Packaging and storage—
Preserve in tight containers, at a temperature not exceeding 21
.
Identification—
B:
Dissolve about 15 mg in 1 mL of sulfuric acid: an orange color is produced, and it disappears on dilution with about 10 volumes of water.
Chloride—
Weigh accurately about 2.0 g into a conical flask, dissolve in 25 mL of alcohol, add 4 mL of nitric acid and 10 mL of water, then add 20 mL of
silver nitrate TS, with stirring, and heat the mixture on a steam bath until the precipitate has coagulated. Transfer the precipitate to a tared filtering crucible, wash with three 10-mL portions of alcohol, dry at 105
for 30 minutes, cool in a desiccator, protected from light, and weigh: the weight of the precipitate is not more than 120 mg, corresponding to not more than 1.5% of chloride.
Free diethylstilbestrol—
Vanadyl sulfate solution—
Dissolve about 100 mg of vanadyl sulfate in 20 mL of sulfuric acid in a 200-mL volumetric flask, cautiously add glacial acetic acid to volume, and mix.
Standard diethylstilbestrol solution—
Dissolve about 10 mg of
USP Diethylstilbestrol RS, accurately weighed, in diluted alcohol, and dilute with diluted alcohol to 100.0 mL.
Procedure—
Transfer about 500 mg of Diethylstilbestrol Diphosphate, accurately weighed, to a beaker containing about 20 mL of water. Add 1 N sodium hydroxide to adjust to a pH of 8.0, determined potentiometrically. Transfer the solution to a separator, and extract with four 20-mL portions of peroxide-free ether. Evaporate the combined ether extracts on a steam bath to about 50 mL, transfer to a glass-stoppered, 50-mL conical flask, and evaporate to dryness. Transfer 5 mL of
Standard diethylstilbestrol solution to a 50-mL conical flask, and evaporate on a steam bath to dryness. Pipet 10 mL of
Vanadyl sulfate solution into each of the flasks containing the residues from the test solution and the
Standard diethylstilbestrol solution, and into a third dry flask, to provide the blank. Heat the loosely stoppered flasks on a steam bath for 1 hour, allow the contents to cool, and determine the absorbances of the solutions from the test preparation and the Standard preparation, relative to that of the blank, in 1-cm cells at the wavelength of maximum absorbance at about 520 nm. Calculate the quantity, in mg, of free diethylstilbestrol in the portion of Diethylstilbestrol Diphosphate taken by the formula:
0.005C(AU / AS),
in which
C is the concentration, in µg per mL, of
USP Diethylstilbestrol RS in the
Standard diethylstilbestrol solution, and
AU and
AS are the absorbances of the solution from Diethylstilbestrol Diphosphate and the Standard preparation, respectively. The limit of free diethylstilbestrol is 0.15%.
Diethylstilbestrol monophosphate—
Transfer about 50 mg of Diethylstilbestrol Diphosphate, accurately weighed, to a beaker containing about 20 mL of water. Add 1 N sodium hydroxide or 1 N phosphoric acid to adjust to a pH of about 2.2, determined potentiometrically. Proceed as directed for
Procedure in the test for
Free diethylstilbestrol, beginning with “Transfer the solution to a separator.” Calculate the total quantity,
T, in mg, of free diethylstilbestrol and diethylstilbestrol monophosphate, as diethylstilbestrol, in the portion of Diethylstilbestrol Diphosphate taken by the formula:
0.005C(AU / AS),
in which the terms are as defined under
Procedure. Calculate the quantity, in mg, of diethylstilbestrol monophosphate in the portion of Diethylstilbestrol Diphosphate taken by the formula:
1.298(
T 
F),
in which
T is as defined above, and
F is the content, in mg, of free diethylstilbestrol in the portion of Diethylstilbestrol Diphosphate taken, calculated from the result of the test for
Free diethylstilbestrol. The limit of diethylstilbestrol monophosphate is 1.5%.
Pyridine—
Cyanogen bromide reagent—
Dissolve 10 g of cyanogen bromide in water to make 100.0 mL.
Buffer solution—
Transfer to a 1000-mL volumetric flask 87 g of dibasic potassium phosphate, 107 g of ammonium chloride, and 6.7 mL of ammonium hydroxide, dilute with water to volume, and mix.
Procedure—
Transfer about 100 mg of Diethylstilbestrol Diphosphate, accurately weighed, to a 50-mL volumetric flask, add 25 mL of sodium hydroxide solution (1 in 500), shake until dissolved, add water to volume, and mix to obtain the Test preparation. Similarly prepare a Standard preparation containing 10 µg of pyridine per mL of sodium hydroxide solution (1 in 1000). Pipet 2 mL of the Test preparation and of the Standard preparation into separate glass-stoppered, 10-mL centrifuge tubes, add 3.0 mL of Buffer solution and 5.0 mL of Cyanogen bromide reagent to each tube, and mix. At 2 minutes, accurately timed, after the addition of Cyanogen bromide reagent measure the absorbance of each mixture relative to that of water at about 420 nm, with a suitable spectrophotometer. The absorbance of the Test preparation is not greater than that of the Standard preparation (0.5%).
Organic volatile impurities, Method V 
467
:
meets the requirements.
Solvent—
Use dimethyl sulfoxide.
Assay—
Assay preparation—
Transfer about 100 mg of Diethylstilbestrol Diphosphate, accurately weighed, to a 250-mL volumetric flask, add 25 mL of alcohol and 5 mL of 1 N sodium hydroxide, dilute with water to volume, and mix. Pipet 5 mL of this solution into a 100-mL volumetric flask, dilute with water to volume, and mix.
Procedure—
Concomitantly determine the absorbances of both solutions in 1-cm cells at the wavelength of maximum absorbance at about 241 nm, with a suitable spectrophotometer. Calculate the quantity, in mg, of C
18H
22O
8P
2 in the portion of Diethylstilbestrol Diphosphate taken by the formula:
5C(AU / AS),
in which
C is the concentration, in µg per mL, of
USP Diethylstilbestrol Diphosphate RS in the
Standard preparation; and
AU and
AS are the absorbances of the
Assay preparation and the
Standard preparation, respectively.
,