A: Transfer the contents of a number of Capsules,equivalent to about 100mg of hydrochlorothiazide,to a 20-mesh sieve.Break up any large lumps with the aid of a spatula,and collect the powder that passes through the sieve.[NOTE—Retain the material on the screen for Identification test B.]Transfer the powder that passed through the sieve to a screw-capped,35-mLcentrifuge tube,add 5mLof solvent hexane,and shake for 5minutes.Centrifuge,and discard the solvent.To the residue in the centrifuge tube add 10mLof 1Nsodium hydroxide,shake,and filter,collecting the filtrate in a separator.Wash the filter with 5mLof water,and collect the washing in the separator.Add 50mLof ether to the separator,shake for 2minutes,and allow the phases to separate.Drain the aqueous layer into a beaker,adjust with 6Nhydrochloric acid to a pHof about 2,induce crystallization by scratching the inner surface of the beaker with a glass rod,and allow to stand until crystallization is complete.Collect the crystals on a filter,and dry at 105for 30minutes.Grind the crystals to a fine powder:the IRabsorption spectrum of a mineral oil dispersion of the powder so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of USP Hydrochlorothiazide RS.

B: Wash the material retained on the screen fromIdentification testAwith a small amount of water,discarding the washings.Transfer the particles remaining on the screen to a glass mortar,add about 5mLof water,and triturate the mixture with a glass pestle.Transfer the suspension,with the aid of about 10mLof water,to a 35-mLscrew-capped centrifuge tube,add about 1mLof 1Nsodium hydroxide,and mix.Add about 15mLof ether,and shake by mechanical means for 5minutes.Centrifuge the mixture,and transfer as much of the ether layer as possible to a second centrifuge tube.Add 0.1mLof hydrochloric acid to the ether extract,and shake.Centrifuge,and discard the ether.Add about 15mLof ether to the residue,and shake by mechanical means for 5minutes.Centrifuge,and discard the ether layer.Dry the residue in vacuum at 45for 30minutes.Grind the crystals to a fine powder:the IRabsorption spectrum of a mineral oil dispersion of the powder so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of USP Propranolol Hydrochloride RS.

C: The chromatogram of theAssay preparation obtained as directed under Assay exhibits major peaks for propranolol hydrochloride and hydrochlorothiazide,the retention times of which correspond to those exhibited in the chromatogram of theStandard preparationobtained as directed under Assay.

pH1.5buffer solution,pH6.8buffer solution,Media,and Apparatus— Proceed as directed in the test for Drug releaseunder Propranolol Hydrochloride Extended-Release Capsules.

Analytical method— Determine the amounts of hydrochlorothiazide (C7H8ClN3O4S2)and propranolol hydrochloride (C16H21NO2·HCl)dissolved,using the following method.

Stock standard solution A— Prepare a solution of USP Propranolol Hydrochloride RSin dilute hydrochloric acid (1in 100)having a known concentration of about 0.4mg per mL.

Stock standard solution B— Dissolve an accurately weighed quantity of USP Hydrochlorothiazide RSin 0.25Nsodium hydroxide to obtain a solution having a concentration of about 25mg per mL.Dilute this solution quantitatively with water to obtain a solution having a known concentration of about 0.5mg per mL.

Standard solution— Prepare,by combining aliquots of Stock standard solutions,Aand B,and diluting with dilute hydrochloric acid (1in 100),solutions bracketing the expected concentration of the samples at the various time points.

Times: 30minutes;1.5hours;4hours;8hours;14hours;24hours.

Procedure— Use an automatic analyzer consisting of a liquid sampler,a proportioning pump,two UVspectrophotometers,and a manifold consisting of the components illustrated in the diagram under Automated Methods of Analysis á16ñ.Start the sampler and conduct determinations at a rate of 30per hour,using a ratio of about 1:1for the sample to wash time.Calculate the amounts of C7H8ClN3O4S2and C16H21NO2·HCl dissolved by comparison with the Standard solution.

Tolerances (Hydrochlorothiazide)— Use the Acceptance Tableunder Dissolution á711ñ.Not less than 80%(Q)of the labeled amount of C7H8ClN3O4S2is dissolved in 30minutes.

