A: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard 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 and a Standard solution of USP Amiloride Hydrochloride RSin methanol,containing 0.2mg per mL,to a thin-layer chromatographic plate (see Chromatography 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 RFvalue of the principal spot in the chromatogram of the test solution corresponds to that obtained from the Standard solution.

Medium: 0.1Nhydrochloric acid;900mL.

Apparatus 2: 50rpm.

Time: 30minutes.

Procedure— Determine the amount of C6H8ClN7O·HCl dissolved from UVabsorbances at the wavelength of maximum absorbance at about 363nm on filtered portions of the solution under test,suitably diluted with 0.1Nhydrochloric acid,in comparison with a Standard solution having a known concentration of USP Amiloride Hydrochloride 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 is dissolved in 30minutes.

Procedure for content uniformity— Transfer 1finely powdered Tablet to a 100-mLvolumetric flask,add 60mLof 0.1Nhydrochloric acid,and shake by mechanical means for 30minutes.Dilute with 0.1Nhydrochloric acid to volume,mix,and centrifuge a portion of the mixture.Dilute an accurately measured portion of the clear supernatant quantitatively to obtain a solution containing about 10µg of amiloride hydrochloride per mL.Concomitantly determine the absorbances of this solution and a Standard solution of USP Amiloride Hydrochloride RSin the same medium having a known concentration of about 10µg per mL,at the wavelength of maximum absorbance at about 363nm,with a suitable spectrophotometer,using 0.1Nhydrochloric acid as the blank.Calculate the quantity,in mg,of C6H8ClN7O·HCl in the Tablet taken by the formula: in which Tis the labeled quantity,in mg,of amiloride hydrochloride in the Tablet,Cis the concentration,in µg per mL,of USP Amiloride Hydrochloride RS,corrected for loss in weight in the Standard solution;Dis the concentration,in µg per mL,of amiloride hydrochloride in the solution from the Tablet,based upon the labeled quantity per Tablet and the extent of dilution;and AUand ASare the absorbances of the solution from the Tablets and the Standard solution,respectively.

Buffer solution— Dissolve 136g of monobasic potassium phosphate in 800mLof water,and adjust by the addition of phosphoric acid,with mixing,to a pHof 3.0.Dilute with water to 1000mL,and mix.

Mobile phase— Prepare a suitable degassed mixture of water,methanol,and Buffer solution(71:25:4).

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 5.0mLof the solution to a 50-mLvolumetric flask,and add 10.0mLof methanol and 2.0mLof 0.1Nhydrochloric acid.Dilute with water to volume,and mix.The concentration of USP Amiloride Hydrochloride RSin the Standard preparationis about 0.1mg per mL.

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 containing 15.0mLof methanol and 2.0mLof 0.1Nhydrochloric 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 the Standard preparation,and record the peak responses as directed for Procedure:the tailing factor of the major peak is not more than 2.0;and the relative standard deviation is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)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 amiloride hydrochloride (C6H8ClN7O·HCl)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Amiloride Hydrochloride RS,corrected for loss in weight in the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.