Estradiol Valerate Principal Spot from the Standard Solution

526 Monographs, Part I slightly soluble in methanol, in ethanol (95) or in ether, Pipet 5 mL each of these solutions, add 5 mL of the and practically insoluble in water. internal standard solution, then add methanol to make 20 mL and use these solutions as the test solution and Identification (1) Take 2 mg of Estradiol Benzoate, the standard solution, respectively. Perform the test add 2 mL of sulfuric acid: a yellowish green color with with 5 μL of the test solution and the standard solution a blue fluorescence is produced and the color of the as directed under Liquid Chromatography according to solution changes to pale orange on the careful addition the following conditions and calculate the ratios, QT of 2 mL of water. and QS, of the peak area of estradiol benzoate to that of (2) Determine the infrared spectra of Estradiol the internal standard for the test solution and the stand- Benzoate and Estradiol Benzoate RS, previously dried, ard solution, respectively. as directed in the potassium bromide disk method under Infrared Spectrophotometry, respectively: both Amount (mg) of estradiol benzoate (C25H28O3) spectra exhibit similar intensities of absorption at the Q = Amount (mg) of Estradiol Benzoate RS × T same wavenumbers. Q S

α 20 ° Specific Optical Rotation [ ]D : + 54 ~ + 58 (after Internal standard solution—A solution of Proges- drying, 0.1 g, acetone, 10 mL, 100 mm). terone in methanol (13 in 80000).

Melting Point 191 ~ 198 ºC. Operating conditions Detector: An ultraviolet absorption photometer Purity (1) 3,17α-Estradiol—Dissolve 10.0 mg each (wavelength: 230 nm). of Estradiol Benzoate and Estradiol Benzoate RS in Column: A stainless steel column, about 4 mm in acetone to make exactly 200 mL and use these solu- internal diameter and about 15 cm in length, packed tions as the test solution and the standard solution, re- with octadecylsilanized silica gel for liquid chromatog- spectively. Pipet 2 mL each of the test solution and the raphy (5 μm in particle diameter). standard solution in separate glass-stoppered test tube, Column temperature: A constant temperature of add boiling stones, evaporate the acetone by heating on about 35 ºC. a water-bath and dry the residue in a desiccator (in Mobile phase: A mixture of acetonitrile and water vacuum, P2O5) for 1 hour. Add 1.0 mL of dilute iron- (7 : 3). phenol TS to each test tube. Stopper the test tubes Flow rate: Adjust the flow rate so that the retention loosely, heat for 30 seconds in a water-bath, shake in a time of Estradiol Benzoate is about 10 minutes. water-bath for several seconds and heat for 2 minutes. System suitability Cool the solutions in ice for 2 minutes, add 4.0 mL of System performance: When the procedure is run diluted sulfuric acid (7 in 20) and mix well: the solu- with 5 μL of the standard solution under the above op- tion obtained from the test solution has no more color erating conditions, the internal standard and estradiol than that from the standard solution. benzoate are eluted in this order with the resolutions (2) Related substances—Dissolve 40 mg of Estra- between their peaks being not less than 9. diol Benzoate in 2 mL of acetone and use this solution System repeatability: When the test is repeated 6 as the test solution. Pipet 1 mL of this solution, add times with 5 μL each of the standard solution under the acetone to make exactly 100 mL and use this solution above operating conditions, the relative standard devia- as the standard solution. Perform the test with these tions of the ratios of the peak area of estradiol benzoate solutions as directed under the Thin-layer chromatog- to that of the internal standard is not more than 1.0 %. raphy. Spot 10 μL each of the test solution and the standard solution on a plate of silica gel with fluores- Containers and Storage Containers—Tight con- cent indicator for thin-layer chromatography, develop tainers. the plate with a mixture of chloroform and Storage—Light-resistant. diethylamine (19 : 1) to a distance of about 15 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): any spot other than the principal spot from the test solution are not more intense than the Estradiol Valerate principal spot from the standard solution. OCOCH2CH2CH2CH3 H3C Loss on Drying Not more than 0.5 % (0.5 g, in vac- H uum, phosphorus (V) oxide, 4 hours). H

