A: Thin-Layer Chromatographic Identification Test 201—

B: The retention time and UV spectrum of the major peaks in the chromatogram of the Assay preparation correspond to those in the chromatogram of the Standard preparation, as obtained in the Assay.

Medium: 0.3% sodium lauryl sulfate in water; 500 mL.

Apparatus 2: 50 rpm.

Time: 30 minutes.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and water (55:45). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Prepare a solution of USP Estradiol RS in alcohol having an accurately known concentration of about 0.02 mg per mL. Prepare a solution of USP Norethindrone Acetate RS in alcohol having an accurately known concentration of about 0.01 mg per mL. Dilute both solutions quantitatively, and stepwise if necessary, with Medium to obtain a solution having a known concentration of both drugs similar to the one expected in the Test solution, assuming complete dissolution.

Test solution— Use portions of the solution under test passed through a suitable 0.45-µm filter.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 241/280 dual-band or wavelength-switchable UV detector and a 4.6-mm × 15-cm column that contains packing L1. The flow rate is about 1.0 mL per minute. Make an investigative run to determine the retention times for estradiol and norethindrone acetate so that the absorption of estradiol at 280 nm and norethindrone acetate at 241 nm can be included in a single run with a switch of the wavelength. Chromatograph replicate injections of the Standard solution, and record the peak area responses as directed for Procedure: the tailing factor 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 150 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the area responses for the major peaks. Calculate the percentage of C18H24O2 and of C22H28O3 in the portion of Tablets taken by the formula: in which rU and rS are the peak responses for the Test solution and the Standard solution, respectively; CS is the concentration, in mg per mL, of estradiol and norethindrone acetate in the Standard solution; 500 is the volume, in mL, of Medium; 100 is the conversion factor to percentage; and LC is the Tablet label claim, in mg, for estradiol and norethindrone acetate.

Tolerances— Not less than 75% (Q) of the labeled amounts of C18H24O2 and C22H28O3 are dissolved in 30 minutes.

Solution A— Prepare a mixture of water and tetrahydrofuran (200:1).

Solution B— Prepare a degassed solution of acetonitrile, water, and tetrahydrofuran (160:40:1).

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

Diluent— Prepare a mixture of water and dehydrated alcohol (1:1).

System suitability solution— Dissolve accurately weighed quantities of USP Estradiol RS, USP Norethindrone Acetate RS, and USP Estrone RS in Diluent to obtain a solution having known concentrations of about 240 µg per mL, 60 µg per mL, and 1 µg per mL, respectively.

Estradiol standard stock solution— Dissolve an accurately weighed quantity of USP Estradiol RS in alcohol to obtain a solution having a known concentration of about 250 µg of estradiol per mL.

Norethindrone acetate standard stock solution— Dissolve an accurately weighed quantity of USP Norethindrone Acetate RS in alcohol to obtain a solution having a known concentration of about 150 µg of norethindrone acetate per mL.

Standard solution— Combine 250 µL of Estradiol standard stock solution and 100 µL of Norethindrone acetate standard stock solution, and dilute with 50.0 mL of Diluent.

Test solution— Accurately weigh and finely powder 20 Tablets. Transfer the equivalent of 12 Tablets to an appropriate flask, and dissolve in a known volume of Diluent to obtain a solution having known concentrations of about 240 µg of estradiol per mL and 120 µg of norethindrone acetate per mL. Filter the solution, if necessary.

Chromatographic system— The liquid chromatograph is equipped with a dual wavelength detector (235 nm and 254 nm) and a 3.9-mm × 30-cm column that contains 4-µm packing L1. The flow rate is about 0.8 mL per minute. The chromatograph is programmed as follows. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the relative retention times are about 1.4 for estrone, about 3.0 for norethindrone acetate, and 1.0 for estradiol; and the resolution, R, between estrone and estradiol is not less than 1.3, measured at 254 nm.

