Hydrochloric acid solution— Dilute 2 mL of 3 N hydrochloric acid with water to 100 mL.

Test preparation— Transfer an accurately measured volume of Injection, equivalent to about 48 mg of trimethoprim and 240 mg of sulfamethoxazole, to a glass-stoppered, 50-mL centrifuge tube. Add 15 mL of Hydrochloric acid solution, and mix. Add 15 mL of chloroform, shake for 30 seconds, and centrifuge at high speed for 3 minutes. Transfer the supernatant layer to a 125-mL separator. Extract the chloroform layer in the centrifuge tube with 15 mL of Hydrochloric acid solution, centrifuge at high speed, and add the extract to the separator. Add 2 mL of sodium hydroxide solution (1 in 10) to the solution in the separator, and extract with three 20-mL portions of chloroform, collecting the organic layer in a 125-mL conical flask. Evaporate the chloroform under a stream of nitrogen to dryness. Dissolve the residue in 1 mL of a mixture of chloroform and methanol (1:1).

Standard preparation A— Using an accurately weighed quantity of USP Trimethoprim RS, prepare a solution in chloroform and methanol (1:1) having a known concentration of 48 mg per mL.

Standard preparation B— Dilute an accurately measured volume of Standard preparation A with a mixture of chloroform and methanol (1:1) to obtain a solution having a known concentration of 240 µg per mL.

Procedure— Apply 10 µL each of the Test preparation, Standard preparation A, and Standard preparation B to separate points on a thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel. Develop the chromatogram in a solvent system consisting of a mixture of chloroform, methanol, and ammonium hydroxide (97:7.5:1) until the solvent front has moved at least 12 cm. Remove the plate from the developing chamber, and air-dry. Locate the bands by viewing under short-wavelength UV light and by spraying with a freshly prepared mixture of ferric chloride solution (1 in 10) and potassium ferricyanide solution (1 in 20) (1:1). Trimethoprim produces a spot at about RF 0.5, and the trimethoprim degradation product produces a spot at about RF 0.6 to 0.7. Any spot from the Test preparation at about RF 0.6 to 0.7 is not greater in size and intensity than the spot produced by Standard preparation B at about RF 0.5, corresponding to not more than 0.5%. [NOTE—There may be spots due to concentrate excipients at about RF 0.1.]

Alcohol–methanol mixture— Mix dehydrated alcohol and methanol (95:5).

Ammonium hydroxide solution— Dilute 1 mL of ammonium hydroxide with Alcohol–methanol mixture to 100 mL.

Modified Ehrlich's reagent— Dissolve 100 mg of p-dimethylaminobenzaldehyde in 1 mL of hydrochloric acid, dilute with alcohol to 100 mL, and mix.

Test preparation— Transfer an accurately measured volume of Injection, equivalent to about 32 mg of trimethoprim and 160 mg of sulfamethoxazole, to a 25-mL graduated cylinder, dilute with Ammonium hydroxide solution to 16 mL, and mix.

Standard preparation A— Transfer about 50 mg of USP Sulfamethoxazole RS, accurately weighed, to a 5-mL volumetric flask, dissolve in and dilute with Ammonium hydroxide solution to volume, and mix.

Standard preparation B— Transfer about 25 mg of USP Sulfanilamide RS, accurately weighed, to a 250-mL volumetric flask, dissolve in and dilute with Ammonium hydroxide solution to volume, and mix. Pipet 5 mL of this solution into a 10-mL volumetric flask, dilute with Ammonium hydroxide solution to volume, and mix.

Standard preparation C— Transfer about 25 mg of USP Sulfanilic Acid RS, accurately weighed, to a 250-mL volumetric flask, dissolve in and dilute with Ammonium hydroxide solution, and mix. Pipet 3 mL of this solution into a 10-mL volumetric flask, dilute with Ammonium hydroxide solution to volume, and mix.

Procedure— Apply 10 µL each of the Test preparation and Standard preparations A, B, and C to separate points on a thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel. Develop the chromatogram in a solvent system consisting of a mixture of Alcohol-methanol mixture, heptane, chloroform and glacial acetic acid (30: 30:30:10) until the solvent front has moved not less than 12 cm. Remove the plate from the developing chamber, air-dry, spray with Modified Ehrlich's reagent, and allow the plate to stand for 15 minutes: sulfamethoxazole produces a spot at about RF 0.7. Any spots from the Test preparation at about RF 0.5 or 0.1 are not greater in size or intensity than spots produced by Standard preparations B and C, respectively, corresponding to not more than 0.5% of sulfanilamide and 0.3% of sulfanilic acid.

(500C/V)(rU / rS),