Standard preparation— Transfer about 25 mg of USP Menadione RS, accurately weighed, to a 100-mL volumetric 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 7 days.

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

Procedure— Transfer 1.0 mL each of the Standard preparation and the Assay preparation to separate 50-mL volumetric flasks, add to each 4 mL of alcohol, and mix. Then to each flask add 1.0 mL of a solution prepared by dissolving 50 mg of 2,4-dinitrophenylhydrazine in 20 mL of a mixture of 2 volumes of 3 N hydrochloric acid and 1 volume of water. Place the flasks in a bath maintained at 70 to 75 for 15 minutes, shaking vigorously every 2 to 3 minutes. Immediately after the heating, cool the flasks to about 25; then add to each 5 mL of alcoholic ammonia, prepared by mixing equal volumes of alcohol and ammonium hydroxide. Shake the flasks thoroughly, add alcohol to make 50.0 mL, mix, allow to stand for 15 minutes, and decant from any separated oil. Determine the absorbances of the solutions, in 1-cm cells at the wavelength of maximum absorbance at about 635 nm, with a suitable spectrophotometer, using a reagent blank to set the instrument. Calculate the quantity, in mg, of C11H8O2 in each mL of the Injection taken by the formula: in which C is the concentration, in µg per mL, of USP Menadione RS in the Standard preparation, V is the volume, in mL, of Injection taken, and AU and AS are the absorbances of the solutions from the Assay preparation and the Standard preparation, respectively.