A: Infrared Absorption 197K.

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.

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: 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%.

0.005C(AU / AS)

1.298(T F)

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%).

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.

Standard preparation— Similarly prepare a solution of USP Diethylstilbestrol Diphosphate RS having a known concentration of about 20 µg per mL.

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 C18H22O8P2 in the portion of Diethylstilbestrol Diphosphate taken by the formula: 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.USP32