A: Infrared Absorption á197Mñ—

B: Infrared Absorption á197Mñ—

Medium: 0.01Nhydrochloric acid;900mL.

Apparatus 1: 100rpm.

Time: 30minutes.

Procedure— Filter a portion of the solution under test,transfer 10.0mLof the filtrate to a suitable capped bottle,and add 5.0mLof water,1.0mLof 5Nsodium hydroxide,and 25.0mLof n-heptane.Cap the bottle,shake by mechanical means for 5minutes,and allow the layers to separate,centrifuging if necessary,to obtain clear upper (n-heptane)and lower (aqueous)extracts.Determine the quantity,in µg,of propranolol hydrochloride (C16H21NO2·HCl)in each mLof the n-heptane extract by employing UVabsorption at the wavelength of maximum absorbance at about 293nm,in comparison with an n-heptane Standard solution obtained by similarly treating and extracting a mixture of 5.0mLof an aqueous solution having a known concentration of USP Propranolol Hydrochloride RSand 5.0mLof a 0.005Nsodium hydroxide solution having a known concentration of USP Hydrochlorothiazide RS,using a blank consisting of the n-heptane extract obtained by similarly treating and extracting 10.0mLof water.Determine the quantity,in µg,of hydrochlorothiazide (C7H8ClN3O4S2)in each mLof the aqueous extract by employing UVabsorption at the wavelength of maximum absorbance at about 273nm,in comparison with the aqueous extract remaining from the preparation of the n-heptane Standard solution,using as a blank the aqueous extract remaining from the preparation of the n-heptane blank extract.Calculate the quantity,in mg,of C16H21NO2·HCl dissolved by multiplying the quantity,in µg,of propranolol hydrochloride in each mLof the n-heptane extract from the solution under test by 2.25.Calculate the quantity,in mg,of C7H8ClN3O4S2dissolved by multiplying the quantity,in µg,of hydrochlorothiazide in each mLof the aqueous extract from the solution under test by 1.44.

Tolerances— Not less than 80%(Q)of the labeled amount of C16H21NO2·HCl and not less than 80%(Q)of the labeled amount of C7H8ClN3O4S2is dissolved in 30minutes.

Procedure for content uniformity— Transfer a Tablet to a suitable container,and add 500.0mLof 0.1Nhydrochloric acid.Shake until the Tablet has disintegrated,sonicate for 30seconds,shake by mechanical means for 30minutes,and then repeat the sonication and shaking.Centrifuge a portion of the solution,and transfer 6.0mLof the clear supernatant and 15.0mLof water to a suitable capped bottle.Add 1.0mLof 5Nsodium hydroxide and 25.0mLof n-heptane,cap the bottle,shake by mechanical means for 5minutes,and allow the layers to separate,centrifuging,if necessary,to obtain clear upper (n-heptane)and lower (aqueous)layers (test solutions).Prepare a similar Standard solution by mixing 6.0mLof 0.1Nhydrochloric acid,3.0mLof water,6.0mLof an aqueous solution having a known concentration of USP Propranolol Hydrochloride RS,and 6.0mLof a 0.04Nsodium hydroxide solution having a known concentration of USP Hydrochlorothiazide RS,and proceeding as directed for the test solutions,beginning with “Add 1.0mLof 5Nsodium hydroxide.”Prepare similar blank n-heptane and aqueous extracts by adding 6.0mLof 0.1Nhydrochloric acid to 15.0mLof water,and proceeding as directed for the test solutions,beginning with “Add 1.0mLof 5Nsodium hydroxide”.Concomitantly determine the absorbances of the n-heptane test solution and the n-heptane Standard solution at the wavelength of maximum absorbance at about 293nm,using the n-heptane blank extract to set the instrument.Calculate the quantity,in mg,of propranolol hydrochloride (C16H21NO2·HCl)in the Tablet taken by the formula: in which Cis the concentration,in µg per mL,of USP Propranolol Hydrochloride RSin the n-heptane Standard solution;and AUand ASare the absorbances at 293nm of the n-heptane test solution and the n-heptane Standard solution,respectively.Concomitantly determine the absorbances of the aqueous test solution and the aqueous Standard solution at the wavelength of maximum absorbance at about 273nm,using the aqueous blank extract to set the instrument.Calculate the quantity,in mg,of hydrochlorothiazide (C7H8ClN3O4S2)in the Tablet taken by the formula: in which Cis the concentration,in µg per mL,of USP Hydrochlorothiazide RSin the aqueous Standard solution;and AUand ASare the absorbances at 273nm of the aqueous test solution and the aqueous Standard solution,respectively.

