Identification
Transfer a quantity of Topical Solution, equivalent to about 0.5 mg of fluocinolone acetonide, to a separator, add 5 mL of water, and extract with 10 mL of chloroform. Withdraw the chloroform layer into a second separator, wash with 10 mL of water, and 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 the Standard solution being applied, and a mixture of chloroform and diethylamine (2:1) being used for development.
Assay
Internal standard solution
Dissolve norethindrone 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 200 µg per mL. Transfer 5.0 mL of this solution, 4.0 mL of
Internal standard solution, 10 mL of propylene glycol, and about 25 mL of acetonitrile to a 50-mL volumetric flask. Mix, cool to room temperature, dilute with acetonitrile to volume, and mix. The final concentration of
USP Fluocinolone Acetonide RS is 20 µg per mL.
Mobile solvent
Prepare a mixture of water and acetonitrile (3:2). Adjust the ratio as necessary to obtain suitable chromatographic performance.
Assay preparation
Transfer an accurately measured volume of Topical Solution, equivalent to about 0.5 mg of fluocinolone acetonide, to a 25-mL volumetric flask. Add 2.0 mL of Internal standard solution and 10 mL of acetonitrile. Mix, cool to room temperature, dilute with acetonitrile to volume, and mix.
Apparatus
Use a suitable 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 containing packing L1 so as to provide a resolution factor,
R, of at least 2.0 between peaks for norethindrone and fluocinolone acetonide.
Procedure
Chromatograph equal volumes of the
Assay preparation and the
Standard preparation, adjusting the system as necessary to obtain peaks of between about 50% and 90% full-scale. Calculate the quantity, in mg, of C
24H
30F
2O
6 in each mL of the Topical Solution taken by the formula:
0.025(C / V)(RU / RS),
in which
C is the concentration, in µg per mL, of
USP Fluocinolone Acetonide RS in the
Standard preparation; V is the volume, in mL, of Solution taken; and
RU and
RS are the ratios of the areas of the fluocinolone acetonide peak to the internal standard peak obtained from the
Assay preparation and the
Standard preparation, respectively.