Mobile phase— Prepare a suitably degassed solution of water,acetonitrile,and 1Mtetrabutylammonium hydroxide (78:22:10).Adjust,dropwise with dilute phosphoric acid (1in 10)to a pHof 7.7±0.2,and mix for 5minutes.If necessary,adjust to a pHof 7.7±0.2,using dilute phosphoric acid (1in 50)or 1Mtetrabutylammonium hydroxide.

Internal standard solution— Dissolve sulfapyridine in acetone to obtain a solution having a concentration of about 10mg per mL.

Standard preparation— Weigh accurately about 29mg of USP Sulfacetamide RS,34mg of USP Sulfathiazole RS,and 37mg of USP Sulfabenzamide RS,and transfer to a 50-mLvolumetric flask.Add 2.0mLof Internal standard solutionand 30mLof acetone,and shake for 10minutes.If necessary,sonicate to effect solution.Dilute with acetone to volume,and mix.Pipet 5mLof this solution into a 50-mLvolumetric flask,evaporate on a steam bath with the aid of a gentle stream of nitrogen to dryness,dissolve the residue in Mobile phase,dilute with Mobile phaseto volume,and mix.

Assay preparation— Using a plastic syringe equipped with a suitable cannula,transfer an accurately weighed quantity of Vaginal Cream,equivalent to about 144mg of sulfacetamide,184mg of sulfabenzamide,and 173mg of sulfathiazole,to a 250-mLvolumetric flask.Add 10.0mLof Internal standard solutionand 100mLof acetone,and warm the flask on a steam bath while swirling the contents to dissolve the cream.Cool to room temperature,dilute with acetone to volume,and mix.Filter the solution through filter paper,discarding the first 10mLof the filtrate.Pipet 5mLof the filtrate so obtained into a 50-mLvolumetric flask,and evaporate on a steam bath with the aid of a gentle stream of nitrogen to dryness.Dissolve the residue in Mobile phase,dilute with Mobile phaseto volume,and mix.Cool the solution in an ice bath for 10minutes,filter the cold solution through filter paper,discarding the first 10mLto 15mLof the filtrate,and collect 5mLfor analysis.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 280-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1.0mLper minute.Chromatograph five replicate injections of the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation is not more than 3.0%,and the resolution,R,between sulfacetamide and sulfapyridine is not less 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 responses for the major peaks.Chromatograms exhibit relative retention times of about 0.8for sulfacetamide,1.0for sulfapyridine,1.8for sulfathiazole,and 2.5for sulfabenzamide.Calculate the quantity,in mg,of C8H10N2O3S,C9H9N3O2S2,and C13H12N2O3Sin the portion of the Vaginal Cream taken by the formula: in which Cis the concentration,in µg per mL,of the appropriate USP Reference Standard in the Standard preparation,and RUand RSare the ratios of the peak responses of the corresponding sulfonamides to those of the internal standard obtained from the Assay preparationand the Standard preparation,respectively.