Residue on Ignition Not more than 0.2 % (0.1 g). H H Assay Weigh accurately about 10 mg each of Estra- HO diol Benzoate and Estradiol Benzoate RS, previously dried and dissolve in methanol to make exactly 20 mL. C23H32O3: 356.50 KP X 527

of estradiol valerate RS and add acetone to make 10 (17β)-3-Hydroxyestra-1,3,5(10)-trien-17-yl pentanoate mL. Pipet 0.1 mL, 0.5 mL, 1 mL and 2 mL of this solu- [979-32-8] tion and add acetone to each to make exactly 10 mL and use these solutions as the standard solutions (1), Estradiol Valerate contains not less than 98.0 % and not (2), (3) and (4). Perform the test with these solutions as more than 102.0 % of estradiol valerate (C23H32O3). directed under the Thin-layer chromatography. Spot 20 μL each of the test solution and the standard solutions Description Estradiol Valerate is a white crystalline (1), (2), (3) and (4) on a plate of silica gel for thin-layer powder, is odorless or has a slightly oily odor. chromatography. Develop the plate with a mixture of Estradiol Valerate is soluble in castor oil, in methanol, cyclohexane and ether (4 : 1) to a distance of about 15 in benzylbenzoate or in 1,4-dioxane, sparingly soluble cm and air-dry the plate. Spray evenly sulfuric acid in in sesame oil or peanut oil and practically insoluble in ethanol (95) (1 in 10) on the plate, heat the plate until water. burn and cool. Examine under ultraviolet light (main wavelength: 254 nm and 366 nm). Compare the inten- Identification Determine the infrared spectra of Es- sities of any spot other than the principal spot from the tradiol Valerate and Estradiol Valerate RS, prepared by test solution with those from the standard solution: the adding chloroform and potassium bromide to Estradiol relative intensity is not more than 2.0 %. Valerate or Estradiol Valerate RS, then grinding and drying at 105 ºC, as directed in the potassium bromide Water Not more than 0.1 % (5 g, direct titration). disk method under Infrared Spectrophotometry: both spectra exhibits similar intensities of absorption at the Assay Weigh accurately about 25 mg each of Estra- same wavenumbers. diol Valerate and Estradiol Valerate RS, dissolve each in the internal standard solution to make exactly 25 mL 20 Specific Optical Rotation [α] : +41 ~ +47° (0.500 and use these solutions as the test solution and the D standard solution, respectively. Perform the test with g after drying, methanol, 20 mL, 100 mm). 10 μL each of the test solution and the standard solu-

tion as directed under Liquid Chromatography accord- Melting Point 143 ~ 150 ºC (Method 1). ing to the following conditions and calculate the ratios, Q and Q , of the peak area of estradiol valerate to that Purity (1) Estradiol—Dissolve 50 mg of Estradiol T S of the internal standard, for the test solution and the Valerate in 10 mL of acetone and use this solution as standard solution, respectively. the test solution. Separately, dissolve 5 mg of Estradiol Valerate RS in 100 mL of acetone and use this solution Amount (mg) of estradiol valerate(C H O ) as the standard solution. Perform the test with the test 23 32 3 Q solution and the standard solution as directed under the = Amount (mg) of Estradiol Valerate RS × T thin-layer chromatography. Spot 5 μL each of the test QS solution and the standard solution on a plate of silica gel for Thin-layer chromatography. Then develop the Internal standard solution—Testosterone benzoate plate with a mixture of cyclohexane and ethyl acetate solution in tetrahydrofuran (2 in 1000). (7 : 3) to a distance of about 15 cm and dry the plate at 90 ºC for 30 minutes. Spray evenly a solution of sulfu- Operating conditions ric acid in methanol (3 in 10) on the plate and heat at Detector: An ultraviolet absorption photometer 90 ºC for 30 min: any spot other than the principal spot (wavelength: 280 nm). from the test solution or the spot corresponding to the Column: A stainless steel column, about 4 mm in Estradiol is not more intense or not larger than the spot internal diameter and about 30 cm in length, packed from the standard solution (not more than 1.0 %). with octadecylsilanized silica gel for liquid chromatog- (2) Free acid—Neutralize 25 mL of ethanol (95), raphy (5 to 10 μm in particle diameter). in a conical flask, with 0.01 mol/L sodium hydroxide Mobile phase: Dissolve 0.8 g of ammonium nitrate VS to a pale blue color, using bromothymol blue TS. in 300 mL of water, add 700 mL of acetonitrile and mix. Accurately weigh about 0.50 g of Estradiol Valerate Flow rate: 2 mL/minute. and dissolve Estradiol Valerate in the neutralized etha- System suitability nol. Titrate rapidly with 0.01 mol/L sodium hydroxide System performance: When the procedure is run VS to a pale blue color. (not more than 0.5 % of valeric with 10 μL of the standard solution, as directed under aid) the above operating conditions, estradiol valerate and the internal standard are eluted in this order with the Each mL of 0.01 mol/L sodium hydroxide VS resolution between their peaks being not less than 3. = 1.021 mg of C5H10O2 System repeatability: When the test is repeated 6 times with 10 μL each of the standard solution under (3) Related substances—Dissolve about 0.1 g of the above operating conditions, the relative standard Estradiol Valerate in 10 mL of acetone and use this deviation of the ratios of the peak area of estradiol solution as the test solution. Separately, weigh 10 mg valerate to that of the internal standard is not more than 528 Monographs, Part I

