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

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

Standard preparation— Weigh accurately about 29 mg of USP Sulfacetamide RS, 34 mg of USP Sulfathiazole RS, and 37 mg of USP Sulfabenzamide RS, and transfer to a 50-mL volumetric flask. Add 2.0 mL of Internal standard solution and 30 mL of acetone, and shake for 10 minutes. If necessary, sonicate to effect solution. Dilute with acetone to volume, and mix. Pipet 5 mL of this solution into a 50-mL volumetric 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 phase to 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 144 mg of sulfacetamide, 184 mg of sulfabenzamide, and 173 mg of sulfathiazole, to a 250-mL volumetric flask. Add 10.0 mL of Internal standard solution and 100 mL of 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 10 mL of the filtrate. Pipet 5 mL of the filtrate so obtained into a 50-mL volumetric 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 phase to volume, and mix. Cool the solution in an ice bath for 10 minutes, filter the cold solution through filter paper, discarding the first 10 mL to 15 mL of the filtrate, and collect 5 mL for analysis.

Chromatographic system (see Chromatography 621)—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 1.0 mL per 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 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Chromatograms exhibit relative retention times of about 0.8 for sulfacetamide, 1.0 for sulfapyridine, 1.8 for sulfathiazole, and 2.5 for sulfabenzamide. Calculate the quantity, in mg, of C8H10N2O3S, C9H9N3O2S2, and C13H12N2O3S in the portion of the Vaginal Cream taken by the formula: in which C is the concentration, in µg per mL, of the appropriate USP Reference Standard in the Standard preparation, and RU and RS are the ratios of the peak responses of the corresponding sulfonamides to those of the internal standard obtained from the Assay preparation and the Standard preparation, respectively.