Norethindrone Acetate and Ethinyl Estradiol Tablets
» Norethindrone Acetate and Ethinyl Estradiol Tablets contain not less than 90.0 percent and not more than 110.0 percent of the labeled amount of norethindrone acetate (C22H28O3), and not less than 88.0 percent and not more than 112.0 percent of the labeled amount of ethinyl estradiol (C20H24O2).
Packaging and storage—
Preserve in well-closed containers.
Identification—
A:
Infrared Absorption 
197K
—
197K—
Test specimen—
Wash the isooctane–toluene solution obtained in the Assay for ethinyl estradiol with 5 mL of water, filter, and evaporate to dryness.
B:
Thin-Layer Chromatographic Identification Test 201—
201—
Adsorbent—
Use either a 0.1-mm or a 0.25-mm layer of chromatographic silica gel mixture as described in the chapter. Activate the plate at 105
for 60 minutes immediately prior to use.
for 60 minutes immediately prior to use.
Test solution—
Crush 1 Tablet in 1 mL of alcohol in a 15-mL conical centrifuge tube, and centrifuge briefly.
Standard solution 1:
an alcohol solution containing in each mL an amount of USP Norethindrone Acetate RS, accurately weighed, corresponding to the labeled quantity of norethindrone acetate per Tablet.
Standard solution 2:
an alcohol solution containing in each mL 50 µg of USP Ethinyl Estradiol RS, accurately weighed.
Application volume:
5 µL.
Developing solvent system:
a mixture of chloroform and glacial acetic acid (95:5).
Procedure—
Proceed as directed in the chapter, except to heat the plate in an oven at 105 for 10 minutes after the solvent evaporates. Remove the plate from the oven, and while it is still hot, spray lightly with dilute sulfuric acid (3 in 4). Observe the plate under long-wavelength UV light: any red fluorescent spot produced by the Test solution at an RF value of about 0.4, and any yellow fluorescent spot produced by the Test solution at an RF value of about 0.2, are not greater in size and intensity than the corresponding spots from Standard solution 1 and Standard solution 2, respectively.
for 10 minutes after the solvent evaporates. Remove the plate from the oven, and while it is still hot, spray lightly with dilute sulfuric acid (3 in 4). Observe the plate under long-wavelength UV light: any red fluorescent spot produced by the Test solution at an RF value of about 0.4, and any yellow fluorescent spot produced by the Test solution at an RF value of about 0.2, are not greater in size and intensity than the corresponding spots from Standard solution 1 and Standard solution 2, respectively.
Change to read:
Dissolution 711—
0.025 M Acetate buffer solution—
Accurately weigh about 5.22 g of anhydrous sodium acetate and 2.2 g of glacial acetic acid into a 4-L volumetric flask, add 3.5 L of water, and mix. Adjust with 1 N sodium hydroxide to a pH of 5.0 ± 0.2, and dilute with water to volume.
Medium:
0.025 M pH 5.0 acetate buffer with 0.15% sodium lauryl sulfate, prepared by accurately weighing about 6 g of sodium lauryl sulfate into a 4-L volumetric flask, adding 1.5 L of 0.025 M Acetate buffer solution, mixing, and diluting with 0.025 M Acetate buffer solution to volume; 600 mL.
Apparatus 2:
75 rpm.
Time:
60 minutes.
Determine the amounts of norethindrone acetate (C22H28O3) and ethinyl estradiol (C20H24O2) dissolved by employing the following method.
Mobile phase—
Prepare a filtered and degassed mixture of 0.2% phosphoric acid, acetonitrile, and tetrahydrofuran (540:380:80). Make adjustments if necessary (see System Suitability under Chromatography 621).
621).
Standard solution—
Dissolve accurately weighed quantities of USP Norethindrone Acetate RS and USP Ethinyl Estradiol RS in a minimum amount of acetonitrile, and dilute quantitatively, and stepwise if necessary, with Medium to obtain a solution having known concentrations equivalent to the expected concentrations of the solution under test.
Test solution—
Withdraw a 2-mL aliquot using a glass pipet or syringe, and centrifuge at about 2000 rpm for about 5 minutes. Use the supernatant.
