A:Infrared Absorption á197Kñ.

B: Dissolve about 15mg in 1mLof sulfuric acid:an orange color is produced,and it disappears on dilution with about 10volumes of water.

Vanadyl sulfate solution— Dissolve about 100mg of vanadyl sulfate in 20mLof sulfuric acid in a 200-mLvolumetric flask,cautiously add glacial acetic acid to volume,and mix.

Standard diethylstilbestrol solution— Dissolve about 10mg of USP Diethylstilbestrol RS,accurately weighed,in diluted alcohol,and dilute with diluted alcohol to 100.0mL.

Procedure— Transfer about 500mg of Diethylstilbestrol Diphosphate,accurately weighed,to a beaker containing about 20mLof water.Add 1Nsodium hydroxide to adjust to a pHof 8.0,determined potentiometrically.Transfer the solution to a separator,and extract with four 20-mLportions of peroxide-free ether.Evaporate the combined ether extracts on a steam bath to about 50mL,transfer to a glass-stoppered,50-mLconical flask,and evaporate to dryness.Transfer 5mLof Standard diethylstilbestrol solutionto a 50-mLconical flask,and evaporate on a steam bath to dryness.Pipet 10mLof Vanadyl sulfate solutioninto 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 1hour,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 520nm.Calculate the quantity,in mg,of free diethylstilbestrol in the portion of Diethylstilbestrol Diphosphate taken by the formula: in which Cis the concentration,in µg per mL,of USP Diethylstilbestrol RSin the Standard diethylstilbestrol solution,and AUand ASare 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 10g of cyanogen bromide in water to make 100.0mL.

Buffer solution— Transfer to a 1000-mLvolumetric flask 87g of dibasic potassium phosphate,107g of ammonium chloride,and 6.7mLof ammonium hydroxide,dilute with water to volume,and mix.

Procedure— Transfer about 100mg of Diethylstilbestrol Diphosphate,accurately weighed,to a 50-mLvolumetric flask,add 25mLof sodium hydroxide solution (1in 500),shake until dissolved,add water to volume,and mix to obtain the Test preparation.Similarly prepare a Standard preparationcontaining 10µg of pyridine per mLof sodium hydroxide solution (1in 1000).Pipet 2mLof the Test preparationand of the Standard preparationinto separate glass-stoppered,10-mLcentrifuge tubes,add 3.0mLof Buffer solutionand 5.0mLof Cyanogen bromide reagentto each tube,and mix.At 2minutes,accurately timed,after the addition of Cyanogen bromide reagentmeasure the absorbance of each mixture relative to that of water at about 420nm,with a suitable spectrophotometer.The absorbance of the Test preparationis not greater than that of the Standard preparation(0.5%).

Solvent— Use dimethyl sulfoxide.

Assay preparation— Transfer about 100mg of Diethylstilbestrol Diphosphate,accurately weighed,to a 250-mLvolumetric flask,add 25mLof alcohol and 5mLof 1Nsodium hydroxide,dilute with water to volume,and mix.Pipet 5mLof this solution into a 100-mLvolumetric flask,dilute with water to volume,and mix.

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

Procedure— Concomitantly determine the absorbances of both solutions in 1-cm cells at the wavelength of maximum absorbance at about 241nm,with a suitable spectrophotometer.Calculate the quantity,in mg,of C18H22O8P2in the portion of Diethylstilbestrol Diphosphate taken by the formula: in which Cis the concentration,in µg per mL,of USP Diethylstilbestrol Diphosphate RSin the Standard preparation;and AUand ASare the absorbances of the Assay preparation and the Standard preparation,respectively.