A: Infrared Absorption 197K.

B: Ultraviolet Absorption 197U.

Test solution: 20 mg per mL, in chloroform.

Mobile phase, System suitability solution, and Chromatographic system— Prepare as directed in the Assay.

Standard solution— Dissolve an accurately weighed quantity of USP Fluorometholone RS in methanol, and dilute quantitatively and stepwise if necessary, with acetonitrile to obtain a solution having a known concentration of about 0.03 mg per mL.

Test solution— Use the Assay preparation.

Blank— Use acetonitrile.

Procedure— Separately inject equal volumes (about 20 µL) of the Blank, the Standard solution, and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. [note—Allow the elution to continue for about two and a half times the elution time of the fluorometholone acetate peak before making the next injection.]

Mobile phase— Prepare a filtered and degassed mixture of water and acetonitrile (60:40). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Fluorometholone Acetate RS in acetonitrile, and dilute quantitatively, and stepwise if necessary, with acetonitrile to obtain a solution having a known concentration of about 1.0 mg per mL.

System suitability solution— Prepare a solution of USP Fluorometholone RS by dissolving a quantity in methanol and diluting in acetonitrile to a final concentration of about 1 mg per mL. Mix equal volumes of this solution and the Standard preparation, and dilute with acetonitrile to a final concentration of about 0.03 mg per mL each for fluorometholone and fluorometholone acetate.

Assay preparation— Transfer about 50 mg of Fluorometholone Acetate, accurately weighed, to a 50-mL volumetric flask, dissolve in and dilute with acetonitrile to volume, and mix.

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 5-µm packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the resolution, R, between fluorometholone and fluorometholone acetate is not less than 10. The column efficiency for fluorometholone acetate is not less than 10,000 theoretical plates, and the tailing factor for fluorometholone acetate is not more than 2.0. Chromatograph the Standard preparation and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections 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, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C24H31FO5 in the portion of Fluorometholone Acetate taken by the formula: in which C is the concentration, in mg per mL, of USP Fluorometholone Acetate RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.