A: The relative retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the Assay for fluorometholone acetate.

B: Allow the Ophthalmic Suspension to settle,and decant 1mLof the supernatant into a test tube.Add 0.1g of sodium sulfate,mix,and centrifuge:the clear supernatant so obtained meets the requirements for Identificationtest Aunder Tobramycin.

Mobile phase ,2,4-Dinitrofluorobenzene reagent,Tris(hydroxymethyl)aminomethane reagent,Standard preparation,Resolution solution,and Chromatographic system—Proceed as directed in the Assayunder Tobramycin.

Assay preparation— Transfer an accurately weighed portion of Ophthalmic Suspension,equivalent to about 4.5mg of tobramycin,to a 50-mLvolumetric flask,dilute with water to volume,and mix.

Derivatization procedure— Proceed as directed in the Assayunder Tobramycin,except to use 10.0mLof Assay preparationinstead of 4.0mL.

Procedure— Proceed as directed in the Assayunder Tobramycin.Calculate the quantity of tobramycin (C18H37N5O9),in mg,in the portion of Ophthalmic Suspension taken by the formula: in which the terms are as defined therein.

Mobile phase— Prepare a suitable mixture of acetonitrile and water (50:50).Pass through a filter having a 1-µm or finer porosity,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).Reduce the proportion of acetonitrile to increase the retention time of fluorometholone acetate.

Resolution solution— Prepare a solution in acetonitrile containing 0.04mg each of USP Fluorometholone RSand USP Fluorometholone Acetate RSper mL.

Standard preparation— Prepare a solution of USP Fluorometholone Acetate RSin acetonitrile having a known concentration of about 0.04mg per mL.

Assay preparation— Transfer an accurately measured volume of Ophthalmic Suspension,freshly mixed and free from air bubbles,equivalent to about 2.5mg of fluorometholone acetate,to a 25-mLvolumetric flask,dilute with acetonitrile to volume,and mix.Transfer 4.0mLof this solution to a 10-mLvolumetric flask,dilute with acetonitrile to volume,and mix.Transfer a portion of this solution to a test tube,and centrifuge for about 15minutes.Use the clear supernatant.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4-mm ×25-cm column that contains packing L1.The flow rate is about 1.5mLper minute.Chromatograph the Resolution solution,and record the peak areas as directed for Procedure:the relative retention times are about 0.7for fluorometholone and 1.0for fluorometholone acetate;and the resolution,R,between fluorometholone and fluorometholone acetate is not less than 2.0.Chromatograph the Standard preparation,and record the areas as directed for Procedure:the capacity factor,k¢,determined from fluorometholone acetate peak is between 1.0and 5.0;the column efficiency is not less than 1000theoretical plates;the tailing factor is not more than 1.35;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas.Calculate the quantity,in mg,of fluorometholone acetate (C24H31FO5)in each mLof Ophthalmic Suspension taken by the formula: in which Cis the concentration,in mg per mL,of USP Fluorometholone Acetate RSin the Standard preparation;Vis the volume,in mL,of Ophthalmic Suspension taken;and rUand rSare the fluorometholone acetate peak areas obtained from the Assay preparationand the Standard preparation,respectively.