A: Transfer a portion of finely powdered Tablets,equivalent to about 50mg of hydrochlorothiazide,to a 50-mLvolumetric flask.Add about 20mLof sodium hydroxide solution (1in 125),and shake vigorously for 15minutes.Dilute with the same solvent to volume,mix,and filter,discarding the first few mLof the filtrate.Transfer 5mLof the filtrate to a 125-mLseparator,and add 5mLof dilute hydrochloric acid (1in 10).Extract with 50mLof ether,filter the ether extract through a small,dry,folded filter paper,and evaporate to dryness.Add 5mLof alcohol,and again evaporate to dryness:the IRabsorption spectrum of a potassium bromide dispersion of the residue so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of USP Hydrochlorothiazide RSpreviously dissolved in alcohol and recovered by evaporating the solution to dryness.

B: The retention time of the major peak in the chromatogram of theAssay preparation corresponds to that in the chromatogram of theStandard preparation,as obtained in theAssay.

Medium: 0.1Nhydrochloric acid;900mL.

Apparatus 1: 100rpm.

Time: 60minutes.

Procedure— Determine the amount of C7H8ClN3O4S2dissolved from UVabsorbances at the wavelength of maximum absorbance at about 272nm on filtered portions of the solution under test,suitably diluted withDissolution Medium,if necessary,in comparison with a Standard solution having a known concentration of USP Hydrochlorothiazide RSin the same Medium.

Tolerances— Not less than 60%(Q)of the labeled amount of C7H8ClN3O4S2is dissolved in 60minutes.

Mobile phase,System suitability solution,and Chromatographic system— Proceed as directed in theAssay.

Test solution— Proceed as directed forAssay preparation in theAssay.

Standard solution— [NOTE—Avolume of acetonitrile not exceeding 10%of the total volume of the solution may be used to dissolve the USP Reference Standard.]Dissolve an accurately weighed quantity of USP Benzothiadiazine Related Compound A RSinMobile phase to obtain a solution having a known concentration of about 1.5µg per mL.

Procedure— Separately inject equal volumes (about 20µL)of theStandard solution and theTest solution into the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of benzothiadiazine related compound Ain the portion of tablets taken by the formula: in whichCis the concentration,in µg per mL,of USP Benzothiadiazine Related Compound A RSin theStandard solution;andrUandrSare the peak responses for benzothiadiazine related compound Aobtained from theTest solution and theStandard solution,respectively:not more than 1.0%is present.

Mobile phase— Prepare a degassed mixture of 0.1Mmonobasic sodium phosphate and acetonitrile (9:1),adjust with phosphoric acid to a pHof 3.0±0.1,and filter.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

System suitability solution— [NOTE—Avolume of acetonitrile not exceeding 10%of the total volume of solution may be used to dissolve the USP Reference Standards.]Dissolve accurately weighed quantities of chlorothiazide and USP Hydrochlorothiazide RSin Mobile phaseto obtain a solution containing about 0.15mg of each per mL.

Standard preparation— [NOTE—Avolume of acetonitrile not exceeding 10%of the total volume of the solution may be used to dissolve the USP Reference Standard.]Dissolve an accurately weighed quantity of USP Hydrochlorothiazide RSin Mobile phaseto obtain a solution having a known concentration of about 0.15mg per mL.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 30mg of hydrochlorothiazide,to a 200-mLvolumetric flask.Add about 20mLof Mobile phase,sonicate for 5minutes,and add about 20mLof acetonitrile.Sonicate for 5minutes,add about 50mLof Mobile phase,and shake by mechanical means for 10minutes.Dilute with Mobile phaseto volume,mix,and filter,discarding the first 10mLof the filtrate.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 2.0mLper minute.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.8for chlorothiazide and 1.0for hydrochlorothiazide;and the resolution,R,between chlorothiazide and hydrochlorothiazide is not less than 2.0.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 1.5%.

Procedure— Separately inject equal volumes (about 20µL)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 hydrochlorothiazide (C7H8ClN3O4S2)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Hydrochlorothiazide RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.