Identification—
Prepare a test solution by placing a quantity of Cream, equivalent to about 5 mg of fluorouracil, in a glass-stoppered conical flask, add 50 mL of alcohol, and shake until dissolved. Dissolve 5 mg of
USP Fluorouracil RS in 50 mL of alcohol to obtain a Standard solution. In 20-µL increments, apply 100 µL each of the Standard solution and the test solution to a line about 3 cm from the bottom edge of a thin-layer chromatographic plate coated with a 0.25-mm layer of chromatographic silica gel mixture, which previously has been dried and activated at 105
for 5 minutes. Develop the chromatogram in a solvent system consisting of a mixture of ethyl acetate, methanol, and ammonium hydroxide (75:25:1), allowing the solvent front to move about 15 cm beyond the initial spotting line. Remove the plate, air-dry for 15 minutes, and examine under short-wavelength UV light: the
RF value of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.
Assay—
Mobile phase
,
Standard preparation, and
Chromatographic system—Proceed as directed in the
Assay under
Fluorouracil.
Assay preparation—
Transfer an accurately weighed portion of Cream, equivalent to about 10 mg of fluorouracil, to a 100-mL volumetric flask, add 20 mL of methanol, and mix on a vortex mixer to dissolve. Dilute with water to volume, and mix. Transfer 1.0 mL of this solution to a 10-mL volumetric flask, dilute with water to volume, mix, and filter.
Procedure—
Separately inject equal volumes (about 10 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of fluorouracil (C
4H
3FN
2O
2) in the portion of Cream taken by the formula:
C(rU / rS),
in which
C is the concentration, in µg per mL, of
USP Fluorouracil RS in the
Standard preparation; and
rU and
rS are the fluorouracil peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
61
—
It meets the requirements of the tests for absence of Staphylococcus aureus and Pseudomonas aeruginosa.