Levonorgestrel and Ethinyl Estradiol Tablets
» Levonorgestrel and Ethinyl Estradiol Tablets contain not less than 90.0 percent and not more than 110.0 percent of the labeled amount of levonorgestrel (C21H28O2) and not less than 90.0 percent and not more than 110.0 percent of the labeled amount of ethinyl estradiol (C20H24O2).
Packaging and storage Preserve in well-closed containers.
A: The retention times of the two major peaks in the chromatogram of the Assay preparation correspond to those of norgestrel and ethinyl estradiol in the Standard preparation, as obtained in the Assay.
B: Finely powder 20 Tablets and transfer a portion of the powder, equivalent to about 4 mg of levonorgestrel, to a suitable container. Add 250 mL of a solvent mixture consisting of isooctane and chloroform (3:1). Sonicate the mixture for 3 minutes, and then stir it by mechanical means for 30 minutes. Filter the mixture and evaporate the filtrate to dryness in a rotating vacuum evaporator. Dissolve the residue in 3 mL of chloroform, and transfer with a pipet to a 60-mL separator containing 18 mL of isooctane. Rinse the evaporator flask with an additional 3-mL portion of chloroform, and add the rinsing to the separator. Add 10 mL of 1 N sodium hydroxide, shake vigorously, and allow the layers to separate. Discard the lower aqueous phase, and filter the organic phase through about 3 g of anhydrous sodium sulfate on filter paper into a 50-mL beaker. Rinse the filter with several small portions of the mixture of isooctane and chloroform (3:1), adding the filtered rinsings to the filtrate, and evaporate under nitrogen on a steam bath to dryness. Dissolve the residue in 1 to 2 mL of hot toluene and transfer to a small glass vial with a pipet. Reduce the volume of the solution to about 0.1 mL under nitrogen with warming. [noteDuring this step, any crystals that deposit on the vial wall should be transferred to the bottom and allowed to redissolve.] Store the vial containing the clear toluene solution at 4 overnight to allow crystallization to occur. Remove and discard the mother liquor with a pipet, rinse the crystals with two 0.5-mL portions of anhydrous ether, and discard the rinsings. Dry the vial containing the rinsed crystals in a vacuum desiccator at 60 for 4 hours: the melting point (see Melting Range or Temperature 741) of the dried crystals of levonorgestrel so obtained, using the Class I method, is not lower than 220.
Medium: polysorbate 80 (5 µg per g) in water; 500 mL.
Apparatus 2: 75 rpm.
Time: 60 minutes.
Determine the amount of C21H28O2 and C20H24O2 dissolved by employing the following procedure.
Mobile phase Prepare a filtered and degassed solution of acetonitrile and water (60:40).
Standard solution [noteA volume of alcohol not exceeding 2% of the final total volume of solution may be used to aid in dissolving the Reference Standards.] Prepare a solution of USP Norgestrel RS and USP Ethinyl Estradiol RS in Medium having accurately known concentrations corresponding approximately to the concentrations that would be obtained by dissolving 1 Tablet in 500 mL of solvent.
Test solution Withdraw 15-mL portions of liquid from each vessel, and pass through a polyvinylidene filter, discarding the first 10 mL of the filtrate.
Chromatographic system (see Chromatography 621)The liquid chromatograph is equipped with a 247-nm detector (for levonorgestrel analysis), a spectrofluorometric detector (for ethinyl estradiol analysis) with an excitation wavelength of 285 nm and an emission wavelength of 310 nm, and a 4-mm × 15-cm column that contains packing L7. The flow rate is about 1 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.7 for ethinyl estradiol and 1.0 for levonorgestrel; and the relative standard deviation is not more than 3.0%.
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 responses for the major peaks. Calculate the percentage of C21H28O2 and C20H24O2 dissolved by the formula:
(0.5C)(100 / K)(rU / rS)in which C is the concentration, in µg per mL, of USP Norgestrel RS or USP Ethinyl Estradiol RS in the Standard solution; K is the labeled amount, in mg per Tablet, of C21H28O2 or C20H24O2; and rU and rS are the peak responses obtained from the Test solution and the Standard solution, respectively.
uncoated tabletsNot less than 80% (Q), of the labeled amount of levonorgestrel (C21H28O2) and 75% (Q) of the labeled amount of ethinyl estradiol (C20H24O2) are dissolved in 60 minutes.
coated tabletsNot less than 60% (Q) of the labeled amounts of levonorgestrel (C21H28O2) and ethinyl estradiol (C20H24O2) is dissolved in 60 minutes.
Uniformity of dosage units 905: meet the requirements.
Procedure for content uniformity Transfer 1 Tablet to a 40-mL centrifuge tube. Add 10.0 mL of Mobile phase and proceed as directed in the Assay.
Mobile phase Prepare a deaerated mixture containing 350 mL of acetonitrile, 150 mL of methanol, and 450 mL of water.
Standard preparation Transfer 15.0 mL of a solution of USP Norgestrel RS in Mobile phase and 3.0 mL of a solution of USP Ethinyl Estradiol RS in Mobile phase, each solution having a concentration of about 0.1 mg per mL, into a 100-mL volumetric flask. Dilute with Mobile phase to volume, and mix. Each mL of this Standard preparation has a known concentration of about 15 µg per mL and 3 µg per mL of USP Norgestrel RS and USP Ethinyl Estradiol RS, respectively.
Assay preparation Transfer a number of Tablets, equivalent to about 3 mg of levonorgestrel, to a 200-mL volumetric flask. Dilute with Mobile phase to volume, sonicate to disintegrate the Tablets, then shake by mechanical means for 20 minutes. Centrifuge, and use the clear, supernatant.
Chromatographic system (see Chromatography 621)The liquid chromatograph is equipped with a 215-nm detector and a 4.6-mm × 15-cm column that contains 5- to 7-µm packing L7. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak response as directed for Procedure: the resolution, R, between the two major peaks, is not less than 2.5; and the relative standard deviation for replicate injections is not more than 2.0.
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 responses for the major peaks. The relative retention times are about 0.7 for ethinyl estradiol and 1.0 for norgestrel. Calculate the quantities, in mg, of levonorgestrel (C21H28O2) and ethinyl estradiol (C20H24O2), respectively, in the portion of Tablets taken for the Assay preparation by the same formula:
0.2C(rU / rS)in which C is the concentration, in µg per mL, of the appropriate USP Reference Standard in the Standard preparation; and rU and rS are the peak responses of the corresponding analyte 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
USP32NF27 Page 2774
Chromatographic columns text is not derived from, and not part of, USP 32 or NF 27.