A: Infrared Absorption á197Kñ.

B: Ultraviolet Absorption á197Uñ—

Solution: 100µg per mL.

Medium: methanol.

C: Prepare a solution in chloroform to contain 1mg of mestranol per mL.Apply 10µLof this solution and 10µLof a solution of USP Mestranol RSin chloroform containing 1mg per mLto a line parallel to and about 2.5cm from the bottom edge of a thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Place the plate in a developing chamber containing and equilibrated with a mixture of 29volumes of chloroform and 1volume of dehydrated alcohol.Develop the chromatogram until the solvent front has moved about 15cm above the line of application.Remove the plate,allow the solvent to evaporate,and spray with Methanol–sulfuric acidprepared as described in the Assay.Heat the plate in an oven at 105for 5minutes,and observe under long-wavelength UVlight:the RFvalue of the principal spot obtained from the solution under test corresponds to that obtained with the Standard solution.

Test solution: 20mg,previously dried,per mL,in dioxane.

Methanol–sulfuric acid— Pipet 30mLof methanol into a 100-mLvolumetric flask contained in an ice bath.Add slowly and cautiously,and with continuous stirring,about 65mLof sulfuric acid,taking care that the temperature remains below 15.Allow the solution to warm to room temperature,and dilute with sulfuric acid to 100mL.

Standard preparation— Dissolve a suitable quantity of USP Mestranol RS,accurately weighed,in chloroform,and dilute quantitatively and stepwise with chloroform to obtain a solution having a known concentration of about 5µg per mL.

Assay preparation— Weigh accurately about 20mg of Mestranol,dissolve in chloroform to make 200.0mL,and mix.Pipet 5mLof this solution into a 100-mLvolumetric flask,add chloroform to volume,and mix.

Procedure— Pipet 4mLeach of the Standard preparationand the Assay preparationinto separate glass-stoppered,25-mLconical flasks.Evaporate the solutions under a slow stream of air,without the aid of heat,to dryness.Dissolve the residue in 0.3mLof methanol.Place the flasks in a water bath maintained at a temperature of 25,and pipet into each,with constant swirling,10mLof Methanol–sulfuric acid.Insert the stoppers in the flasks.At 6minutes,accurately timed,after the addition of the Methanol–sulfuric acid,concomitantly determine the absorbances of the solutions obtained from the Assay preparationand the Standard preparationat the wavelength of maximum absorbance at about 545nm,with a suitable spectrophotometer,using Methanol–sulfuric acidas the blank.Calculate the quantity,in mg,of C21H26O2in the Mestranol taken by the formula: in which Cis the concentration,in µg per mL,of USP Mestranol RSin the Standard preparation;and AUand ASare the absorbances of the solutions from the Assay preparationand the Standard preparation,respectively.