Estradiol Benzoate
Estra-1,3,5(10)-triene-3,17-diol, (17 ![]() Estradiol 3-benzoate ![]() ![]() ![]() » Estradiol Benzoate contains not less than 97.0 percent and not more than 103.0 percent of C25H28O3, calculated on the dried basis.
Packaging and storage
Preserve in tight, light-resistant containers.
Labeling
Label it to indicate that it is for veterinary use only. Label it to indicate whether it is coarse grade or fine grade.
Identification
B:
The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.
Specific rotation
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Test solution:
10 mg per mL, previously dried, in dioxane.
Loss on drying
Dry it at 100
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Residue on ignition
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Particle size
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Suspension fluid
To a mixture of glycerin and water (60:40, w/w) add a sufficient quantity of polysorbate 20 to obtain a solution having a concentration of 125 µL of polysorbate 20 per 100 g of solution.
Test suspension
Saturate the Suspension fluid by adding about 100 mg of fine Estradiol Benzoate per 100 g of Suspension fluid, and sonicate for about 10 minutes. Filter the resulting suspension through a 0.45-µm nylon filter. To the filtrate, add about 50 mg of Estradiol Benzoate per mL of the filtered, saturated Suspension fluid, and mix on a vortex mixer until dispersed (about 1 minute).
Procedure
Using a suitable multi-wavelength particle size analyzer,1 determine the particle size distribution within the Test suspension, analyzing the results in the range from 5 µm to 600 µm. Not more than 50% of the particles are less than 30 µm, and not less than 90% of the particles are less than 450 µm. The mean diameter of fine grade Estradiol Benzoate is not more than 100 µm, and the mean diameter of coarse grade Estradiol Benzoate is not less than 100 µm and not more than 200 µm.
Limit of methanol and dichloromethane
Internal standard solution
Prepare a solution that contains 0.1% (v/v) ethyl acetate in pyridine. [noteInject 2 µL of the pyridine into the Chromatographic system to confirm that it contains no peaks that would interfere with the analysis. ]
Standard solution
Transfer 50 µL of methylene chloride and 50 µL of methanol to a 50-mL volumetric flask, and dilute with Internal standard solution to volume. Mix well.
Test solution
Accurately weigh about 100 mg of Estradiol Benzoate into a low-actinic glass vial. Dissolve in 1.0 mL of Internal standard solution, and mix.
Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 2.0 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak areas for methanol, methylene chloride, and ethyl acetate. Calculate the percentage (w/w) of each residual solvent in the portion of Estradiol Benzoate taken by the formula:
100CI (DI / CU)(RU / RS)
in which CI is the concentration, in µL per mL, of the solvent of interest in the Standard solution; DI is the density, in g per mL, of the solvent of interest; CU is the concentration, in mg per mL, of Estradiol Benzoate in the Test solution; and RU and RS are the peak response ratios of the solvent of interest to the internal standard obtained from the Test solution and the Standard solution, respectively: the sum of the percentages of methanol and methylene chloride is not more than 0.20%.
Chromatographic purity
Adsorbent:
a 0.25-mm layer of chromatographic silica gel mixture.
Developing solvent system
Use a mixture of toluene and ethyl acetate (70:30).
Ammonium molybdate solution
Dissolve 5 g of ammonium molybdate in 100 mL of 10% (v/v) sulfuric acid.
Diluent
Prepare a solution containing a mixture of methylene chloride and alcohol (2:1).
Standard solution
Dissolve an accurately weighed quantity of USP Estradiol Benzoate RS in Diluent to obtain a solution containing about 5 mg per mL.
Test solution 1
Dissolve an accurately weighed quantity of Estradiol Benzoate in Diluent to obtain a solution containing about 5 mg per mL.
Test solution 2
Transfer 200 µL of Test solution 1 to a 10-mL volumetric flask, and dilute with Diluent to volume.
Procedure (see Thin-Layer Chromatography under Chromatography
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Assay
Mobile phase
Prepare a filtered and degassed mixture of acetonitrile and water (7:3). Make adjustments if necessary (see System Suitability under Chromatography
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System suitability preparation
Transfer an accurately weighed quantity of about 20.0 mg each of USP Estradiol Benzoate RS and estradiol 17-acetate into a 100-mL volumetric flask. Add 70 mL of acetonitrile, and sonicate until dissolved. Add 25 mL of water, mix well, and allow to equilibrate to ambient temperature. Dilute with water to volume, and mix.
Standard preparation
Transfer an accurately weighed quantity of about 20.0 mg of USP Estradiol Benzoate RS into a 100-mL volumetric flask. Add 70 mL of acetonitrile, and sonicate until dissolved. Add 25 mL of water, mix well, and allow to equilibrate to ambient temperature. Dilute with water to volume, and mix.
Assay preparation
Transfer an accurately weighed quantity of about 20.0 mg of Estradiol Benzoate into a 100-mL volumetric flask. Add 70 mL of acetonitrile, and sonicate until dissolved. Add 25 mL of water, mix well, and allow to equilibrate to ambient temperature. Dilute with water to volume, and mix.
Chromatographic system (see Chromatography
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Procedure
Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C25H28O3 in the portion of Estradiol Benzoate taken by the formula:
100C(rU / rS)
in which C is the concentration, in mg per mL, of USP Estradiol Benzoate RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.
1
A suitable multi-wavelength particle size analyzer is model LS 13 320, obtained from Beckman Coulter, Inc., Fullerton, CA, or equivalent.
Auxiliary Information
Please check for your question in the FAQs before contacting USP.
USP35NF30 Page 3100
Pharmacopeial Forum: Volume No. 33(2) Page 229
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