1.5 %. make exactly 25 mL and use this solution as the standard solution. Proceed as directed for the Assay under Containers and Storage Containers—Tight con- Estradiol Valerate. tainers. Storage—Light-resistant. Amount (mg) of estradiol valerate (C23H32O3) Q = Amount (mg) of Estradiol Valerate RS × T

QS Estradiol Valerate Injection Internal standard solution—Testosterone benzoate Estradiol Valerate Injection is an oily solution for injec- solution in tetrahydrofuran (8 in 1000). tion. Estradiol Valerate Injection contains not less than 90.0 % and not more than 115.0 % of the labeled Containers and Storage Containers—Hermetic amount of estradiol valerate (C23H32O3: 356.50). containers. Storage—Light-resistant. Method of Preparation Prepare as directed under Injections, with Estradiol Valerate. Estriol Description Estradiol Valerate Injection appears as pale yellow oil liquid. OH CH 3 H Identification Add 0.5 mL of Estradiol Valerate In- OH jection in 10 mL of hexane and 10 mL of 80 % metha- H nol in a separatory funnel and shake for 2 minutes. Al- H low to separate two layers. Add 1 mL of Folin’s TS H H (dilute 1 mL with 2 mL of water before use) and 3 mL of sodium carbonate VS (1 in 5): a blue color is ob- HO served. C18H24O3: 288.38 Purity Estradiol—Weigh accurately a portion of Estradiol Valerate, add acetone to make a solution con- (16α,17β)-Estra-1,3,5(10)-triene-3,16,17-triol [50-27-1] taining 30 % of estradiol according to the labeled amount. To 1.0 mL of this solution add oil labeled as Estriol, when dried, contains not less than 97.0 % and vehicle to make exactly 10 mL and use this solution as not more than 102.0 % of estriol (C18H24O3). the Estradiol solution. Perform the test with this solution and Estradiol Valerate Injection as directed under Description Estriol is a white, crystalline powder the Thin-layer chromatography. Spot 5 μL each of so- and is odorless. lutions on a plate coated with Thin-layer chromatog- Estriol is sparingly soluble in methanol, slightly solu- raphy silica gel and proceed as directed in the Purity ble in ethanol (95) or in 1,4-dioxane, and practically for Estradiol in Estradiol Valerate: not more than 3.0 %. insoluble in water or in ether.

Sterility Test It meets the requirement. Identification (1) Dissolve 10 mg of Estriol in 100 mL of ethanol (95) by warming and use this solution as Foreign Insoluble Matter Test It meets the require- the test solution. Evaporate 1 mL of this solution on a ment. water-bath to dryness, add 5 mL of a solution of sodium p-phenolsulfonate in phosphoric acid (1 in 50), heat Insoluble Particulate Matter Test for Injections It at 150 ºC for 10 minutes and cool: a red-purple color meets the requirement. develops. (2) Dissolve 10 mg each of Estriol and Estriol RS Determination of Volume of Injection in Containers separately in 100 mL ethanol (95) by warming and It meets the requirement. determine absorption spectra as directed under Ultravi- olet-visible Spectrophotometry: both spectra exhibit Assay Pipet a volume of Estradiol Valerate injection, similar intensities of absorption at the same wave- equivalent to about 20 mg of estradiol valerate lengths. (3) Determine the infrared spectra of Estriol and (C23H32O3) according to the labeled amount. Rinse the pipet with tetrahydrofuran and add 5.0 mL of the inter- Estriol RS, previously dried, as directed in the potassi- nal standard solution, dilute with tetrahydrofuran to um bromide disk method under Infrared Spectropho- make exactly 25 mL and use this solution as the test tometry, respectively: both spectra exhibit similar in- solution. Separately, weigh accurately about 20 mg of tensities of absorption at the same wavenumbers. Estradiol Valerate RS, add 5.0 mL of the internal standard solution and dilute with tetrahydrofuran to