Chromatographic system—
The liquid chromatograph is equipped with a 242-nm detector and a fluorescent detector with an excitation wavelength set at 210 nm and an emission wavelength set at 310 nm, a 6-mm × 40-mm column that contains 3-µm packing L1, and a 4-mm × 12.5-mm guard column that contains 5-µm packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the column efficiency is not less than 500 theoretical plates for ethinyl estradiol and not less than 1400 theoretical plates for norethindrone acetate; the tailing factor for each analyte is not more than 2.0 
for both norethindrone acetate and ethinyl estradiol;USP32 and the relative standard deviation for each analyte is not more than 2.5%.
for both norethindrone acetate and ethinyl estradiol;USP32 and the relative standard deviation for each analyte is not more than 2.5%.
Procedure—
Separately inject equal volumes (about 200 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses.
Tolerances—
Not less than 80% (Q) of the labeled amounts of C22H28O3 and C20H24O2 is dissolved in 60 minutes.
Uniformity of dosage units 905:
meet the requirements.
905:
meet the requirements.
Procedure for content uniformity for ethinyl estradiol—
Place 1 Tablet in a 125-mL separator, add 5 mL of water, and shake until the Tablet has disintegrated completely. Add 25.0 mL of a mixture of toluene and isooctane (3:2), shake thoroughly, allow to settle, and remove and discard the aqueous phase. Transfer 20.0 mL of the isooctane–toluene solution to a second 125-mL separator, avoiding mechanical transfer of any of the aqueous phase. Transfer 8.0 mL of a solution of USP Ethinyl Estradiol RS in toluene, containing in each mL an amount of ethinyl estradiol equal to one-tenth of the labeled quantity per Tablet, to a third 125-mL separator, and add 12 mL of isooctane. To the second and third separators add 8.0 mL of a 1 in 25 solution of sodium hydroxide in dilute alcohol (1 in 10), shake, allow to settle, and transfer the sodium hydroxide solution from each separator into separate suitable containers. Proceed as directed in the Assay for ethinyl estradiol, beginning with “Add, dropwise, 5.0 mL of the sodium hydroxide extract”, but determine the absorbances of the final solutions using 5-cm cells. Calculate the quantity, in µg, of C20H24O2 in the Tablet taken by the formula:
10C(AU / AS)
in which C is the concentration, in µg per mL, of the Standard solution in toluene; and AU and AS are the absorbances of the test solution and the Standard solution, respectively.
Procedure for content uniformity for norethindrone acetate—
Transfer 1 finely powdered Tablet to a 100-mL volumetric flask with the aid of about 75 mL of alcohol, heat to boiling, and allow to remain at a temperature just below the boiling temperature for about 15 minutes, with occasional swirling. Cool to room temperature, dilute with alcohol to volume, and mix. Centrifuge a portion of the contents at about 2000 rpm until the solution becomes clear. If necessary, dilute a portion of the supernatant quantitatively with alcohol to provide a solution containing about 10 µg per mL of norethindrone acetate. Concomitantly determine the absorbances of this solution and of a solution of USP Norethindrone Acetate RS in alcohol having a known concentration of about 10 µg per mL in 1-cm cells at the wavelength of maximum absorbance at about 240 nm, with a suitable spectrophotometer, using alcohol as the blank. Calculate the quantity, in mg, of C22H28O3 in the Tablet taken by the formula:
(T/D)C(AU / AS)
in which T is the labeled quantity, in mg, of norethindrone acetate in the Tablet; D is the concentration, in µg per mL, of norethindrone acetate in the test solution, based on the labeled quantity per Tablet and the extent of dilution; C is the concentration, in µg per mL, of USP Norethindrone Acetate RS in the Standard solution; and AU and AS are the absorbances of the solution from the Tablet and the Standard solution, respectively.
