A: Transfer a quantity of finely powdered Tablets,equivalent to about 100mg of hydralazine hydrochloride,to a glass-stoppered flask.Add 40mLof dilute hydrochloric acid (1in 12),shake by mechanical means for 5minutes,and filter,discarding the first few mLof the filtrate.Transfer 20mLof the filtrate to a separator.Extract with a 10-mLportion of methylene chloride,and discard the methylene chloride.To the aqueous phase add 2mLof sodium nitrite solution (14in 1000)and 10mLof methylene chloride,and shake by mechanical means for 5minutes.Allow the layers to separate,and drain the methylene chloride through sodium sulfate that has been pre-washed with methylene chloride,into a 50-mLbeaker.Evaporate over gentle heat with the aid of nitrogen 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 Hydralazine Hydrochloride RS,similarly treated (presence of hydralazine hydrochloride).

B: Transfer a quantity of finely powdered Tablets,equivalent to about 1mg of reserpine,to a 50-mLcentrifuge tube.Add 20mLof cyclohexane,shake by mechanical means for 15minutes,centrifuge,and discard the cyclohexane.Repeat the extraction with two additional 20-mLportions of cyclohexane,shaking by mechanical means for 2minutes each time.To the residue add 10mLof chloroform,shake for 2minutes,and filter through a medium-porosity,sintered-glass funnel into another 50-mLcentrifuge tube.Extract the chloroform with 10mLof 1.0Nhydrochloric acid,discarding the aqueous acid layer.Extract the chloroform with 10mLof 0.5Nsodium hydroxide.Centrifuge for 5minutes,and withdraw the chloroform with a syringe,passing the chloroform through cotton into a 50-mLvolumetric flask containing 40mLof methanol.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).

C: The UVabsorption spectrum of the solution from the Assay preparation,obtained as directed for Procedurein the Assay for hydrochlorothiazide,exhibits maxima and minima at the same wavelengths as that of the solution from the Standard preparation,prepared as directed in the Assay for hydrochlorothiazide,similarly measured (presence of hydrochlorothiazide).

Procedure for content uniformity—

Standard solution— Weigh accurately 25mg of USP Benzothiadiazine Related Compound A RS,dissolve in 5mLof methanol contained in a 50-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 the powdered Tablets prepared for the Assay for hydrochlorothiazide,accurately weighed and equivalent to about 100mg of hydrochlorothiazide,to a 100-mLvolumetric flask,and add a mixture of 20mLof methanol and 20mLof water.Shake continuously for 5to 10minutes,dilute with water to volume,mix,and filter.Use the filtrate as the Test solution.

Procedure— Pipet 5mLeach of the Standard solutionand the Test 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),allow to stand for 5minutes with frequent swirling,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 the Standard solutionand the Test 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 the Test solutiondoes not exceed that of the solution from the Standard solution,corresponding to not more than 2.0%of diazotizable substances.

Standard preparation— Dissolve about 25mg of USP Reserpine RS,accurately weighed,in chloroform in a 50-mLvolumetric flask,and dilute with chloroform to volume (Solution 1).Pipet 10mLof Solution 1into a 50-mLvolumetric flask,and dilute with chloroform to volume.Protect the solution from light.

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 separator or a stoppered,50-mLcentrifuge tube containing 10mLof citric acid solution (1in 50).Shake vigorously until the powder is completely suspended.Add 10mLof chloroform,and proceed as directed for Procedure,beginning with “shake thoroughly for 2minutes.”

Procedure— [NOTE—Conduct the entire procedure quickly,without exposure to direct sunlight.Perform the extractions in a suitable separator or in a suitably stoppered,50-mLcentrifuge tube,and separate by centrifuging,withdrawing the portion to be retained into a hypodermic syringe fitted with a square-tipped,14-gauge,15-cm needle.]Pipet 10mLof the Standard preparationinto the extraction vessel,add 10mLof citric acid solution (1in 50),and shake thoroughly for 2minutes.Separate and withdraw the chloroform.Wash the citric acid solution with two 10-mLportions of chloroform,adding the washings to the main chloroform solution.To the combined chloroform solutions add 10mLof citric acid solution (1in 50),shake for 2minutes,and separate and withdraw the chloroform.To the combined chloroform solutions add 10mLof sodium bicarbonate solution (1in 100),shake for 2minutes,and separate.Withdraw the chloroform,filtering it through a pledget of cotton,into a 50-mLvolumetric flask containing 14.0mLof methanol.Extract the aqueous bicarbonate layer in the extraction vessel with two 2-mLportions of chloroform,passing each portion successively through the filter into the volumetric flask.Add chloroform to volume,and mix.Pipet duplicate 5-mLaliquots of the chloroform-methanol solutions into separate,10-mLvolumetric flasks.Add 2.0mLof a 1in 10solution of hydrochloric acid in methanol to each flask.To one flask of each pair of duplicates (representing the extracted Standard preparationand the extracted Assay preparation)add 1.0mLof a 3in 1000solution of sodium nitrite in dilute methanol (1in 2).To the second flask of each pair (constituting the blanks)add 1mLof dilute methanol (1in 2).Mix,and allow to stand for 30minutes.Add 0.5mLof ammonium sulfamate solution (1in 20)to each flask,add methanol to volume,mix,and allow to stand for 10minutes.Determine the absorbance of each solution in a 1-cm cell at the wavelength of maximum absorbance at about 390nm,with a suitable spectrophotometer,relative to the absorbance of a mixture of methanol,chloroform,and water (5.4:3.6:1).

