A: The chromatogram of the Assay preparationobtained as directed in the Assayexhibits a peak for chlorothiazide,the retention time of which corresponds to that exhibited by the Standard preparation.

B: Powder 1Tablet,and fuse it with a pellet of sodium hydroxide:the ammonia fumes produced turn moistened red litmus paper blue,and the residue responds to the test for Sulfite á191ñ.

Medium: 0.05MpH8.0phosphate buffer (see Buffer Solutionsin the section Reagents,Indicators,and Solutions);900mL.

Apparatus 2: 75rpm.

Time: 60minutes.

Procedure— Determine the amount of C7H6ClN3O4S2dissolved from UVabsorbances at the wavelength of maximum absorbance at about 294nm of filtered portions of the solution under test,suitably diluted with Dissolution Medium,if necessary,in comparison with a Standard solution having a known concentration of USP Chlorothiazide RSin the same medium.

Tolerances— Not less than 75%(Q)of the labeled amount of C7H6ClN3O4S2is dissolved in 60minutes.

Mobile phase— Prepare a filtered and degassed solution of 0.08Mmonobasic sodium phosphate (adjusted with phosphoric acid to a pHof 2.9±0.1)and methanol (95:5),making adjustments if necessary (seeSystem Suitabilityunder Chromatography á621ñ).

Standard preparation— Transfer an accurately weighed quantity of about 25mg of USP Chlorothiazide RSto a 50-mLvolumetric flask,add 5mLof 0.05Mmonobasic sodium phosphate solution,followed by 10mLof acetonitrile to the flask,and sonicate with occasional shaking for about 3minutes.Dilute with water to volume,mix,and filter to obtain a Standard preparationhaving a known concentration of about 0.5mg of USP Chlorothiazide RSper mL.

Assay preparation— [NOTE—Prepare fresh daily.]Weigh and finely powder not less than 20Tablets.Weigh accurately a portion of the powder,equivalent to about 250mg of chlorothiazide,and transfer to a 500-mLvolumetric flask.Add 50mLof 0.05Mmonobasic sodium phosphate solution,and shake by mechanical means for about 15minutes followed by sonication for about 2minutes.Add 100mLof acetonitrile,sonicate for about 3minutes,dilute with water to volume,mix,and filter.

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 L1and is fitted with a guard column.The flow rate is about 2mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed under Procedure:the capacity factor (k¢)is not less than 4.3,the tailing factor (T)for chlorothiazide is not more than 2.0,the theoretical plate count (N)for chlorothiazide is not less than 1300,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 15µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C7H6ClN3O4S2in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Chlorothiazide RSin the Standard preparation,and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.