A: Infrared Absorption á197Kñ.

B: Ultraviolet Absorption á197Uñ—

Solution: 10µg per mL.

Medium: methanol.

C: Prepare a solution in methanol containing 500µg per mL.Apply 100µLof this solution and 100µLof a methanol solution of USP Fluorometholone RScontaining 500µg per mLto a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 250-µm layer of chromatographic silica gel mixture.Allow the spots to dry,and develop the chromatogram in a solvent system consisting of a mixture of methylene chloride and acetone (4:1)until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the developing chamber,mark the solvent front,and allow the solvent to evaporate.Locate the spots on the plate by examination under short-wavelength UVlight:the RFvalue of the principal spot obtained from the specimen solution corresponds to that obtained from the Standard solution.

Test solution: 10mg per mL,in pyridine.

Mobile phase— Prepare a suitable filtered solution of methanol and water (60:40)such that the retention time of fluorometholone is about 3minutes.

Standard preparation— Dissolve an accurately weighed quantity of USP Fluorometholone RSin Mobile phaseto obtain a solution having a known concentration of about 100µg per mL.

Assay preparation— Transfer about 20mg of Fluorometholone,accurately weighed,to a 200-mLvolumetric flask,add Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—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 2mLper 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 10µL)of the Standard preparationand the Assay preparationinto 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 C22H29FO4in the portion of Fluorometholone taken by the formula: in which Cis the concentration,in µg per mL,of USP Fluorometholone RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.