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

B: Vigorously shake 700mg of finely ground Tablet powder with 50mLof a 1in 50solution of ammonium hydroxide in methanol for 3minutes,and filter.Separately apply 10µLeach of the test solution,a Standard solution of USP Sulfadoxine RSsimilarly prepared,containing 10mg per mL,and a Standard solution of USP Pyrimethamine RSsimilarly prepared,containing 0.5mg per mL,to a suitable thin-layer chromatographic plate (see Chromatography á621ñ)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 1in 20solution 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 UVlight:the RFvalues of the principal spots from the solution under test correspond to the RFvalues of the principal spots from the corresponding Standard solutions.

Medium: pH6.8phosphate buffer,prepared as directed under Buffer Solutionsin the section Reagents,Indicators,and Solutions;1000mL.

Apparatus 2: 75rpm.

Time: 30minutes.

Procedure— Determine the amounts of C12H14N4O4Sand C12H13ClN4dissolved,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 C12H14N4O4Sand C12H13ClN4is dissolved in 30minutes.

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

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

Standard stock solution— Transfer about 500mg,accurately weighed,of USP Sulfadoxine RSand 25mg,accurately weighed,of USP Pyrimethamine RSto a 100-mLvolumetric flask,dissolve in 35mLof acetonitrile,dilute with Mobile phaseto volume,and mix.

Standard preparation 1— Pipet 25mLof Standard stock solutionand 2mLof Internal standard solutioninto a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Standard preparation 2— Pipet 2mLof Standard stock solutionand 10mLof Internal standard solutioninto a 250-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Assay preparations— Weigh and finely powder not less than 20Tablets.Transfer an accurately weighed portion of the finely ground powder,equivalent to about 500mg of sulfadoxine and 25mg of pyrimethamine,to a 100-mLvolumetric flask,add 35mLof acetonitrile,shake for 30minutes,dilute with Mobile phaseto volume,mix,and filter.Pipet 25mLof the filtrate and 2mLof Internal standard solutioninto a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix (Assay preparation 1).Pipet 2mLof the filtrate and 10mLof the Internal standard solutioninto a 250-mLvolumetric flask,dilute with Mobile phaseto volume,and mix (Assay preparation2).

Chromatographic system (see Chromatography á621ñ)—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 2.0mLper 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 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 10µL)of the Standard preparationsand the Assay preparationsinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 0.7for sulfadoxine and 1.0for phenacetin and 1.3for pyrimethamine.Calculate the quantity,in mg,of sulfadoxine in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Sulfadoxine RSin Standard preparation 2,and RUand RSare the relative peak response ratios obtained from Assay preparation 2and 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 RSin Standard preparation 1,and R¢Uand R¢Sare the relative peak response ratios obtained from Assay preparation 1and Standard preparation 1,respectively.