Norethindrone and Mestranol Tablets
» Norethindrone and Mestranol Tablets contain not less than 90.0 percent and not more than 110.0 percent of the labeled amount of norethindrone (C20H26O2), and not less than 90.0 percent and not more than 110.0 percent of the labeled amount of mestranol (C21H26O2).
Packaging and storage—
Preserve in well-closed containers.
Identification—
Crush 1 Tablet in 1 mL of alcohol in a 15-mL conical centrifuge tube, and centrifuge briefly. Apply 10 µL of this test solution and 10 µL each of solutions containing, respectively, about 1 mg per mL of USP Norethindrone RS in alcohol and about 50 µg per mL of USP Mestranol RS in alcohol 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 equal volumes of ethyl acetate and cyclohexane in a suitable chamber, previously equilibrated with the solvent mixture, until the solvent front has moved about three-fourths of the length of the plate. Remove the plate, air-dry, and observe under short-wavelength UV light: the principal spot from the test solution appears at the same RF value as the principal spot from USP Norethindrone RS, at about RF 0.6. Spray the plate with a sulfuric acid and methanol mixture prepared by cautiously adding and mixing sulfuric acid in small increments to 30 mL of chilled anhydrous methanol in a 100-mL volumetric flask. Adjust to room temperature, dilute with sulfuric acid to volume, and mix. Heat the plate at 105 for 10 minutes: the pink spot from the test solution appears at the same RF value as the pink spot from USP Mestranol RS (about RF 0.8).
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 equal volumes of ethyl acetate and cyclohexane in a suitable chamber, previously equilibrated with the solvent mixture, until the solvent front has moved about three-fourths of the length of the plate. Remove the plate, air-dry, and observe under short-wavelength UV light: the principal spot from the test solution appears at the same RF value as the principal spot from USP Norethindrone RS, at about RF 0.6. Spray the plate with a sulfuric acid and methanol mixture prepared by cautiously adding and mixing sulfuric acid in small increments to 30 mL of chilled anhydrous methanol in a 100-mL volumetric flask. Adjust to room temperature, dilute with sulfuric acid to volume, and mix. Heat the plate at 105 for 10 minutes: the pink spot from the test solution appears at the same RF value as the pink spot from USP Mestranol RS (about RF 0.8).
Dissolution 711—
[note—Exercise care in filtering solutions containing mestranol 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.]
711—
[note—Exercise care in filtering solutions containing mestranol 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 lauryl sulfate in 0.1 N hydrochloric acid; 500 mL.
Apparatus 2:
75 rpm.
Time:
60 minutes.
Determine the amounts of norethindrone (C20H26O2) and mestranol (C21H26O2) dissolved, employing the following method.
Mobile phase—
Prepare a degassed and filtered mixture of water and acetonitrile (60:40). Make adjustments if necessary (see System Suitability under Chromatography 621).
621).
Chromatographic system
(see Chromatography 621)—The liquid chromatograph is equipped with a 205-nm detector and a 4.6-mm × 25-cm column that contains packing L10. 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 Mestranol RS in Dissolution Medium having known concentrations similar to those expected in the solution under test, and record the peak responses as directed for Procedure: the relative standard deviation is not more than 3.0%. The minimum number of theoretical plates for the mestranol peak is 4000, and the tailing factors for the norethindrone and mestranol peaks do not exceed 1.5.
621)—The liquid chromatograph is equipped with a 205-nm detector and a 4.6-mm × 25-cm column that contains packing L10. 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 Mestranol RS in Dissolution Medium having known concentrations similar to those expected in the solution under test, and record the peak responses as directed for Procedure: the relative standard deviation is not more than 3.0%. The minimum number of theoretical plates for the mestranol peak is 4000, and the tailing factors for the norethindrone and mestranol peaks do not exceed 1.5.
Procedure—
Separately inject equal volumes (about 200 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 0.4 for norethindrone and 1.0 for mestranol. Calculate the quantities of norethindrone and mestranol dissolved by comparison of the corresponding peak responses obtained from the Standard solution and the test solutions.
Tolerances—
Not less than 75% (Q) of the labeled amount of C20H26O2 and 75% (Q) of the labeled amount of C21H26O2 are dissolved in 60 minutes.
Uniformity of dosage units 905:
meet the requirements for Content Uniformity with respect to norethindrone and to mestranol.
905:
meet the requirements for Content Uniformity with respect to norethindrone and to mestranol.
Assay—
Mobile phase—
Prepare a filtered and degassed mixture of acetonitrile and water (50:50). Make adjustments if necessary (see System Suitability under Chromatography 621).
621).
Internal standard solution—
Transfer about 80 mg of progesterone into a 100-mL volumetric flask, add 50 mL of acetonitrile, dilute with water to volume, and mix.
Mestranol standard stock solution—
Dissolve an accurately weighed quantity of USP Mestranol RS in acetonitrile, and dilute quantitatively and stepwise with acetonitrile to obtain a solution having a known concentration of about 0.05 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 mg per mL.
Standard preparation—
Transfer 2.0 mL of Internal standard solution into a 100-mL volumetric flask. Add accurately measured volumes of Mestranol 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-fiftieth of the labeled amounts of the corresponding ingredients in the Tablets. Add 50 mL of water, dilute with acetonitrile to volume, and mix.
Assay preparation—
Transfer 10 Tablets to a 250-mL volumetric flask, add 50 mL of water, and shake by mechanical means until the Tablets are completely disintegrated. Add 10.0 mL of Internal standard solution and 165 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. Transfer 5.0 mL of this solution to a 10-mL volumetric flask, add 1.0 mL of acetonitrile, dilute with water to volume, and mix.
Chromatographic system
(see Chromatography 621)—The liquid chromatograph is equipped with a 200-nm detector and a 4.6-mm × 15-cm column that contains packing L7. The flow rate is about 1.0 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency determined from the mestranol peak is not less than 6000 theoretical plates, the resolution, R, between the progesterone and mestranol peaks is not less than 5.0, and the relative standard deviation for six replicate injections is not more than 2.0% (both peaks).
621)—The liquid chromatograph is equipped with a 200-nm detector and a 4.6-mm × 15-cm column that contains packing L7. The flow rate is about 1.0 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency determined from the mestranol peak is not less than 6000 theoretical plates, the resolution, R, between the progesterone and mestranol peaks is not less than 5.0, 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 2.5 for mestranol and 1.0 for norethindrone. Calculate the quantities, in mg, of norethindrone (C20H26O2) and mestranol (C21H26O2) in each Tablet taken by the formula:
50C(RU / RS)
in which C is the concentration, in mg per mL, of the appropriate USP Reference Standard in the 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.
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 3107
Chromatographic columns text is not derived from, and not part of, USP 32 or NF 27.