Standard preparation— Transfer about 25mg of USP Menadione RS,accurately weighed,to a 100-mLvolumetric flask,dissolve in a mixture of equal volumes of alcohol and ether,dilute with the same mixture to volume,and mix.Keep the solution tightly closed in a dark,cool place,and use it within 7days.

Assay preparation— Transfer an accurately measured volume of Injection,equivalent to about 25mg of menadione,to a 100-mLvolumetric flask,dilute with a mixture of equal volumes of ether and alcohol to volume,and mix.

Procedure— Transfer 1.0mLeach of the Standard preparationand the Assay preparationto separate 50-mLvolumetric flasks,add to each 4mLof alcohol,and mix.Then to each flask add 1.0mLof a solution prepared by dissolving 50mg of 2,4-dinitrophenylhydrazine in 20mLof a mixture of 2volumes of 3Nhydrochloric acid and 1volume of water.Place the flasks in a bath maintained at 70to 75for 15minutes,shaking vigorously every 2to 3minutes.Immediately after the heating,cool the flasks to about 25;then add to each 5mLof alcoholic ammonia,prepared by mixing equal volumes of alcohol and ammonium hydroxide.Shake the flasks thoroughly,add alcohol to make 50.0mL,mix,allow to stand for 15minutes,and decant from any separated oil.Determine the absorbances of the solutions,in 1-cm cells at the wavelength of maximum absorbance at about 635nm,with a suitable spectrophotometer,using a reagent blank to set the instrument.Calculate the quantity,in mg,of C11H8O2in each mLof the Injection taken by the formula: in which Cis the concentration,in µg per mL,of USP Menadione RSin the Standard preparation,Vis the volume,in mL,of Injection taken,and AUand ASare the absorbances of the solutions from the Assay preparationand the Standard preparation,respectively.