Identification—
Transfer a volume of well-mixed Injectable Suspension, equivalent to about 10 mg of estradiol, to a flask, render it acid to
bromophenol blue TS with dilute hydrochloric acid (1 in 12), mix thoroughly, and place in an ice bath for 15 minutes. Filter the acidified suspension with suction through a sintered-glass funnel. Wash the crystals of estradiol so isolated with five successive 5-mL portions of water, and dry the funnel and contents at 105
to constant weight. The estradiol so obtained responds to
Identification test
A and meets the requirements of the test for
Melting range under
Estradiol.
Assay—
Standard preparation—
Dissolve a suitable quantity of
USP Estradiol RS, accurately weighed, in methanol, and dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a known concentration of about 40 µg per mL.
Assay preparation—
Transfer an accurately measured volume of well-mixed Injectable Suspension, equivalent to about 1 mg of estradiol, to a 100-mL beaker, and add water, if necessary, to obtain a volume of about 5 mL. Add 6 g of purified siliceous earth, mix, and pack the mixture tightly into a 20- × 200-mm chromatographic tube containing in its base a pledget of fine glass wool. Dry-rinse the beaker with about 1 g of purified siliceous earth, add the rinsing to the packed column, and wipe out the beaker with a pledget of glass wool used to top the column. Elute the column with 50 mL of ether that previously has been saturated with water, and collect the eluate in a glass-stoppered, 125-mL conical flask. Evaporate with the aid of gentle heat and a current of air to dryness, add 25.0 mL of methanol to the residue, and mix.
Procedure—
Transfer 1.0 mL each of the
Standard preparation and the
Assay preparation to separate glass-stoppered, 16- × 150-mm test tubes, and evaporate with the aid of gentle heat and a current of air to dryness. Using a suitable syringe, add 1.0 mL of iron-phenol TS to each tube and to a third, similar tube to provide the blank. Suspend the tubes in a vigorously boiling water bath, mixing them simultaneously after heating for 5 minutes. Remove the tubes after heating in the water bath for a total of 35 minutes, and immediately cool in an ice-water bath. Remove from the ice bath, add 10.0 mL of dilute sulfuric acid (1 in 3) to each tube, mix to obtain homogeneous solutions, and allow to reach room temperature. Concomitantly determine the absorbances of the solutions in 1-cm cells at the wavelength of maximum absorbance at about 520 nm, with a suitable spectrophotometer, against the blank. Calculate the quantity, in mg, of C
18H
24O
2 in each mL of the Injectable Suspension taken by the formula:
(0.025C / V)(AU / AS),
in which
C is the concentration, in µg per mL, of
USP Estradiol RS in the
Standard preparation,
V is the volume, in mL, of Injectable Suspension taken, and
AU and
AS are the absorbances of the solutions from the
Assay preparation and the
Standard preparation, respectively.
85
—
It contains not more than 250.0 USP Endotoxin Units per mg of estradiol.