Packaging and storage—
Preserve in well-closed containers.
Identification—
A:
Infrared Absorption 
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—
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.
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.
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.
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-liter volumetric flask, add 3.5 liters 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-liter volumetric flask, adding 1.5 liters 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).
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
Dissolution 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 as the Test solution.
Chromatographic system—
The liquid chromatograph is equipped with a 242-nm detector and a fluorescent detector with an excitation wavelength set at 210-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.
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 heights.
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.
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 C
20H
24O
2 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 C
22H
28O
3 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 assure 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 (C
22H
28O
3) 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 (C
20H
24O
2) 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.
