.[NOTE—During this step,any crystals that deposit on the vial wall should be transferred to the bottom and allowed to redissolve.]Store the vial containing the clear toluene solution at 4overnight to allow crystallization to occur.Remove and discard the mother liquor with a pipet,rinse the crystals with two 0.5-mLportions of anhydrous ether,and discard the rinsings.Dry the vial containing the rinsed crystals in a vacuum desiccator at 60for 4hours:the melting point (see Melting Range or Temperature á741ñ)of the dried crystals of levonorgestrel so obtained,using the Class Imethod,is not lower than 220.

Medium: polysorbate 80(5µg per g)in water;500mL.

Apparatus 2: 75rpm.

Time: 60minutes.

Mobile phase— Prepare a filtered and degassed solution of acetonitrile and water (60:40).

Standard solution— [NOTE—Avolume of alcohol not exceeding 2%of the final total volume of solution may be used to aid in dissolving the Reference Standards.]Prepare a solution of USP Norgestrel RSand USP Ethinyl Estradiol RSin Mediumhaving accurately known concentrations corresponding approximately to the concentrations that would be obtained by dissolving 1Tablet in 500mLof solvent.

Test solution— Withdraw 15-mLportions of liquid from each vessel,and pass through a polyvinylidene filter,discarding the first 10mLof the filtrate.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 247-nm detector (for levonorgestrel analysis),a spectrofluorometric detector (for ethinyl estradiol analysis)with an excitation wavelength of 285nm and an emission wavelength of 310nm,and a 4-mm ×15-cm column that contains packing L7.The flow rate is about 1mLper minute.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.7for ethinyl estradiol and 1.0for levonorgestrel;and the relative standard deviation is not more than 3.0%.

Procedure— Separately inject equal volumes (about 100µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the percentage of C21H28O2and C20H24O2dissolved by the formula: in which Cis the concentration,in µg per mL,of USP Norgestrel RSor USP Ethinyl Estradiol RSin the Standard solution;Kis the labeled amount,in mg per Tablet,of C21H28O2or C20H24O2;and rUand rSare the peak responses obtained from the Test solutionand the Standard solution,respectively.

Tolerances—

Procedure for content uniformity— Transfer 1Tablet to a 40-mLcentrifuge tube.Add 10.0mLof Mobile phaseand proceed as directed in the Assay.

Mobile phase— Prepare a deaerated mixture containing 350mLof acetonitrile,150mLof methanol,and 450mLof water.

Standard preparation— Transfer 15.0mLof a solution of USP Norgestrel RSin Mobile phaseand 3.0mLof a solution of USP Ethinyl Estradiol RSin Mobile phase,each solution having a concentration of about 0.1mg per mL,into a 100-mLvolumetric flask.Dilute with Mobile phaseto volume,and mix.Each mLof this Standard preparationhas a known concentration of about 15µg per mLand 3µg per mLof USP Norgestrel RSand USP Ethinyl Estradiol RS,respectively.

Assay preparation— Transfer a number of Tablets,equivalent to about 3mg of levonorgestrel,to a 200-mLvolumetric flask.Dilute with Mobile phaseto volume,sonicate to disintegrate the Tablets,then shake by mechanical means for 20minutes.Centrifuge,and use the clear,supernatant.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 215-nm detector and a 4.6-mm ×15-cm column that contains 5-to 7-µm packing L7.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the peak response as directed for Procedure:the resolution,R,between the two major peaks,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 50µL)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 0.7for ethinyl estradiol and 1.0for norgestrel.Calculate the quantities,in mg,of levonorgestrel (C21H28O2)and ethinyl estradiol (C20H24O2),respectively,in the portion of Tablets taken for the Assay preparationby the same formula: in which Cis the concentration,in µg per mL,of the appropriate USP Reference Standard in the Standard preparation,and rUand rSare the peak responses of the corresponding analyte obtained from the Assay preparationand the Standard preparation,respectively.