Identification—
Transfer a quantity of the Cream, equivalent to about 0.5 mg of fluocinolone acetonide, to a centrifuge tube, disperse it in 5 mL of water, add 10 mL of chloroform, shake, and centrifuge. Remove and discard the aqueous layer, add 10 mL of water to the tube, shake, and centrifuge. Dry about 2 mL of the chloroform extract over about 200 mg of anhydrous sodium sulfate: the dried extract responds to the
Thin-Layer Chromatographic Identification Test 
201
, 50 µL of the dried chloroform extract and 50 µL of a Standard solution containing about 50 µg per mL of
USP Fluocinolone Acetonide RS being applied, and a mixture of chloroform and diethylamine (2:1) being used for development.
Assay—
Internal standard solution—
Dissolve
USP Norethindrone RS in acetonitrile to obtain a solution containing about 200 µg per mL.
Standard preparation—
Dissolve an accurately weighed quantity of
USP Fluocinolone Acetonide RS in acetonitrile to obtain a solution having a known concentration of about 300 µg per mL. Transfer 5.0 mL of this solution, 6.0 mL of
Internal standard solution, and 15.0 mL of water to a 50-mL volumetric flask. Dilute with acetonitrile to volume, and mix. The
Standard preparation contains 30 µg of
USP Fluocinolone Acetonide RS per mL.
Mobile solvent—
Prepare a mixture of water and acetonitrile (5:3). Adjust the ratio as necessary to obtain suitable chromatographic performance.
Assay preparation—
Dissolve an accurately weighed portion of Cream, equivalent to about 0.75 mg of fluocinolone acetonide, in about 10 mL of acetonitrile by heating on a steam bath. Transfer the mixture to a 25-mL volumetric flask with the aid of three 2-mL portions of acetonitrile. Add 3.0 mL of Internal standard solution and 5.0 mL of water, cool, and mix. Dilute with acetonitrile to volume, mix, and cool in an ice bath. Centrifuge or filter the mixture to obtain a clear solution.
Apparatus—
Use a high-pressure liquid chromatograph (see
Chromatography 621) of the general type equipped with a detector for monitoring UV absorbance at about 254 nm, and capable of providing a flow rate of about 2 mL per minute for the
Mobile solvent. Use a column that contains packing L1.
Procedure—
Chromatograph equal volumes of the
Assay preparation and the
Standard preparation. Three replicate injections of the
Standard preparation show a resolution factor of not less than 2.0 between the peaks for norethindrone and fluocinolone acetonide and a relative standard deviation of not more than 1.5%. Calculate the quantity, in mg, of C
24H
30F
2O
6 in the portion of Cream taken by the formula:
0.025C(RU / RS),
in which
C is the concentration, in µg per mL, of
USP Fluocinolone Acetonide RS in the
Standard preparation; and
RU and
RS are the ratios of the peak areas of fluocinolone acetonide and norethindrone obtained from the
Assay preparation and the
Standard preparation, respectively.
