Standard preparation— Dissolve a suitable quantity of USP Estradiol RS,accurately weighed,in methanol,and dilute quantitatively,and stepwise if necessary,with methanol to obtain a solution having a known concentration of about 40µg per mL.

Assay preparation— Transfer an accurately measured volume of well-mixed Injectable Suspension,equivalent to about 1mg of estradiol,to a 100-mLbeaker,and add water,if necessary,to obtain a volume of about 5mL.Add 6g of purified siliceous earth,mix,and pack the mixture tightly into a 20-×200-mm chromatographic tube containing in its base a pledget of fine glass wool.Dry-rinse the beaker with about 1g of purified siliceous earth,add the rinsing to the packed column,and wipe out the beaker with a pledget of glass wool used to top the column.Elute the column with 50mLof ether that previously has been saturated with water,and collect the eluate in a glass-stoppered,125-mLconical flask.Evaporate with the aid of gentle heat and a current of air to dryness,add 25.0mLof methanol to the residue,and mix.

Procedure— Transfer 1.0mLeach of the Standard preparationand the Assay preparationto separate glass-stoppered,16-×150-mm test tubes,and evaporate with the aid of gentle heat and a current of air to dryness.Using a suitable syringe,add 1.0mLof iron-phenol TSto each tube and to a third,similar tube to provide the blank.Suspend the tubes in a vigorously boiling water bath,mixing them simultaneously after heating for 5minutes.Remove the tubes after heating in the water bath for a total of 35minutes,and immediately cool in an ice-water bath.Remove from the ice bath,add 10.0mLof dilute sulfuric acid (1in 3)to each tube,mix to obtain homogeneous solutions,and allow to reach room temperature.Concomitantly determine the absorbances of the solutions in 1-cm cells at the wavelength of maximum absorbance at about 520nm,with a suitable spectrophotometer,against the blank.Calculate the quantity,in mg,of C18H24O2in each mLof the Injectable Suspension taken by the formula: in which Cis the concentration,in µg per mL,of USP Estradiol RSin the Standard preparation,Vis the volume,in mL,of Injectable Suspension taken,and AUand ASare the absorbances of the solutions from the Assay preparationand the Standard preparation,respectively.