Sodium acetate solution— Dissolve 27.2g of sodium acetate trihydrate in 50mLof water,bring to room temperature,and dilute with water to 100mL.

Ferric ammonium sulfate solution— Dissolve 1.8g of ferric ammonium sulfate in 4mLof dilute hydrochloric acid (1in 12),and dilute with water to 100mL.Filter,and use the clear filtrate.Prepare fresh.

1,10-Phenanthroline solution— Shake 300mg of 1,10-phenanthroline with 100mLof water for 1hour.Filter,and use the clear filtrate.Prepare fresh.

Standard preparation— Dissolve about 50mg of USP Hydralazine Hydrochloride RS,accurately weighed,in water in a 100-mLvolumetric flask,and dilute with water to volume.Pipet 10mLof this solution into a 100-mLvolumetric flask,dilute with water to volume,and mix.

Assay preparation— Weigh and finely powder not less than 20Tablets.Transfer to a 200-mLvolumetric flask an accurately weighed portion of the powder,equivalent to about 100mg of hydralazine hydrochloride,add 100mLof water,and shake by mechanical means for 30minutes.Dilute with water to volume,mix,and filter through paper,discarding the first 15mLof the filtrate.Pipet 10mLof the clear filtrate into a 100-mLvolumetric flask,and dilute with water to volume.

Procedure— Pipet 10mLeach of the Assay preparation,the Standard preparation,and water to provide the blank,into separate,200-mLvolumetric flasks.To each flask add 5mLof acetic acid solution (12in 100),5mLof Sodium acetate solution,2mLof 1,10-Phenanthroline solution,and 1mLof Ferric ammonium sulfate solution,mix,and allow to stand in the dark for 30minutes.Dilute with water to volume,and mix.Concomitantly determine the absorbances of both solutions in 1-cm cells at the wavelength of maximum absorbance at about 510nm,with a suitable spectrophotometer,against the blank.Calculate the quantity,in mg,of C8H8N4·HCl in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Hydralazine Hydrochloride RSin the Standard preparation,and AUand ASare the absorbances of the solutions from the Assay preparationand the Standard preparation,respectively.

Methanolic sodium hydroxide solution— Dissolve 40g of sodium hydroxide in 125mLof water,cool,and dilute with methanol to 500mL.Filter before use.

Column preparation— Weigh about 10g of sulfonic acid cation exchange resin into a 250-mLbeaker.Add 75mLof methanol,and stir the mixture with a magnetic stirrer for 30minutes.Place a glass wool plug at the lower end of a glass chromatographic column (15mm ×45-cm long),equipped with a stopcock to regulate the eluant flow.Transfer and pack the slurry in portions into the prepared column to a height of 10cm.If air is trapped,remove by tapping,stirring,or back-flushing the column.Place a glass wool plug on top of the resin bed after packing.Wash the resin with consecutive 100-mLportions of a 1in 5solution of hydrochloric acid in methanol,and of methanol,then of Methanolic sodium hydroxide solutionand of methanol,respectively,using a flow rate of approximately 4mLper minute.Wash the sodium form of the resin with 150mLof a 1in 5solution of hydrochloric acid in methanol,followed by not less than 100mLof methanol.The UVspectrum of the last few mLof the methanol washing conforms to that of the methanol.

Standard preparation— Mix about 40mg of USP Hydrochlorothiazide RS,accurately weighed,with 4mLof water in a 200-mLvolumetric flask.Add 150mLof methanol,warm gently on a steam bath for a few minutes,and shake by mechanical means for 20minutes.Dilute with methanol to volume,and mix.

Assay preparation— Weigh and finely powder not less than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 20mg of hydrochlorothiazide,to a 100-mLvolumetric flask.Add 2mLof water,mix,and add 75mLof methanol.Warm gently on a steam bath for a few minutes,and shake by mechanical means for 20minutes.Dilute with methanol to volume,and mix.Centrifuge a portion of the mixture,and use the clear solution as the Assay preparation.

Procedure— Keeping the column stopcocks closed,pipet 4mLof the clear Assay preparationon the top of the resin bed of 1column,and pipet 4mLof the Standard preparationon the top of the second column.Place a 100-mLvolumetric flask beneath each of the columns,and collect the eluate.Adjust the flow rate to approximately 2mLper minute,and allow the solution to flow until it just disappears into the upper glass wool plug.Close the stopcock,carefully add 25mLof methanol,and further elute each column with the same flow rate as before.Repeat with two additional 25-mLportions of methanol.Dilute the contents of each flask with methanol to volume.Concomitantly determine the absorbances of both solutions at the wavelength of maximum absorbance at about 271nm,with a suitable spectrophotometer,using methanol 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 solution from the Assay preparationand the Standard preparation,respectively.