Test solution— Transfer 25Tablets to a suitable container,add 50mLof water,and sonicate until the tablets disintegrate (if needed,remove any coating with water before sonication).Place the sample in a separatory funnel,add 25mLof ether,and shake well to extract the actives.Using a glass pipet,transfer the ether layer to a clean beaker,and evaporate to about 10mL.

Standard solution— Dissolve an accurately weighed quantity of USP Ethinyl Estradiol RSin methanol to obtain a solution containing about 0.03mg per mL.

Application volume: 30µL.

Developing solvent system: a mixture of chloroform and alcohol (96:4).

Procedure— Proceed as directed in the chapter,and then air-dry.Spray the plate with a mixture of methanol and sulfuric acid (50:50),place in an oven at 105for about 5minutes,and examine the plate:meet the requirements.

Solution A: acetonitrile and 20mMpotassium phosphate buffer,pH6.0(50:50).

Solution B: acetonitrile and 20mMpotassium phosphate buffer,pH6.0(80:20).

Mobile phase— Use variable mixtures ofSolution AandSolution Bas directed underChromatographic system.Make adjustments if necessary (seeSystem Suitability underChromatography á621ñ).

Diluent— Prepare a mixture of acetonitrile and water (50:50).

Standard stock solution— Dissolve an accurately weighed quantity of USP Ethinyl Estradiol RSin methanol,and dilute quantitatively,and stepwise if necessary,with methanol to obtain a solution having a known concentration of about 0.3mg per mL.

Standard solution— Quantitatively dilute portions ofStandard stock solution withDiluent to obtain a solution containing about 0.12µg per mLof USP Ethinyl Estradiol RS.

Test solution 1— Transfer 20Tablets into a 200-mLvolumetric flask.Add about 120mLofDiluent,and shake for about 30minutes.Dilute withDiluent to volume,and mix.Centrifuge a portion of the dissolution sample,and use the clear supernatant.

Test solution 2— Dilute a portion of theTest solution 1withDiluent to obtain a solution containing about 0.6µg per mLof ethinyl estradiol.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 210-nm UVdetector and a spectrofluorometric detector with an excitation wavelength of 285nm and an emission wavelength of 310nm;a 4.6-mm ×15-cm column that contains packing L11;and a 4.6-mm ×12.5-mm guard column that contains packing L11.The chromatograph is programmed as follows: Chromatograph theStandard solution,and record the peak responses as directed forProcedure:the tailing factor is not more than 2.0for ethinyl estradiol;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Inject a volume (about 200µL)ofTest solution 2into the chromatograph,record the chromatograms,and measure the peak heights for the major peaks obtained within 20minutes.Calculate the percentage of 17b-ethinyl estradiol in the portion of Tablets taken by the formula: in whichrUis the height of any peak at the relative retention time of 1.16;andrSis the peak height of ethinyl estradiol obtained with the spectrofluorometric detector.Inject a volume (about 200µL)ofTest solution 1into the chromatograph,record the chromatograms,and measure the peak heights for the major peaks obtained within 20minutes.Calculate the percentage of estrone in the portion of Tablets taken by the formula: in whichrUis the height of any peak at the relative retention time of 1.2;rSis the peak height of ethinyl estradiol obtained with the UVdetector at 210nm;andEis the percentage of 17b-ethinyl estradiol obtained in the Tablets.Calculate the percentage of any other impurity taken by the formula: in whichrUis the height of any peak other than those mentioned above;andrSis the peak height of ethinyl estradiol obtained with the UVdetector.Not more than 0.5%of 17b-ethinyl estradiol is found;not more than 0.5%of estrone is found;not more than 0.1%of any unknown impurity is found;and not more than 2.0%of total impurities is found.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and 20mMpotassium phosphate buffer,pH6.0(50:50).Make adjustments if necessary (seeSystem Suitability underChromatography á621ñ).

Diluent— Prepare a mixture of acetonitrile and water (50:50).

Standard stock solution— Dissolve an accurately weighed quantity of USP Ethinyl Estradiol RSin methanol,and dilute quantitatively,and stepwise if necessary,with methanol to obtain a solution having a known concentration of about 0.3mg per mL.

Standard preparation— Dilute an appropriate aliquot of theStandard stock solution withDiluent to obtain a solution having a known concentration of about 0.12µg per mLof USP Ethinyl Estradiol RS.

Assay preparation— Transfer 20Tablets into a 200-mLvolumetric flask.Add about 120mLofDiluent,and shake for about 30minutes.Dilute withDiluent to volume,and mix.Centrifuge a portion of the solution so obtained,and transfer to a 50-mLvolumetric flask an accurately measured volume having a final concentration of about 0.12µg per mLof ethinyl estradiol.Dilute withDiluent to volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a spectrofluorometric detector with an excitation wavelength of 285nm and an emission wavelength of 310nm,a 4.6-mm ×15-cm column that contains packing L11,and a 4.6-mm ×1.25-mm guard column that contains packing L11.The flow rate is about 2mLper minute.Chromatograph theStandard preparation,and record the peak responses as directed forProcedure:the tailing factor for ethinyl estradiol is not more than 2.0;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 200µL)of theStandard preparation and theAssay preparation into the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C20H24O2in the portion of Tablets taken by the formula: in whichCis the concentration,in mg per mL,of USPethinyl estradiol RSin theStandard preparation;Vis the volume of the aliquot of solution taken for theAssay preparation;andrUandrSare the peak responses obtained for ethinyl estradiol from theAssay preparation and theStandard preparation,respectively.