Assay for norethindrone acetate—
Place 20 Tablets in a 125-mL separator, add 20 mL of water, and shake until the Tablets have disintegrated completely. Extract with three 30-mL portions of chloroform, filtering each extract through chloroform-moistened cotton into a round-bottom, 250-mL flask. Evaporate the combined extracts under vacuum to dryness, with the aid of gentle heat (not more than 40). Cool, add 5 mL of water and 100.0 mL of a mixture of isooctane and toluene (3:2), insert the stopper, and shake for 2 to 3 minutes. Transfer an accurately measured volume of the supernatant isooctane–toluene solution, containing about 1.5 mg of norethindrone acetate, to a round-bottom, 250-mL flask, and similarly evaporate under vacuum to dryness, taking care to ensure complete removal of residual toluene. [note—Retain the remaining isooctane–toluene solution for the Assay for ethinyl estradiol.] Dissolve the residue in 100.0 mL of alcohol, and mix. Concomitantly determine the absorbances of this solution and a solution of USP Norethindrone Acetate RS in the same medium, having a known concentration of about 15 µg per mL, in 1-cm cells at the wavelength of maximum absorbance at about 240 nm, with a suitable spectrophotometer, using alcohol as the blank. Calculate the quantity, in mg, of norethindrone acetate (C22H28O3) in the accurately measured volume of isooctane–toluene solution of the Tablets taken by the formula:
). Cool, add 5 mL of water and 100.0 mL of a mixture of isooctane and toluene (3:2), insert the stopper, and shake for 2 to 3 minutes. Transfer an accurately measured volume of the supernatant isooctane–toluene solution, containing about 1.5 mg of norethindrone acetate, to a round-bottom, 250-mL flask, and similarly evaporate under vacuum to dryness, taking care to ensure complete removal of residual toluene. [note—Retain the remaining isooctane–toluene solution for the Assay for ethinyl estradiol.] Dissolve the residue in 100.0 mL of alcohol, and mix. Concomitantly determine the absorbances of this solution and a solution of USP Norethindrone Acetate RS in the same medium, having a known concentration of about 15 µg per mL, in 1-cm cells at the wavelength of maximum absorbance at about 240 nm, with a suitable spectrophotometer, using alcohol as the blank. Calculate the quantity, in mg, of norethindrone acetate (C22H28O3) in the accurately measured volume of isooctane–toluene solution of the Tablets taken by the formula:
0.1C(AU / AS)
in which C is the concentration, in µg per mL, of USP Norethindrone Acetate RS in the Standard solution; and AU and AS are the absorbances of the solution from the Tablets and the Standard solution, respectively.
Assay for ethinyl estradiol—
Transfer 25.0 mL of the isooctane–toluene solution prepared from the Tablets as directed in the Assay for norethindrone acetate to a 125-mL separator, avoiding mechanical transfer of any of the aqueous phase. Transfer 10.0 mL of a toluene solution of USP Ethinyl Estradiol RS, containing in each mL a known amount of ethinyl estradiol equal to one-half of the labeled quantity per Tablet, to another 125-mL separator containing 15.0 mL of isooctane. Add 10.0 mL of a 1 in 25 solution of sodium hydroxide in dilute alcohol (1 in 10) to each separator, and shake gently for 3 minutes. Allow to settle, and transfer the sodium hydroxide solution from each separator into separate suitable containers. [note—Retain the isooctane–toluene solution for Identification test A.] Add, dropwise, 5.0 mL of the sodium hydroxide extract from the Tablets to 25.0 mL of dilute sulfuric acid (4 in 5) contained in a 150-mL beaker and previously chilled in an ice bath. Stir the acid solution continuously during the addition with the aid of a magnetic stirrer, and keep it in the ice bath. [note—Stir the acid solution rapidly and introduce the alkaline solution near the perimeter of the rapidly swirling acid solution, rather than near the vortex. Add the alkaline solution slowly, dropwise.] Treat 5.0 mL of the sodium hydroxide solution from the Standard in the same manner, and allow the solutions to reach room temperature. Concomitantly determine the absorbances of both solutions in 1-cm cells at the wavelength of maximum absorbance at about 536 nm, with a suitable spectrophotometer, using water as the blank. [note—Use 2-cm cells for Tablets labeled to contain 30 µg or less of ethinyl estradiol.] Calculate the quantity, in µg, of ethinyl estradiol (C20H24O2) in the portion of isooctane–toluene solution taken by the formula:
10C(AU / AS)
in which C is the concentration, in µg per mL, of USP Ethinyl Estradiol RS in the Standard solution; and AU and AS are the absorbances of the solutions from the Tablets and the Reference Standard, respectively.
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
Chromatographic Column—
| Topic/Question | Contact | Expert Committee |
| Monograph | Daniel K. Bempong, Ph.D.
Senior Scientist 1-301-816-8143 |
(MDPS05) Monograph Development-Pulmonary and Steroids |
| Reference Standards | Lili Wang, Technical Services Scientist 1-301-816-8129 RSTech@usp.org |
|
| 711 |
Margareth R.C. Marques, Ph.D.
Senior Scientist 1-301-816-8106 |
(BPC05) Biopharmaceutics05 |
USP32–NF27 Page 3109
Pharmacopeial Forum: Volume No. 33(3) Page 432
Chromatographic columns text is not derived from, and not part of, USP 32 or NF 27.