Procedure— Separately inject equal volumes (about 100 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of any estradiol impurity in the portion of Tablets taken by the formula: in which F is the relative response factor of any estradiol impurity relative to estradiol; CS and CT are the concentrations of the Standard solution and the Test solution, respectively; ri is the peak area at 235 nm for each impurity obtained from the Test solution; and rS is the peak area at 235 nm obtained from the Standard solution. The Tablets meet the requirements given in Table 1. Calculate the percentage of any norethindrone acetate related impurities in the portion of Tablets taken by the formula: in which F is the relative response factor of any norethindrone acetate related impurity relative to norethindrone acetate; CS and CT are the concentrations of the Standard solution and the Test solution, respectively; ri is the peak area at 254 nm for each impurity obtained from the Test solution; and rS is the peak area at 254 nm obtained from the Standard solution. The Tablets meet the requirements given in Table 2. Not more than 0.5% of any individual unknown impurity is found and not more than 1.0% of total impurities (from Table 1 and Table 2) is found. [note—For the calculation of the percentage of any individual unknown impurity, rS is equal to the peak response of the smaller of the two major peaks in the Standard solution, and CS is the corresponding concentration in the Standard solution.]

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and water (55:45) (see Chromatography 621).

Diluent— Prepare a mixture of water and dehydrated alcohol (1:1).

Estrone standard stock solution— Transfer about 6.00 mg of USP Estrone RS, accurately weighed, to a 50-mL volumetric flask, and dissolve in 10 mL of dehydrated alcohol. Dilute with dehydrated alcohol to volume, and mix.

Estradiol standard stock solution— Prepare a solution of USP Estradiol RS in dehydrated alcohol having a known concentration of 0.25 mg per mL.

Norethindrone acetate standard stock solution— Prepare a solution of USP Norethindrone Acetate RS in dehydrated alcohol having a known concentration of 0.15 mg per mL.

Resolution solution— Transfer 800 µL of Estradiol standard stock solution, 600 µL of Norethindrone acetate standard stock solution, and 200 µL of Estrone standard stock solution to a suitable flask containing 10.0 mL of Diluent.

Standard preparation— Prepare a solution of Estradiol standard stock solution and Norethindrone acetate standard stock solution in Diluent having an accurately known concentration of about 20 µg per mL and 10 µg per mL, respectively.

Assay preparation— Add 12 Tablets into a measured amount of Diluent to obtain a solution having an estradiol concentration of about 20 µg per mL and a norethindrone acetate concentration of about 10 µg per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a diode array detector and a 4.6-mm × 15-cm column that contains packing L1. The flow rate is about 1.0 mL per minute. Perform an investigational run to determine the retention times for estradiol and norethindrone acetate. Thus, the absorption of estradiol at 280 nm and norethindrone acetate at 254 nm can be included in a single run by altering the wavelength. Chromatograph the Resolution solution, and record the peak areas as directed for Procedure: the resolution, R, between estradiol and estrone acetate is not less than 1.8. Chromatograph the Standard preparation, and record the peak area as directed for Procedure: the relative standard deviation for replicate injections is not more than 3%.

Procedure— Separately inject equal volumes (about 50 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the estradiol and norethindrone acetate peaks. Calculate the quantity, in mg, of estradiol (C18H24O2) in each of the Tablets taken by the formula: in which V is the volume, in mL, of Diluent taken to prepare the Assay preparation; C is the concentration, in mg per mL, of USP Estradiol RS in the Standard preparation; and rU and rS are the peak areas obtained from the Assay preparation and the Standard preparation, respectively. Calculate the quantity, in mg, of norethindrone acetate (C22H28O3) in each of the Tablets taken by the formula: in which V is the volume, in mL, of Diluent used in the Assay preparation; C is the concentration, in mg per mL, of USP Norethindrone Acetate RS in the Standard preparation; and rU and rS are the peak areas obtained from the Assay preparation and the Standard preparation, respectively.