Identification—
Mix 1 mL of the well-shaken Ophthalmic Suspension with 2 mL of a mixture of methanol and water (3:2) until a solution is obtained. Apply 20-µL portions of this solution and of a Standard solution of
USP Fluorometholone RS in the same solvent containing 500 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 and previously activated by heating at 80
for 5 minutes. Allow the spots to dry, and develop the chromatogram in a solvent system consisting of methylene chloride and acetone (4:1) until the solvent front has moved not less than 15 cm. Remove the plate from the developing chamber, mark the solvent front, and allow to air-dry. Examine the plate under short-wavelength UV light: the
RF value and intensity of the principal spot obtained from the test solution correspond to those obtained from the Standard solution.
Assay—
Mobile phase—
Prepare a suitable filtered solution of methanol and water (60:40) such that the retention time of fluorometholone is about 3 minutes.
Standard preparation—
Using a suitable quantity of
USP Fluorometholone RS, accurately weighed, prepare a solution in methanol containing 0.5 mg per mL. Pipet 10 mL of this solution and 5 mL of water into a 50-mL volumetric flask. Dilute with methanol to volume, and mix to obtain a
Standard preparation having a known concentration of about 100 µg per mL.
Assay preparation—
Pipet a volume of well-shaken Ophthalmic Suspension, equivalent to about 5 mg of fluorometholone, into a 50-mL volumetric flask, dilute with methanol to volume, and mix. Pass through a 5-µm membrane filter, and use the clear filtrate.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 2 mL per minute. Chromatograph six replicate injections of the
Standard preparation, and record the peak responses as directed for
Procedure: the relative standard deviation is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 10 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, using a suitable microsyringe or sampling valve, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of fluorometholone (C
22H
29FO
4) in the portion of Ophthalmic Suspension taken by the formula:
0.05C(rU / rS),
in which
C is the concentration, in µg per mL, of
USP Fluorometholone RS in the
Standard preparation; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
