Dissolve the residue from the benzene extract in a small quantity of chloroform, warming, if necessary, and completely transfer the solution, with the aid of a few mL of chloroform, to a 20- × 150-mm test tube. Carefully evaporate the chloroform on a steam bath with the aid of a current of air. Add 100 mg of trimethylacethydrazide ammonium chloride and 500 µL of glacial acetic acid to the test tube, insert the stopper loosely, and heat in a boiling water bath for 5 minutes. Cool the reaction mixture in an ice bath, dissolve in a small volume of cold water, and completely transfer, with the aid of a small volume of water, to a 125-mL separator containing 25 mL of cold water. Neutralize the solution to litmus with 1 N sodium hydroxide (approximately 6 mL), and wash at once with three 15-mL portions of chloroform. Combine the chloroform washings in another separator, and wash them with 5 mL of water. Discard the chloroform, and add the wash water to the first separator. Add 2 mL of dilute sulfuric acid (1 in 2), and allow to remain at room temperature for 2 hours. Add 15 mL of chloroform, shake vigorously for 1 minute, and allow the layers to separate. Transfer the chloroform layer to another separator, and repeat the extraction of the water layer with three additional, successive 15-mL portions of chloroform. Wash the combined chloroform extracts with 5 mL of water, filter through chloroform-washed cotton into a beaker, evaporate to a small volume, and transfer completely, with the aid of several small portions of chloroform, to a tared 25-mL beaker. Evaporate on a steam bath with the aid of a current of air to dryness, and dry the residue of estrone in a vacuum desiccator to constant weight: the weight of the residue, corrected for the residue of a reagent blank similarly prepared, indicates the amount of C18H22O2 in the volume of Injection taken.