Estradiol and Norethindrone Acetate Tablets
DEFINITION
Estradiol and Norethindrone Acetate Tablets contain NLT 90.0% and NMT 110.0% of the labeled amount of estradiol (C18H24O2) and NLT 90.0% and NMT 110.0% of the labeled amount of norethindrone acetate (C22H28O3).
IDENTIFICATION
•  A. Thin-Layer Chromatographic Identification Test 201
Standard solution:  0.5 mg/mL of USP Estradiol RS and 0.25 mg/mL of USP Norethindrone Acetate RS in dehydrated alcohol
Sample solution:  Place 2 Tablets into a 10-mL vial, and add 0.2 mL of water. When the Tablets are partially disintegrated, add a few glass beads, and shake vigorously to disintegrate. Add 4.0 mL of dehydrated alcohol, and shake. [Note—Centrifuge until the supernatant is clear before application to the plate. ]
Adsorbent:  0.25-mm chromatographic silica gel plate
Application volume:  2 µL
Developing solvent system:  Chloroform and acetone (9:1)
Analysis 
Samples:  Standard solution and Sample solution
Proceed as directed in the chapter, using the Developing solvent system. Apply the Samples and develop the plate. After removal of the plate, mark the solvent front, and allow the solvent to evaporate. Place the plate on a heating plate at 100 for 15 min. Allow the plate to cool, and then immerse it in a mixture of dehydrated alcohol and concentrated sulfuric acid (95:5). Place the plate on a piece of thick horizontal paper until it is almost dry. Heat the plate at 100 until it has fully developed. Examine under UV light at 365 nm.
Acceptance criteria:  The color and RF value of the principal spots of the Sample solution correspond to those of the Standard solution.
•  B. The retention time of the major peaks of the Sample solution correspond to those of the Standard solution, as obtained in the Assay.
ASSAY
•  Procedure
Mobile phase:  Acetonitrile and water (11:9)
Diluent:  Dehydrated alcohol and water (1:1)
Estrone standard stock solution:  0.12 mg/mL of USP Estrone RS in dehydrated alcohol
Estradiol standard stock solution:  0.25 mg/mL of USP Estradiol RS in dehydrated alcohol
Norethindrone acetate standard stock solution:  0.15 mg/mL of USP Norethindrone Acetate RS in dehydrated alcohol
Standard solution:  20 µg/mL of USP Estradiol RS from the Estradiol standard stock solution and 10 µg/mL of USP Norethindrone Acetate RS from the Norethindrone acetate standard stock solution in Diluent
System suitability solution:  Combine 800 µL of the Estradiol standard stock solution, 600 µL of the Norethindrone acetate standard stock solution, 200 µL of the Estrone standard stock solution, and 10.0 mL of Diluent.
Sample solution:  Add 12 Tablets into a measured amount of Diluent to obtain a solution having an estradiol concentration of 20 µg/mL and a norethindrone acetate concentration of 10 µg/mL.
Chromatographic system  
Mode:  LC
Detector:  UV Dual wavelength (254 nm/280 nm) or equivalent
[Note—The absorption of estradiol at 280 nm and norethindrone acetate at 254 nm can be included in a single run by altering the wavelength. ]
Column:  4.6-mm × 15-cm; packing L1
Flow rate:  1 mL/min
[Note—Perform an investigational run to determine the retention times for estradiol and norethindrone acetate. ]
Injection size:  50 µL
System suitability 
Samples:  Standard solution and System suitability solution
Suitability requirements 
Resolution:  NLT 1.8 between estradiol and estrone, System suitability solution
Relative standard deviation:  NMT 3%, Standard solution
Analysis 
Samples:  Standard solution and Sample solution
[Note—Measure the areas for the estradiol and norethindrone acetate peaks. ]
Calculate the quantity, as a percentage, of C18H24O2 in each of the Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak area from the Sample solution
rS== peak area from the Standard solution
CS== concentration of USP Estradiol RS in the Standard solution (µg/mL)
CU== nominal concentration of estradiol in the Sample solution (µg/mL)
Calculate the quantity, as a percentage, of C22H28O3 in each of the Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak area from the Sample solution
rS== peak area from the Standard solution
CS== concentration of USP Norethindrone Acetate RS in the Standard solution (µg/mL)
CU== nominal concentration of norethindrone acetate in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–110.0% of the labeled amount of C18H24O2 and 90.0%–110.0% of the labeled amount of C22H28O3
PERFORMANCE TESTS
•  Dissolution 711
Medium:  0.3% sodium lauryl sulfate; 500 mL
Apparatus 2:  50 rpm
Time:  30 min for Tablets labeled to contain 1 mg of estradiol and 0.5 mg of norethindrone acetate, and 50 min for Tablets labeled to contain 0.5 mg of estradiol and 0.1 mg of norethindrone acetate
Mobile phase:  Acetonitrile and water (11:9)
Standard stock solution A:  20 µg/mL of USP Estradiol RS in alcohol or in a mixture of alcohol and water
Standard stock solution B:  10 µg/mL of USP Norethindrone Acetate RS in alcohol or in a mixture of alcohol and water
Standard solution:  Dilute suitable quantities of Standard stock solution A and Standard stock solution B in Medium or a mixture of alcohol and water to obtain a final concentration of both analytes similar to the expected concentration of the Sample solution.
