Levonorgestrel and Ethinyl Estradiol Tablets
DEFINITION
Levonorgestrel and Ethinyl Estradiol Tablets contain NLT 90.0% and NMT 110.0% of the labeled amount of levonorgestrel (C21H28O2) and NLT 90.0% and NMT 110.0% of the labeled amount of ethinyl estradiol (C20H24O2).
IDENTIFICATION
•  A. The retention times of the two major peaks of the Sample solution correspond to those of levonorgestrel and ethinyl estradiol in the Standard solution, as obtained in the Assay.
•  B. Finely powder 20 Tablets and transfer a portion of the powder, equivalent to 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 min, and then stir it by mechanical means for 30 min. 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 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 1–2 mL of hot toluene, and transfer with a pipet to a small glass vial. Reduce the volume of the solution to 0.1 mL under nitrogen with warming. [Note—During 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 h.
Acceptance criteria:  The melting point of the dried crystals of levonorgestrel so obtained is not lower than 220, using the procedure described under Melting Range or Temperature 741, Class I.
ASSAY
•  Procedure
Mobile phase:  Acetonitrile, methanol, and water (35:15:45)
Standard solution:  15 µg/mL of USP Levonorgestrel RS and 3 µg/mL of USP Ethinyl Estradiol RS in Mobile phase
Sample solution:  Transfer a number of Tablets, equivalent to 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 min. Centrifuge, and use the clear supernatant.
Chromatographic system 
Mode:  LC
Detector:  UV 215 nm
Column:  4.6-mm × 15-cm; 5- to 7-µm packing L7
Flow rate:  1 mL/min
Injection size:  50 µL
System suitability 
Sample:  Standard solution
[Note—The relative retention times for ethinyl estradiol and levonorgestrel are about 0.7 and 1.0, respectively. ]
Suitability requirements 
Resolution:  NLT 2.5 between the two major peaks
Relative standard deviation:  NMT 2.0%
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of C21H28O2 and C20H24O2 in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak response of the corresponding analyte from the Sample solution
rS== peak response of the corresponding analyte from the Standard solution
CS== concentration of the appropriate USP Reference Standard in the Standard solution (µg/mL)
CU== nominal concentration of the corresponding analyte in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–110.0% of the labeled amount of C21H28O2, 90.0%–110.0% of the labeled amount of C20H24O2
PERFORMANCE TESTS
•  Dissolution 711: Determine the amount of C21H28O2 and C20H24O2 dissolved by employing the following method.
Medium:  Polysorbate 80 (5 µg/g) in water; 500 mL
Apparatus 2:  75 rpm
Time:  60 min
Mobile phase:  Acetonitrile and water (6:4)
Standard solution:  Prepare a solution of USP Levonorgestrel RS and USP Ethinyl Estradiol RS in Medium having known concentrations corresponding approximately to the concentrations that would be obtained by dissolving 1 Tablet in 500 mL of Medium.
[Note—A volume of alcohol not exceeding 2% of the final total volume of solution may be used to aid in dissolving the Reference Standards. ]
Sample 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 
Mode:  LC
Detector:  UV 247 nm (for levonorgestrel analysis); a spectrofluorometric detector (for ethinyl estradiol analysis), with an excitation wavelength of 285 nm, and an emission wavelength of 310 nm
Column:  4-mm × 15-cm; packing L7
Flow rate:  1 mL/min
Injection size:  100 µL
System suitability 
Sample:  Standard solution
[Note—The relative retention times for ethinyl estradiol and levonorgestrel are about 0.7 and 1.0, respectively. ]
Suitability requirements 
Relative standard deviation:  NMT 3.0%
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of C21H28O2 and C20H24O2 dissolved:
Result = (rU/rS) × (CS/CU) × 100
rU== peak response of the corresponding analyte from the Sample solution
rS== peak response of the corresponding analyte from the Standard solution
CS== concentration of the appropriate USP Reference Standard in the Standard solution (µg/mL)
CU== nominal concentration of the corresponding analyte in the Sample solution (µg/mL)
Tolerances 
Uncoated Tablets:  NLT 80% (Q) of the labeled amount of C21H28O2, and 75% (Q) of the labeled amount of C20H24O2 is dissolved.
Coated Tablets:  NLT 60% (Q) of the labeled amount of C21H28O2 and C20H24O2 is dissolved.
•  Uniformity of Dosage Units 905: Meet the requirements
Sample solution:  Place 1 Tablet in a 40-mL centrifuge tube, add 10.0 mL of Mobile phase, and proceed as directed in the Assay.
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in well-closed containers.
•  USP Reference Standards 11
USP Ethinyl Estradiol RS Click to View Structure
USP Levonorgestrel RS Click to View Structure
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
Topic/Question Contact Expert Committee
Monograph Mary S. Waddell
Scientific Liaison
1-301-816-8124
(SM42010) Monographs - Small Molecules 4
711 Margareth R.C. Marques, Ph.D.
Senior Scientific Liaison
1-301-816-8106
(GCDF2010) General Chapters - Dosage Forms
Reference Standards RS Technical Services
1-301-816-8129
rstech@usp.org
USP35–NF30 Page 3674
Pharmacopeial Forum: Volume No. 36(1) Page 117