Arsenomolybdate spray reagent— Dissolve 25 g of ammonium molybdate in 450 mL of water, add 21 mL of sulfuric acid, and mix. Add 3 g of dibasic sodium arsenate heptahydrate dissolved in 25 mL of water, mix, and incubate at 37 ± 2 for 24 to 48 hours. Store, protected from light.

Procedure— Transfer a portion of Vaginal Cream, equivalent to about 8 mg of estropipate, to a container. Add 20 mL of methanol, stir to obtain a homogeneous mixture, and pass through filter paper. Apply 30 µL of the filtrate and 30 µL of a Standard solution of USP Estropipate RS in methanol containing about 400 µg per mL to a suitable thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture. Allow the spots to dry, and develop the chromatogram in a solvent system consisting of a mixture of chloroform and methanol (10:8) until the solvent front has moved about 14 cm from the origin. Remove the plate from the developing chamber, mark the solvent front, and allow the solvent to evaporate. Locate the spots by lightly spraying with Arsenomolybdate spray reagent and by heating at 105 for about 20 minutes: the RF values of the principal spots obtained from the test solution correspond to those obtained from the Standard solution.

Diluent— Prepare a mixture of water and methanol (1:1).

Resolution solution— Dissolve a suitable quantity of 4¢-nitroacetophenone in methanol to obtain a solution having a known concentration of about 1.0 mg per mL. Transfer 5.0 mL of the solution so obtained to a 200-mL volumetric flask, dilute with Diluent to volume, and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Estropipate RS in Diluent, and dilute quantitatively, and stepwise if necessary, with Diluent to obtain a solution having a known concentration of about 1.0 mg per mL. Transfer 5.0 mL of the solution so obtained to a 100-mL volumetric flask, dilute with 0.05 M piperazine to volume, and mix. Transfer 5.0 mL of the solution so obtained to a 25-mL volumetric flask, add 5.0 mL of the Resolution solution, dilute with 0.05 M piperazine to volume, mix, and filter.

Assay preparation— Transfer an accurately weighed portion of Vaginal Cream, equivalent to about 1.5 mg of estropipate, to a 125-mL separator, add 50 mL of chloroform and 20 mL of 0.05 M piperazine, and extract for 3 minutes. Collect the organic extract in another separator, and extract the aqueous layer with an additional 20-mL portion of chloroform. Combine the organic phases, wash with a 20-mL portion of 0.05 M piperazine for 3 minutes, and collect the aqueous washings. Discard the organic layer. Rinse the separators with 0.05 M piperazine, and add the rinse to the aqueous washings. Transfer the aqueous extract and the aqueous washings to a 100-mL volumetric flask, dilute with 0.05 M piperazine to volume, and mix. Transfer 15.0 mL of the solution so obtained to a 25-mL volumetric flask, dilute with 0.05 M piperazine to volume, mix, and filter.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 213-nm detector and 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1 mL per minute. [note—The injection of a diluted solution of 4¢-nitroacetophenone can be used to better identify the peak locus.] Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 0.8 for 4¢-nitroacetophenone and 1.0 for estropipate; the resolution, R, between 4¢-nitroacetophenone and estropipate is not less than 2.0; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms for not less than 30 minutes for the Standard preparation and 90 minutes for the Assay preparation, and measure the responses for the major peaks. Calculate the quantity, in mg, of estropipate (C18H22O5S·C4H10N2) in the portion of Vaginal Cream taken by the formula: in which C is the concentration, in mg per mL, of USP Estropipate RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.