Identification—
Crush 1 Tablet in 1 mL of alcohol in a 15-mL conical centrifuge tube, and warm to 50
for 10 minutes with gentle swirling. Cool, and centrifuge to obtain a clear solution. Apply 20 µL of this test solution and 20 µL of an alcohol solution containing, in each mL, about 1 mg of
USP Norethindrone RS and about 50 µg of
USP Ethinyl Estradiol RS at equidistant points along a line about 2.5 cm from the bottom of a thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of chromatographic silica gel and previously activated by heating at 105
for 30 minutes. Develop the chromatogram in a mixture of benzene and ethyl acetate (4:1) in a suitable chamber, previously equilibrated with the solvent mixture, until the solvent front has moved about 10 cm. Remove the plate, and air-dry. Spray the plate with sulfuric acid-methanol, prepared by cautiously adding 70 mL of sulfuric acid in small increments to 30 mL of methanol chilled in an ice-bath, and mixing: the spots from the test solution have the same
RF values as the spots from
USP Ethinyl Estradiol RS (about 0.7) and from
USP Norethindrone RS (about 0.4).
Dissolution 711—
[NOTE—Exercise care in filtering and handling solutions containing ethinyl estradiol to prevent adsorptive loss of the drug. Centrifugation may be used instead of filtration with nonadsorptive membrane filters. Withdraw dissolution aliquots with glass or polytef pipets or syringes that have been checked for adsorptive loss. Use glass dissolution vessels and polytef-coated or solid polytef paddles.
]
Medium:
0.09% sodium dodecyl sulfate in 0.1 N hydrochloric acid; 500 mL.
Apparatus 2:
75 rpm.
Time:
60 minutes.
Determine the amounts of norethindrone (C20H26O2) and ethinyl estradiol (C20H24O2) dissolved, employing the following method.
Mobile phase—
Prepare a degassed and filtered mixture of 0.02
M pH 6.0 phosphate buffer and acetonitrile (65:35). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 200-nm detector, and a 5-mm × 8.3-cm column that contains 3-µm packing L1. The flow rate is about 1 mL per minute. Chromatograph replicate injections of a filtered portion of a Standard solution of
USP Norethindrone RS and
USP Ethinyl Estradiol RS in
Dissolution Medium having concentrations similar to those expected in the solution under test.
[NOTE—A volume of methanol not exceeding 4% of the total final volume of the Standard solution may be used in preparing the Standard solution.
] Record the peak responses as directed under
Procedure: the relative standard deviation is not more than 3.0%, the minimum number of theoretical plates for the ethinyl estradiol peak is not less than 7000, the resolution,
R, for norethindrone and ethinyl estradiol is not less than 1.5, and the tailing factor does not exceed 2.0 for either peak.
Procedure—
Separately inject equal volumes (about 100 µL) of the Standard solution and a filtered portion of the solution under test into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times are about 0.9 for norethindrone and 1.0 for ethinyl estradiol. Calculate the quantities of norethindrone (C20H26O2) and ethinyl estradiol (C20H24O2) dissolved by comparison of the corresponding peak responses obtained from the Standard solution and the solution under test.
Tolerances—
Not less than 75% (Q) of each of the labeled amounts of C20H26O2 and C20H24O2, respectively, are dissolved in 60 minutes.
Assay—
Mobile phase—
Prepare a filtered and degassed mixture of acetonitrile and water (60:40). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Internal standard solution—
Transfer about 15 mg of valerophenone into a 250-mL volumetric flask, add 125 mL of acetonitrile, dilute with water to volume, and mix.
Ethinyl estradiol standard stock solution—
Dissolve an accurately weighed quantity of
USP Ethinyl Estradiol RS in acetonitrile, and dilute quantitatively and stepwise with acetonitrile to obtain a solution having a known concentration of about 0.09 mg per mL.
Norethindrone standard stock solution—
Using an accurately weighed quantity of
USP Norethindrone RS, prepare a solution in acetonitrile having a known concentration of about 1.25 mg per mL.
Mixed standard preparation—
Transfer 5.0 mL of
Internal standard solution into a 100-mL volumetric flask. Add accurately measured volumes of
Ethinyl estradiol standard stock solution and
Norethindrone standard stock solution so that the final known concentrations, in mg per mL, of the Reference Standards correspond numerically to about one-twentieth of the labeled amounts of the corresponding ingredients in the Tablets. Add (26
X) mL of acetonitrile,
X being the total volume of the
standard stock solution taken. Dilute with a mixture of acetonitrile and water (45 in 100) to volume, and mix.
Assay preparation—
Transfer 10 Tablets to a 100-mL volumetric flask, add 20 mL of water, and shake by mechanical means until the tablets are completely disintegrated. Add 10.0 mL of Internal standard solution and 60 mL of acetonitrile, and mix. Sonicate for about 2 minutes. Dilute with acetonitrile to volume, and mix. Allow solid particles to settle, or centrifuge if necessary to obtain a slightly turbid solution. Dilute 5.0 mL of this solution with a mixture of acetonitrile and water (45 in 100) to 10.0 mL, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 200-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1.0 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed under
Procedure: the column efficiency determined from the internal standard peak is not less than 8000 theoretical plates, the resolution,
R, between the norethindrone and ethinyl estradiol peaks is not less than 2.0, and the relative standard deviation for six replicate injections is not more than 2.0% (both peaks).
Procedure—
Separately inject equal volumes (about 25 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times are about 0.9 for ethinyl estradiol and 1.0 for norethindrone. Calculate the quantities, in mg, of norethindrone (C
20H
26O
2) and ethinyl estradiol (C
20H
24O
2) in each Tablet taken by the formula:
20C(RU / RS),
in which
C is the concentration, in mg per mL, of the appropriate USP Reference Standard in the
Mixed standard preparation, and
RU and
RS are the peak response ratios, at corresponding retention times, obtained from the
Assay preparation and the
Standard preparation, respectively.