Tetrabutylammonium hydroxide solution,Buffer,Mobile phase,and Chromatographic system— Proceed as directed underAssay.

Standard solution— Dissolve an accurately weighed quantity of USP Benzothiadiazine Related Compound A RSin methanol to obtain a solution having a known concentration of about 0.5mg per mL.Transfer an accurately measured volume of this solution,and dilute quantitatively,and stepwise if necessary,withMobile phase to obtain a solution having a known concentration of about 0.5µg per mL.

Test solution— Use theAssay preparation prepared as directed in theAssay.

Chromatographic system— Proceed as directed underAssay,except to chromatograph theStandard solution:the relative standard deviation for replicate injections is not more than 5.0%.

Procedure— Separately inject equal volumes (about 50µL)of theStandard solution andTest solution into the chromatograph,record the chromatograms,and measure the peak areas.Calculate the percentage 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;Lis the amount,in mg,of hydrochlorothiazide in the portion of Tablets taken,based on the labeled amount;andrUandrSare the peak areas of benzothiadiazine related compound Aobtained from theTest solution andStandard solution,respectively:not more than 1.0%is found.

Tetrabutylammonium hydroxide solution— Use a suitable aqueous or methanolic solution having a known concentration of tetrabutylammonium hydroxide.

Buffer— Dissolve 6.8g of monobasic potassium phosphate in 1000mLof water in a 2000-mLvolumetric flask.Add 3.4mLof phosphoric acid and a volume ofTetrabutylammonium hydroxide solutionequivalent to about 2.6g of tetrabutylammonium hydroxide,dilute with water to volume,and mix.Adjust,if necessary,with phosphoric acid or 10Npotassium hydroxide to a pHof 2.5±0.1,and pass through a filter having a 0.5-µm or finer porosity.

Mobile phase— Prepare a suitable mixture ofBufferand methanol (850:150).Make adjustments if necessary (seeSystem SuitabilityunderChromatography á621ñ)so that the retention time of propranolol is between 12and 25minutes.

Standard hydrochlorothiazide stock solution— Transfer about 25mg of USP Hydrochlorothiazide RS,accurately weighed,to a 100-mLvolumetric flask,add 15mLof methanol,and mix to dissolve.Dilute withBufferto volume,and mix.

Standard propranolol hydrochloride stock solution— Dissolve an accurately weighed quantity of USP Propranolol Hydrochloride RSinMobile phaseto obtain a solution having a known concentration of about 0.25Jmg per mL,Jbeing the ratio of the labeled quantity,in mg,of propranolol hydrochloride to the labeled quantity,in mg,of hydrochlorothiazide per Tablet.

Standard preparation— Transfer 5.0mLofStandard hydrochlorothiazide stock solutionand 5.0mLofStandard propranolol hydrochloride stock solutionto a 25-mLvolumetric flask,dilute withMobile phaseto volume,and mix.This solution contains about 50µg of hydrochlorothiazide and 50Jµg of propranolol hydrochloride per mL.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 25mg of hydrochlorothiazide,to a 500-mLvolumetric flask.Add 5mLof water,mix,and allow to stand for 5minutes,with occasional swirling.Add 75mLof methanol,mix,and sonicate for 10minutes,with occasional swirling,adding ice to the bath,if necessary,to maintain the temperature at not more than 20.Add about 350mLofBufferto the flask,and sonicate for 10minutes,with occasional swirling,maintaining the temperature of the bath at not more than 20.Dilute withBufferto volume,and mix.Centrifuge a portion of this solution,if necessary,to obtain a clear solution (Assay preparation).

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 270-nm detector and a 4-mm ×15-cm column that contains 5-µm packing L1.The flow rate is about 1.5mLper minute.Chromatograph theStandard preparation,and record the peak responses as directed forProcedure:the column efficiency determined from the propranolol peak is not less than 2500theoretical plates;the tailing factor for the propranolol and hydrochlorothiazide peaks is not more than 1.5;and the relative standard deviation for replicate injections is not more than 2.0%.[NOTE—The relative retention times are about 0.25for benzothiadiazine related compound A,0.4for hydrochlorothiazide,and 1.0for propranolol.]

Procedure— Separately inject equal volumes (about 50µL)of the Standard preparation and theAssay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantities,in mg,of propranolol hydrochloride (C16H21NO2·HCl)and hydrochlorothiazide (C7H8ClN3O4S2)in the portion of Tablets taken by the same formula: in whichCis the concentration,in µg per mL,of the appropriate Reference Standard in theStandard preparation;andrUandrSare the peak responses of the corresponding analyte obtained from theAssay preparationand theStandard preparation,respectively.