Identification—
Evaporate 10.0 mL of the
Assay preparation obtained in the
Assay to dryness, and dissolve the residue in 1 mL of chloroform: it responds to the
Thin-layer Chromatographic Identification Test 
201
, 50 µL of the test solution and 50 µL of the 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 a suitable quantity of
USP Norethindrone RS in methanol to obtain a solution containing about 850 µg per mL.
Diluted internal standard solution—
Transfer 5.0 mL of Internal standard solution to a 250-mL flask. Dilute with methanol to volume, and mix.
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 10.0 mL of this solution and 2.0 mL of
Internal standard solution to a 100-mL volumetric flask. Dilute with methanol to volume, and mix. The concentration of
USP Fluocinolone Acetonide RS in the
Standard preparation is 20 µg per mL.
Mobile solvent—
Prepare a mixture of acetonitrile and water (1:1). Adjust the ratio as necessary to obtain suitable chromatographic performance.
Assay preparation—
Transfer an accurately weighed portion of Ointment, equivalent to about 0.7 mg of fluocinolone acetonide, to a 50-mL, round-bottom centrifuge tube. Add 35.0 mL of Diluted internal standard solution, emulsify using an ultrasonic probe, and centrifuge to bring the insoluble matter to the bottom. The clear supernatant is the Assay preparation.
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 50-cm × 4-mm column that contains packing L1 so as to provide a resolution factor,
R (see
Chromatography 621), of at least 2.0 between peaks for norethindrone and fluocinolone acetonide. Three replicate injections of the
Standard preparation show a relative standard deviation of not more than 1.5%.
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% of full-scale. Calculate the quantity, in mg, of C
24H
30F
2O
6 in the portion of Ointment taken by the formula:
0.035C(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 the internal standard obtained from the
Assay preparation and the
Standard preparation, respectively.
