Mobile phase— Prepare a filtered and degassed mixture of water,methanol,and glacial acetic acid (89:10:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Transfer about 50mg of USP Sulfacetamide Sodium RS,accurately weighed,to a 40-mLcentrifuge tube.Add 10.0mLof dilute methanol (1in 5),insert the stopper,and mix,using a vortex mixer,for about 3minutes to dissolve the Reference Standard.Add 7.5mLof heptane,insert the stopper,and mix,using a vortex mixer,for another 3minutes.Centrifuge to effect separation of the phases.Withdraw and discard the upper heptane layer.Transfer 3.0mLof the bottom layer to a 500-mLvolumetric flask,add dilute methanol (1in 5)to volume,and mix.

Assay preparation— Transfer an accurately weighed quantity of Ophthalmic Ointment,equivalent to about 100mg of sulfacetamide sodium,to a 40-mLcentrifuge tube.Add 15.0mLof heptane,insert the stopper,and mix,using a vortex mixer,for about 3minutes to dissolve the Ophthalmic Ointment.Add 20.0mLof dilute methanol (1in 5),insert the stopper,and mix,using a vortex mixer,for 3minutes.Centrifuge to effect separation of the phases.Withdraw and discard the upper heptane layer.Transfer 3.0mLof the bottom layer to a 500-mLvolumetric flask,add dilute methanol (1in 5)to volume,and mix.

System suitability preparation— Dissolve 3mg of sulfanilamide in 100mLof the Standard preparation,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 1.5mLper minute.Chromatograph the Standard preparationand the System suitability preparation,and record the peak responses as directed for Procedure:the column efficiency determined from the analyte peak is not less than 1500theoretical plates,the resolution,R,between the sulfacetamide and sulfanilamide peaks is not less than 3,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 90µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C8H9N2NaO3S·H2Osulfacetamide sodium in the portion of Ophthalmic Ointment taken by the formula: in which 254.24and 236.23are the molecular weights of sulfacetamide sodium monohydrate and anhydrous sulfacetamide sodium,respectively,Cis the concentration,in µg per mL,of sulfacetamide sodium,calculated on the anhydrous basis,in the Standard preparation,and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.