Tolerances (Propranolol Hydrochloride)— The percentages of the labeled amount of C16H21NO2·HCl dissolved at the times specified conform to Acceptance Table 1under Drug Release á724ñ.

Procedure for content uniformity—

Tetrabutylammonium hydroxide solution,Buffer,Mobile phase,Standard preparation,and Chromatographic system— Prepare as directed in theAssay.

Standard solution— Transfer about 25mg of USP Benzothiadiazine Related Compound A RS,accurately weighed,to a 200-mLvolumetric flask,add 30mLof methanol,and swirl to dissolve.Dilute withBufferto volume,and mix.Dilute an accurately measured volume of this solution quantitatively,and stepwise if necessary,withMobile phaseto obtain a solution having a known concentration of about 0.5µg per mL.

Test solution— Use theAssay preparationprepared as directed in theAssay.

Procedure— Proceed as directed forProcedurein theAssay,except to inject equal volumes (about 50µL)of theStandard solutionand theTest solution.Calculate the percentage of benzothiadiazine related compound Ain the Capsules taken by the formula: in whichCis the concentration,in µg per mL,of USP Benzothiadiazine Related Compound A RSin theStandard solution;Nis the number of Capsules taken to prepare theTest solution;Lis the labeled amount,in mg,of hydrochlorothiazide in each Capsule taken;andrUandrSare the peak responses of benzothiadiazine related compound Aobtained from theTest solutionand theStandard solution,respectively:not more than 1.0%is present.

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

Buffer— Dissolve 31.25g of monobasic potassium phosphate in 500mLof water in a 1000-mLvolumetric flask.Add 18.75mLof phosphoric acid,mix,and add a volume ofTetrabutylammonium hydroxide solutionequivalent to about 13g of tetrabutylammonium hydroxide.Dilute with water to volume,and mix.Dilute 100mLof this solution with water to obtain 1000mLof solution,adjusting,if necessary,with phosphoric acid or 10Npotassium hydroxide to a pHof 2.4±0.1.

Mobile phase— Prepare a suitable mixture ofBufferand methanol (850:150).Make adjustments if necessary (seeSystem SuitabilityunderChromatography á621ñ).

Standard hydrochlorothiazide stock solution— Transfer about 25mg of USP Hydrochlorothiazide RS,accurately weighed,to a 100-mLvolumetric flask,add 15mLof methanol,and sonicate for 5minutes,adding ice to the bath,if necessary,to maintain the temperature at not more than 20.Dilute withBufferto volume,and mix.Use this solution within 3days.

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 Capsule.

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.Use this solution within 3days.

Assay preparation— Carefully open an accurately counted number of Capsules,equivalent to about 500mg of hydrochlorothiazide,and transfer the contents and the Capsule shells to a 500-mLvolumetric flask.Add 5.0mLof water to the flask,and allow to stand for 5minutes.Dilute with methanol to volume,mix,and sonicate for 10minutes,adding ice to the bath,if necessary,to maintain the temperature at not more than 20.Remove the flask from the bath,and shake it occasionally for 1hour.Centrifuge a portion of the contents of the flask,if necessary,to obtain a clear solution.Transfer 5.0mLof the clear solution to a 100-mLvolumetric flask,add 10.0mLof methanol,dilute withBufferto volume,and mix.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 220-nm detector and a 4-mm ×15-cm column that contains 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 when calculated by the formula: the tailing factor for the hydrochlorothiazide and propranolol peaks is not more than 1.5;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50µL)of theStandard preparationand theAssay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.The retention time for propranolol is between 12and 25minutes,and the relative retention times are about 0.25for benzothiadiazine related compound A,0.4for hydrochlorothiazide,and 1.0for propranolol.Calculate the quantities,in mg,of hydrochlorothiazide (C7H8ClN3O4S2)and propranolol hydrochloride (C16H21NO2·HCl)in each Capsule taken by the same formula: in whichCis the concentration,in µg per mL,ofUSP Propranolol Hydrochloride RSor USP Hydrochlorothiazide RSin theStandard preparation;Nis the number of Capsules taken to prepare theAssay preparation;andrUandrSare the peak responses of the corresponding analyte obtained from theAssay preparationand theStandard preparation,respectively.