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

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

Application volume: 30 µL.

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 105 for about 5 minutes, and examine the plate: meet the requirements.

Solution A: acetonitrile and 20 mM potassium phosphate buffer, pH 6.0 (50:50).

Solution B: acetonitrile and 20 mM potassium phosphate buffer, pH 6.0 (80:20).

Mobile phase— Use variable mixtures of Solution A and Solution B as directed under Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

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

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

Standard solution— Quantitatively dilute portions of Standard stock solution with Diluent to obtain a solution containing about 0.12 µg per mL of USP Ethinyl Estradiol RS.

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

Test solution 2— Dilute a portion of the Test solution 1 with Diluent to obtain a solution containing about 0.6 µg per mL of ethinyl estradiol.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 210-nm UV detector and a spectrofluorometric detector with an excitation wavelength of 285 nm and an emission wavelength of 310 nm; 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 the Standard solution, and record the peak responses as directed for Procedure: the tailing factor is not more than 2.0 for ethinyl estradiol; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Inject a volume (about 200 µL) of Test solution 2 into the chromatograph, record the chromatograms, and measure the peak heights for the major peaks obtained within 20 minutes. Calculate the percentage of 17-ethinyl estradiol in the portion of Tablets taken by the formula: in which rU is the height of any peak at the relative retention time of 1.16; and rS is the peak height of ethinyl estradiol obtained with the spectrofluorometric detector. Inject a volume (about 200 µL) of Test solution 1 into the chromatograph, record the chromatograms, and measure the peak heights for the major peaks obtained within 20 minutes. Calculate the percentage of estrone in the portion of Tablets taken by the formula: in which rU is the height of any peak at the relative retention time of 1.2; rS is the peak height of ethinyl estradiol obtained with the UV detector at 210 nm; and E is the percentage of 17-ethinyl estradiol obtained in the Tablets. Calculate the percentage of any other impurity taken by the formula: in which rU is the height of any peak other than those mentioned above; and rS is the peak height of ethinyl estradiol obtained with the UV detector. Not more than 0.5% of 17-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.

(Official January 1, 2007)

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and 20 mM potassium phosphate buffer, pH 6.0 (50:50). Make adjustments if necessary (see System Suitability under Chromatography 621).

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

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

Standard preparation— Dilute an appropriate aliquot of the Standard stock solution with Diluent to obtain a solution having a known concentration of about 0.12 µg per mL of USP Ethinyl Estradiol RS.

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

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a spectrofluorometric detector with an excitation wavelength of 285 nm and an emission wavelength of 310 nm, 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 2 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: 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 200 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C20H24O2 in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP ethinyl estradiol RS in the Standard preparation; V is the volume of the aliquot of solution taken for the Assay preparation; and rU and rS are the peak responses obtained for ethinyl estradiol from the Assay preparation and the Standard preparation, respectively.