» Diethylstilbestrol Injection is a sterile solution of Diethylstilbestrol in a suitable vegetable oil. It contains not less than 90.0 percent and not more than 110.0 percent of the labeled amount of C18H20O2.
Packaging and storage Preserve in light-resistant, single-dose or multiple-dose containers, preferably of Type I glass.
USP Reference standards 11
USP Endotoxin RS
Identification The absorption spectrum, in the range of 250 to 450 nm, of the yellow solution obtained in the Assay after irradiation of the Assay preparation exhibits inflections only at the same wavelengths as that of the solution obtained after irradiation of the Standard preparation.
Bacterial endotoxins 85 It contains not more than 0.7 USP Endotoxin Unit per mg of diethylstilbestrol.
Other requirements It meets the requirements under Injections 1.
Standard preparation Dissolve an accurately weighed quantity of USP Diethylstilbestrol RS in alcohol, and dilute quantitatively and stepwise with alcohol to obtain a solution having a known concentration of about 20 µg per mL. Mix 25.0 mL of this solution with an equal volume of dibasic potassium phosphate solution (1 in 55).
Assay preparation Transfer an accurately measured volume of Injection, equivalent to not less than 1 mg and not more than 50 mg of diethylstilbestrol, to a 125-mL separator containing 75 mL of isooctane. Extract the solution with one 20-mL and two 10-mL portions of 1 N sodium hydroxide. Wash the combined alkaline extracts with two 10-mL portions of chloroform. Transfer the alkaline solution to a 150-mL beaker, and with the careful addition of 2.5 M phosphoric acid adjust the pH of the solution to 9.5. Transfer the adjusted solution, with the aid of a small volume of water, to a 125-mL separator, and extract with four 20-mL portions of chloroform. Filter the extracts into a 100-mL volumetric flask through a chloroform-wetted pledget of cotton, washing the filter with several small portions of chloroform to adjust the solution to volume. If necessary, dilute the solution quantitatively and stepwise with chloroform so that it contains about 10 µg of diethylstilbestrol per mL. Transfer 20 mL of this solution to a 50-mL conical flask, and evaporate with the aid of gentle heating in a current of air to about 5 mL. Complete the evaporation of the solvent in the air current without further application of heat. Dissolve the residue in 10.0 mL of alcohol, add 10.0 mL of dibasic potassium phosphate solution (1 in 55), and mix.
Procedure [CautionProtect the eyes from direct rays of UV light throughout this procedure. ] Transfer 4 mL of the Standard preparation to a stoppered, 1-cm quartz cell, place about 5 cm from a low-pressure, short-wave mercury lamp rated at from 2 to 20 watts, and irradiate for about 5 minutes. Place the cell in the sample compartment of a spectrophotometer, and measure the absorbance at the wavelength of maximum absorbance at about 418 nm, using water as the blank. Continue irradiation for successive 1- to 3-minute intervals, measuring at 418 nm until the maximum absorbance (about 0.7) has been obtained. If necessary, adjust the geometry of the irradiation apparatus so as to obtain maximum, reproducible absorbance at 418 nm. Similarly irradiate a 4-mL portion of the Assay preparation, recording the absorbance at 418 nm, at successive short intervals until maximum absorbance is obtained. Concomitantly determine the absorbances of the Assay preparation and the Standard preparation in 1-cm cells at 418 nm, using water as the blank, and subtract these values from those for the respective irradiated solutions to obtain the corrected maximum absorbances. Calculate the quantity, in µg, of diethylstilbestrol (C18H20O2) in each mL of the Assay preparation taken by the formula:
C(AU / AS)in which C is the concentration, in µg per mL, of USP Diethylstilbestrol RS in the Standard preparation, and AU and AS are the corrected maximum absorbances of the irradiated Assay preparation and Standard preparation, respectively.
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USP35NF30 Page 2896