Identification—
Dissolve the residue obtained in the
Assay in sufficient alcohol to obtain a solution containing 500 µg of estrone in each mL. Transfer to an acetylation flask, and evaporate to dryness. Add 10 mg of hydroxylamine hydrochloride, 0.20 mL of glacial acetic acid, and 5 mL of alcohol, and reflux for 5 hours. Dilute with 5 mL of water, filter, and recrystallize the precipitate from hot alcohol: the estrone oxime so obtained melts between 236
and 242
, the procedure for
Class I being used (see
Melting Range or Temperature 
741
).
Assay—
[NOTE—Use only water as a lubricant for the separators used in this assay, and complete the assay without interruption other than at the stage of obtaining the dry residue from the benzene extract.
] Transfer a volume of Injection, equivalent to about 10 mg of estrone, to a suitable separator containing 25 mL, or not less than twice the volume of the Injection taken, of solvent hexane. Add 10 mL of sodium hydroxide solution (1 in 10), shake vigorously for 2 minutes, and allow the layers to separate completely. Transfer the aqueous layer to a second 125-mL separator, and repeat the extraction of the solvent hexane with two additional, successive 10-mL portions of the sodium hydroxide solution, adding each extract to the second separator. Complete the alkaline extraction as quickly as possible, since long standing in strongly alkaline solution may cause decomposition of the estrone. Wash the combined alkaline extracts with 25 mL of solvent hexane. Using dilute sulfuric acid (1 in 2), acidify the combined alkaline extracts until acid to litmus. Cool thoroughly, add 25 mL of benzene, shake carefully for 1 minute, and allow the layers to separate. Transfer the acid layer to another 125-mL separator, and extract with a second 25-mL portion of benzene. Discard the acid layer. Extract the benzene layers with two 5-mL portions of
sodium carbonate TS and two 5-mL portions of water. Discard the aqueous layers. Transfer the benzene solutions to a beaker with the aid of benzene, and evaporate on a steam bath with the aid of a current of air to dryness.
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.
