A: Transfer a quantity of powdered Tablets,equivalent to 1mg of reserpine,to a stoppered,50-mLcentrifuge tube.Add 20mLof citric acid solution (1in 50),and shake until the powder is suspended.Extract the mixture with two 20-mLportions of chloroform,centrifuge,and withdraw the chloroform,filtering each extract through a pledget of cotton into a 50-mLvolumetric flask.Dilute with chloroform to volume,and mix:the UVabsorption spectrum of the solution exhibits maxima and minima at the same wavelengths as that of a similar solution of USP Reserpine RS,concomitantly measured (presence of reserpine).

B: Transfer a quantity of powdered Tablets,equivalent to about 50mg of hydrochlorothiazide,to a test tube containing 10mLof acetone,agitate for 5minutes,and centrifuge.Use the clear supernatant as the Test solution.Separately apply 10µLeach of the Test solution and a Standard solution of USP Hydrochlorothiazide RSin acetone containing 5mg per mLto a thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture and previously washed with methanol.Develop the chromatogram in a solvent system consisting of a mixture of ethyl acetate and isopropyl alcohol (17:3)until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the 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 (presence of hydrochlorothiazide).

Medium: mixture of 0.1Nhydrochloric acid and n-propyl alcohol (3:2);900mL.

Apparatus 2: 50rpm.

Times: 45minutes,60minutes.

Determination of dissolved reserpine—

Determination of dissolved hydrochlorothiazide—

Procedure for content uniformity for reserpine—

Procedure for content uniformity for hydrochlorothiazide—

Standard solution— Accurately weigh 25mg of USP Benzothiadiazine Related Compound A RS,dissolve in 5mLof methanol contained in a 100-mLvolumetric flask,dilute with water to volume,and mix.Pipet 4mLof this solution into a 100-mLvolumetric flask,dilute with water to volume,and mix.

Test solution— Transfer a portion of finely powdered Tablets,accurately weighed and equivalent to about 100mg of hydrochlorothiazide,to a 100-mLvolumetric flask,and add 20mLof methanol and 20mLof water.Shake continuously for 5to 10minutes,dilute with water to volume,mix,and filter.Use the filtrate as theTest solution.

Procedure— Pipet 5mLeach of theStandard solutionand theTest solutioninto separate,50-mLvolumetric flasks.Pipet 5mLof water into a third 50-mLvolumetric flask to provide the blank.To each flask add 1mLof freshly prepared sodium nitrite solution (1in 100)and 5mLof dilute hydrochloric acid (1in 12),and allow to stand for 5minutes.Add 2mLof ammonium sulfamate solution (1in 50),shake vigorously,allow to stand for 5minutes,then add 2mLof freshly prepared disodium chromotropate solution (1in 100)and 10mLof sodium acetate TS.Dilute with water to volume,and mix.Concomitantly determine the absorbances of the solutions from theStandard solutionand theTest solutionin 1-cm cells at the wavelength of maximum absorbance at about 500nm,with a suitable spectrophotometer,against the blank.The absorbance of the solution from theTest solutiondoes not exceed that of the solution from theStandard solution,corresponding to not more than 1.0%of diazotizable substances.

Standard preparation— Dissolve about 25mg of USP Reserpine RS,accurately weighed,in 1mLof chloroform contained in a 50-mLvolumetric flask,dilute with methanol to volume,and mix.Dilute a portion of this solution quantitatively and stepwise with chloroform and methanol solution (1:1)to obtain a solution having a known concentration of about 0.2µg of reserpine per mL.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer to a stoppered,50-mLcentrifuge tube an accurately weighed portion of the powder,equivalent to about 1mg of reserpine,add 25.0mLof a mixture of chloroform and methanol solution (1:1),shake by mechanical means for 15minutes,and centrifuge.Dilute a portion of the clear supernatant quantitatively and stepwise with a mixture of chloroform and methanol solution (1:1)to obtain a solution having a concentration of about 0.2µg of reserpine per mL.

Procedure— Separately transfer 5.0mLof the Assay preparation,5.0mLof the Standard preparation,and 5.0mLof a mixture of chloroform and methanol solution (1:1)to provide the reagent blank,respectively,to three 25-mLvolumetric flasks.To each flask add 0.5mLof hydrochloric acid,1.0mLof a 3in 1000solution of sodium nitrite in dilute methanol (1in 2),mix,and allow to stand for 30minutes.Add 1mLof ammonium sulfamate solution (1in 20)to each flask,add chloroform and methanol solution (1:1)to volume,mix,and allow to stand for 10minutes.Concomitantly determine the fluorescence intensities of the solutions in a suitable spectrophotometer arranged to deliver activation radiation at 405nm and to measure the resultant fluorescence at the emission wavelength of about 500nm.Calculate the quantity,in mg,of C33H40N2O9in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Reserpine RSin the Standard preparation,and IUand ISare the fluorescence intensities of the solutions from the Assay preparationand the Standard preparation,respectively.

Standard preparation— Dissolve an accurately weighed quantity of USP Hydrochlorothiazide RSin 0.1Nsodium hydroxide,and dilute quantitatively and stepwise with 0.1Nsodium hydroxide to obtain a solution having a known concentration of about 10µg of hydrochlorothiazide per mL.Use a freshly prepared solution.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Weigh accurately a portion of the powder,equivalent to about 50mg of hydrochlorothiazide,and transfer to a 500-mLvolumetric flask.Add 200mLof 0.1Nsodium hydroxide,shake by mechanical means for 15minutes,dilute with the same solvent to volume,and mix.Filter a portion of the solution through paper,discarding the first 15mLof the filtrate,and transfer 10.0mLof the clear filtrate to a 100-mLvolumetric flask.Dilute with 0.1Nsodium hydroxide to volume,and mix.

Procedure— Concomitantly determine the absorbances of the Standard preparationand the Assay preparationin 1-cm cells at the wavelength of maximum absorbance,at about 274nm,with a suitable spectrophotometer,using 0.1Nsodium hydroxide as the blank.Calculate the quantity,in mg,of C7H8ClN3O4S2in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Hydrochlorothiazide RSin the Standard preparation,and AUand ASare the absorbances of the solutions from the Assay preparationand the Standard preparation,respectively.