Sample solution:  Pass a portion of the solution through a filter of 0.45-µm pore size.
Chromatographic system 
Mode:  LC
Detector:  UV 241 nm for norethindrone acetate and 280 nm for estradiol
Column:  4.6-mm × 15-cm; packing L1
Flow rate:  1 mL/min
Injection volume:  150 µL
System suitability 
Sample:  Standard solution
Suitability requirements 
Tailing factor:  NMT 1.5
Relative standard deviation:  NMT 2.0%
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of C18H24O2 and of C22H28O3 dissolved:
Result = (rU/rS) × (CS/CU) × 100
rU== peak response of estradiol or norethindrone acetate from the Sample solution
rS== peak response of estradiol or norethindrone acetate from the Standard solution
CS== concentration of USP Estradiol RS or USP Norethindrone Acetate RS in the Standard solution (mg/mL)
CU== nominal concentration of estradiol or norethindrone acetate in the Sample solution (mg/mL) (based on the label claim)
Tolerances:  For Tablets labeled to contain 1 mg of estradiol and 0.5 mg of norethindrone acetate: NLT 75% (Q) of the labeled amounts of C18H24O2 and C22H28O3 is dissolved in 30 min. For Tablets labeled to contain 0.5 mg of estradiol and 0.1 mg of norethindrone acetate: NLT 75% (Q) of the labeled amounts of C18H24O2 and C22H28O3 is dissolved in 50 min.
•  Uniformity of Dosage Units 905: Meet the requirements
IMPURITIES
Organic Impurities 
•  Procedure
Solution A:  Tetrahydrofuran and water (1:200)
Solution B:  Acetonitrile, tetrahydrofuran, and water (160:1:40)
Mobile phase:  See the gradient table below.
Time
(min)
Solution A
(%)
Solution B
(%)
0 80 20
2 65 35
35 20 80
49 20 80
50 80 20
60 80 20
Diluent:  Dehydrated alcohol and water (1:1)
System suitability solution:  240 µg/mL of USP Estradiol RS, 60 µg/mL of USP Norethindrone Acetate RS, and 1 µg/mL of USP Estrone RS in Diluent
Estradiol standard stock solution:  250 µg/mL of USP Estradiol RS in alcohol
Norethindrone acetate standard stock solution:  150 µg/mL of USP Norethindrone Acetate RS in alcohol
Standard solution:  Combine 250 µL of the Estradiol standard stock solution and 100 µL of the Norethindrone acetate standard stock solution, and dilute with 50.0 mL of Diluent.
