A: Transfer a quantity of powdered Tablets,equivalent to about 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 so obtained 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 chlorothiazide,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 Chlorothiazide 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 chlorothiazide).

Medium: mixture of pH8.0phosphate buffer (see Buffer Solutionsin the section Reagents,Indicators,and Solutions)and n-propyl alcohol (3:2);900mL.

Apparatus 2: 75rpm.

Time: 60minutes.

Determination of dissolved reserpine—

STANDARDPREPARATION —Dissolve about 34mg of USP Reserpine RS,accurately weighed,in a 50-mLvolumetric flask containing 5mLof chloroform,dilute with n-propyl alcohol to volume (Solution 1),and mix.Pipet 1mLof Solution 1into a 50-mLvolumetric flask,dilute with n-propyl alcohol to volume (Solution 2),and mix.Pipet 1mLof Solution 2into a 100-mLvolumetric flask,dilute with Dissolution Mediumto volume,and mix to obtain the Standard preparation.

PROCEDURE —Filter a portion of the solution under test through paper,and transfer 5.0mLof the clear filtrate to a 25-mLvolumetric flask.Pipet 5mLof the Standard preparationinto a separate 25-mLvolumetric flask.Treat each flask as follows:Add 5drops of hydrochloric acid and 5mLof a mixture of water,alcohol,and sulfuric acid (29:20:1),and mix.Add by pipet 5mLof sodium nitrite solution (3in 1000),dilute with alcohol to volume,mix,and allow to stand for 30minutes.Concomitantly determine the fluorescences of the solution under test and the Standard preparationin 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 amount of reserpine (C33H40N2O9)dissolved.

Determination of dissolved chlorothiazide—

STANDARDPREPARATION —Transfer about 27mg of USP Chlorothiazide RS,accurately weighed,to a 50-mLvolumetric flask containing 5mLof methanol,swirl to dissolve,dilute with Dissolution Mediumto volume,and mix.Pipet 2mLof this solution into a 100-mLvolumetric flask,dilute with Dissolution Mediumto volume,and mix.

PROCEDURE —Determine the amount of chlorothiazide (C7H6ClN3O4S2)dissolved from UVabsorbances at the wavelength of maximum absorbance at about 292nm of filtered portions of the solution under test,suitably diluted with Dissolution Medium,in comparison with the Standard preparation.

Tolerances— Not less than 75%(Q)of the labeled amount of C33H40N2O9is dissolved in 60minutes,and not less than 75%(Q)of the labeled amount of C7H6ClN3O4S2is dissolved in 60minutes.

Procedure for content uniformity for reserpine—

Standard preparation— Prepare as directed for Standard preparationin the Assay for reserpine.

Test preparation— Weigh 1Tablet,grind to a fine powder,and transfer to a stoppered,50-mLcentrifuge tube.Add 25.0mLof chloroform and methanol solution (1:1),shake by mechanical means for 15minutes,and centrifuge.Pipet 4mLof the clear supernatant into a 100-mLvolumetric flask,dilute with the chloroform and methanol solution to volume,and mix.

Procedure— Proceed as directed for Procedurein the Assay for reserpine.Calculate the quantity,in mg,of C33H40N2O9,in the Tablet taken by the formula: in which Wtis the weight,in mg,of the Tablet,WUis the weight,in mg,of the portion of Tablet taken,Tis the labeled quantity,in mg,of reserpine in the Tablet,Cis the concentration,in µg per mL,of USP Reserpine RSin the Standard preparation,Dis the concentration,in µg per mL,of reserpine in the Test preparation,based upon the labeled quantity per Tablet and the extent of dilution,and IUand ISare the fluorescence intensities of the solutions from the Test preparationand the Standard preparation,respectively.

Procedure for content uniformity for chlorothiazide—

Standard preparation— Prepare as directed for Standard preparationunder Assay for chlorothiazide.

Test preparation— Transfer 1Tablet to a 500-mLvolumetric flask,add 300mLof 0.1Nsodium hydroxide,and sonicate,swirling the flask intermittently,until the Tablet is dissolved.Dilute with 0.1Nsodium hydroxide to volume,mix,and filter,discarding the first 15mLof the filtrate.Dilute a portion of the clear filtrate quantitatively with 0.1Nsodium hydroxide to obtain a solution having a concentration of about 10µg of chlorothiazide per mL.

Procedure— Proceed as directed for Procedurein the Assay for chlorothiazide.Calculate the quantity,in mg,of C7H6ClN3O4S2,in the Tablet taken by the formula: in which Tis the labeled quantity,in mg,of chlorothiazide in the Tablet,Cis the concentration,in µg per mL,of USP Chlorothiazide RSin the Standard preparation,Dis the concentration,in µg per mL,of chlorothiazide in the Test preparation,based upon the labeled quantity per Tablet and the extent of dilution,and AUand ASare the absorbances of the solutions from the Test preparationand the Standard preparation,respectively.

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 less than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 1mg of reserpine,to a stoppered,50-mLcentrifuge tube,add 25.0mLof 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 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 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 Chlorothiazide RSin 0.1Nsodium hydroxide,and dilute quantitatively and stepwise with 0.1Nsodium hydroxide to obtain a solution having a known concentration of about 10µg per mL.Use a freshly prepared solution.

Assay preparation— Weigh and finely powder not less than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 250mg of chlorothiazide,to a 500-mLvolumetric flask,add about 300mLof 0.1Nsodium hydroxide,and shake by mechanical means for 15minutes.Dilute with 0.1Nsodium hydroxide to volume,and mix.Filter through paper,discarding the first 15mLof the filtrate.Pipet 2mLof the clear filtrate into 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 292nm,with a suitable spectrophotometer,using 0.1Nsodium hydroxide as the blank.Calculate the quantity,in mg,of C7H6ClN3O4S2,in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Chlorothiazide RSin the Standard preparation,and AUand ASare the absorbances of the Assay preparationand the Standard preparation,respectively.