A: The retention times of the major peaks in the chromatogram of theAssay preparationcorrespond to those in the chromatogram of theStandard preparation,as obtained in the Assay.

B: Transfer an amount of finely ground Tablets,equivalent to 5mg of amiloride hydrochloride,to a 25-mLvolumetric flask,add methanol to volume,mix,and filter.Separately apply 10µLeach of the filtrate,a Standard solution of USP Amiloride Hydrochloride RSin methanol containing 0.2mg per mL,and a Standard solution of USP Hydrochlorothiazide RSin methanol containing 2mg per mLto a thin-layer chromatographic plate (seeChromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Develop the chromatogram in a solvent system consisting of a mixture of tetrahydrofuran and 3Nammonium hydroxide (22:3)until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the developing chamber,air-dry,and examine under short-wavelength UVlight:the RFvalues of the amiloride hydrochloride and hydrochlorothiazide spots obtained from the test solution correspond to those obtained from the corresponding Standard solutions.

Medium: 0.1Nhydrochloric acid;900mL.

Apparatus 2: 50rpm.

Time: 30minutes.

Procedure— Determine the amounts of amiloride hydrochloride (C6H8ClN7O·HCl)and hydrochlorothiazide (C7H8ClN3O4S2)dissolved by employing UVabsorption at the wavelengths of maximum absorbance at about 363nm for amiloride hydrochloride and 270nm for hydrochlorothiazide (corrected for interference from amiloride hydrochloride on the basis of the absorbances of amiloride hydrochloride at 270nm and 363nm)on filtered portions of the solution under test,suitably diluted with 0.1Nhydrochloric acid,in comparison with Standard solutions having known concentrations of USP Amiloride Hydrochloride RSand USP Hydrochlorothiazide RSin the same Medium.An amount of methanol not to exceed 2%of the total volume of the Standard solution may be used to dissolve the amiloride hydrochloride.

Tolerances— Not less than 80%(Q)of the labeled amount of C6H8ClN7O·HCl and 75%(Q)of the labeled amount of C7H8ClN3O4S2are dissolved in 30minutes.

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

Test solution— Proceed as directed forAssay preparation in theAssay.

Standard solution— Dissolve an accurately weighed quantity of USP Benzothiadiazine Related Compound A RSinMobile phase to obtain a solution having a known concentration of about 10µg per mL.

Procedure— Separately inject equal volumes (about 20µL)of theStandard solutionand theTest solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in µg,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 of benzothiadiazine related compound Aobtained from theTest solution and theStandard solution,respectively:not more than 1.0%is present.

Buffer solutionand Mobile phase— Prepare as directed in theAssay underAmiloride Hydrochloride Tablets.

Standard preparation— Dissolve a suitable quantity of USP Amiloride Hydrochloride RSin methanol to obtain a solution having a known concentration of about 1.0mg of amiloride hydrochloride per mL.Transfer 10.0mLof this solution to a 100-mLvolumetric flask containing 100mg of USP Hydrochlorothiazide RS,accurately weighed,and 20.0mLof methanol.Add 4.0mLof 1Nhydrochloric acid,dilute with water to volume,and mix.The concentrations of USP Amiloride Hydrochloride RSand USP Hydrochlorothiazide RSin theStandard preparationare about 0.1mg per mLand 1mg per mL,respectively.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 5mg of amiloride hydrochloride,to a 50-mLvolumetric flask.Add 15.0mLof methanol,and 2.0mLof 1Nhydrochloric acid.Sonicate for 10minutes,dilute with water to volume,sonicate for an additional 10minutes,mix,and filter.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 286-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph five replicate injections of theStandard preparation,and record the peak responses as directed forProcedure:the relative retention times are about 0.7for hydrochlorothiazide and 1.0for amiloride hydrochloride;the resolution,R,between hydrochlorothiazide and amiloride hydrochloride is not less than 2.0;and the relative standard deviation is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of theStandard preparation and theAssay preparation into the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of amiloride hydrochloride (C6H8ClN7O·HCl)in the portion of Tablets taken by the formula: in whichCis the concentration,in mg per mL,of USP Amiloride Hydrochloride RS,corrected for loss in weight in theStandard preparation;andrUandrSare the peak responses for amiloride hydrochloride obtained from theAssay preparation and theStandard preparation,respectively.Similarly calculate the quantity,in mg,of hydrochlorothiazide (C7H8ClN3O4S2)in the portion of Tablets taken by the formula: in whichCis the concentration,in mg per mL,of USP Hydrochlorothiazide RSin theStandard preparation;andrUandrSare the peak responses of hydrochlorothiazide obtained from theAssay preparation and theStandard preparation,respectively.