Sample solution:  A quantity equivalent to 240 µg/mL of estradiol and 120 µg/mL of norethindrone acetate from NLT 20 finely ground Tablets in Diluent
Chromatographic system  
Mode:  LC
Detector:  UV 235 and 254 nm
Column:  3.9-mm × 30-cm; 4-µm packing L1
Flow rate:  0.8 mL/min
Injection size:  100 µL
System suitability 
Sample:  System suitability solution
[Note—The relative retention times for estradiol, estrone, and norethindrone acetate are about 1.0, 1.4, and 3.0, respectively. ]
Suitability requirements 
Resolution:  NLT 1.3 between estrone and estradiol, measured at 254 nm
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of any estradiol impurity in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × (1/F) × 100
rU== peak response area at 235 nm for each impurity from the Sample solution
rS== peak response area at 235 nm from the Standard solution
CS== concentration of the Standard solution (µg/mL)
CU== concentration of the Sample solution (µg/mL)
F== relative response factor (see Impurity Table 1 or Impurity Table 2)
Calculate the percentage of any norethindrone acetate related impurities in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × (1/F) × 100
rU== peak response area at 254 nm for each impurity from the Sample solution
rS== peak response area at 254 nm from the Standard solution
CS== concentration of the Standard solution (µg/mL)
CU== concentration of the Sample solution (µg/mL)
F== relative response factor (see Impurity Table 1 or Impurity Table 2)
Acceptance criteria:  The Tablets meet the requirements given in either Impurity Table 1 or Impurity Table 2.
Impurity Table 1. Tablets Labeled as Containing 1 mg of
Estradiol and 0.5 mg of Norethindrone Acetate
Name Relative
Retention
Time
Relative
Response
Factor
Acceptance
Criteria,
NMT (%)
Estradiol related impurities
6-Hydroxyestradiol 0.47 1.0 1.0
6-Hydroxyestradiol 0.51 1.0 1.0
6-Ketoestradiol 0.62 1.0 1.0
6-Dehydroestradiol 0.95 1.0 1.0
Estradiol 1.0
Any other single
estradiol related
impurity
1.0 0.5
Total estradiol related impurities 2.0
Norethindrone acetate related impurities
6-Hydroxynorethindrone acetate 0.58 1.0 1.0
Norethindrone 0.66 1.0 1.0
6-Ketonorethindrone acetate 0.79 0.56 1.0
6-Dehydronorethindrone acetate 0.97 0.45 1.0
Norethindrone
acetate
1.0
Any other single norethindrone
acetate related
impurity
1.0 0.5
Total norethindrone acetate related
impurities
2.0
Impurity Table 2. Tablets Labeled as Containing 0.5 mg of
Estradiol and 0.1 mg of Norethindrone Acetate
Name Relative
Retention
Time
Relative
Response
Factor
Acceptance
Criteria,
NMT (%)
Estradiol related impurities
6-Hydroxy-estradiol 0.51 1.0 1.0
Estradiol 1.0
Any other single
estradiol related
impurity
1.0 1.0
Total estradiol related impurities 2.5
Norethindrone acetate related impurities
6-Hydroxy-norethindrone acetate 0.58 1.0 1.5
Norethindrone 0.66 1.0 1.0
6-Keto-norethindrone acetate 0.79 0.56 2.5
6-Dehydro-norethindrone acetate 0.97 0.45 1.0
Norethindrone
acetate
1.0
Any other single norethindrone
acetate related
impurity
1.0 1.0
Total norethindrone acetate related
impurities
4.0
SPECIFIC TESTS
•  Microbial Enumeration Tests 61 and Tests for Specified Microorganisms 62: The total aerobic microbial count does not exceed 1000 cfu/g, and the total combined molds and yeasts count does not exceed 100 cfu/g. The Tablets meet the requirements of the tests for the absence of Salmonella species and Escherichia coli.
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in well-closed containers, and store at controlled room temperature.
•  USP Reference Standards 11
USP Estradiol RS Click to View Structure
USP Estrone RS Click to View Structure
USP Norethindrone Acetate RS Click to View Structure
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
Topic/Question Contact Expert Committee
Monograph Domenick Vicchio, Ph.D.
Senior Scientific Liaison
1-301-998-6828
(SM42010) Monographs - Small Molecules 4
711 Margareth R.C. Marques, Ph.D.
Senior Scientific Liaison
1-301-816-8106
(GCDF2010) General Chapters - Dosage Forms
61 Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison
1-301-816-8339
(GCM2010) General Chapters - Microbiology
62 Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison
1-301-816-8339
(GCM2010) General Chapters - Microbiology
Reference Standards RS Technical Services
1-301-816-8129
rstech@usp.org
USP35–NF30 Page 3108
Pharmacopeial Forum: Volume No. 35(5) Page 1139