A: The retention times of the major peaks in the chromatogram of the Assay preparation correspond to those of the Standard preparations of sulfadoxine and pyrimethamine, relative to the internal standard, as obtained in the Assay.

B: Vigorously shake 700 mg of finely ground Tablet powder with 50 mL of a 1 in 50 solution of ammonium hydroxide in methanol for 3 minutes, and filter. Separately apply 10 µL each of the test solution, a Standard solution of USP Sulfadoxine RS similarly prepared, containing 10 mg per mL, and a Standard solution of USP Pyrimethamine RS similarly prepared, containing 0.5 mg per mL, to a suitable thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture. Dry the spots in a current of warm air, and develop the plate in a solvent system consisting of a mixture of heptane, chloroform, a 1 in 20 solution of methanol in alcohol, and glacial acetic acid (4:4:4:1). Allow the solvent front to move about two-thirds of the length of the plate, remove the plate, dry, and examine under short-wavelength UV light: the RF values of the principal spots from the solution under test correspond to the RF values of the principal spots from the corresponding Standard solutions.

Medium: pH 6.8 phosphate buffer, prepared as directed under Buffer Solutions in the section Reagents, Indicators, and Solutions; 1000 mL.

Apparatus 2: 75 rpm.

Time: 30 minutes.

Procedure— Determine the amounts of C12H14N4O4S and C12H13ClN4 dissolved, employing the procedure set forth in the Assay, making any necessary modifications.

Tolerances— Not less than 60% (Q) of the labeled amount of each of C12H14N4O4S and C12H13ClN4 is dissolved in 30 minutes.

(Official January 1, 2007)

Mobile phase— Prepare a suitable degassed and filtered mixture of dilute glacial acetic acid (1 in 100) and acetonitrile (4:1).

Internal standard solution— Prepare a solution of phenacetin in acetonitrile having a concentration of 1 mg per mL.

Standard stock solution— Transfer about 500 mg, accurately weighed, of USP Sulfadoxine RS and 25 mg, accurately weighed, of USP Pyrimethamine RS to a 100-mL volumetric flask, dissolve in 35 mL of acetonitrile, dilute with Mobile phase to volume, and mix.

Standard preparation 1— Pipet 25 mL of Standard stock solution and 2 mL of Internal standard solution into a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Standard preparation 2— Pipet 2 mL of Standard stock solution and 10 mL of Internal standard solution into a 250-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Assay preparations— Weigh and finely powder not less than 20 Tablets. Transfer an accurately weighed portion of the finely ground powder, equivalent to about 500 mg of sulfadoxine and 25 mg of pyrimethamine, to a 100-mL volumetric flask, add 35 mL of acetonitrile, shake for 30 minutes, dilute with Mobile phase to volume, mix, and filter. Pipet 25 mL of the filtrate and 2 mL of Internal standard solution into a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix (Assay preparation 1). Pipet 2 mL of the filtrate and 10 mL of the Internal standard solution into a 250-mL volumetric flask, dilute with Mobile phase to volume, and mix (Assay preparation 2).

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 2.0 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 2.5%, and the resolution factor between sulfadoxine and phenacetin is not less than 1.0, and between pyrimethamine and phenacetin is not less than 1.0.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparations and the Assay preparations into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times are about 0.7 for sulfadoxine and 1.0 for phenacetin and 1.3 for pyrimethamine. Calculate the quantity, in mg, of sulfadoxine in the portion of Tablets taken by the formula: in which C is the concentration, in µg per mL, of USP Sulfadoxine RS in Standard preparation 2, and RU and RS are the relative peak response ratios obtained from Assay preparation 2 and Standard preparation 2, respectively. Calculate the quantity, in mg, of pyrimethamine in the portion of Tablets taken by the formula: in which C¢ is the concentration, in µg per mL, of USP Pyrimethamine RS in Standard preparation 1, and R¢U and R¢S are the relative peak response ratios obtained from Assay preparation 1 and Standard preparation 1, respectively.