A:Thin-Layer Chromatographic Identification Test á201ñ—

B: The retention times of the major peaks in the chromatograms of the Bisoprolol fumarate assay preparationand the Hydrochlorothiazide assay preparationcorrespond to those in the chromatogram of the Standard preparation,as obtained in the Assay.

Medium: 0.1Nhydrochloric acid;900mL.

Apparatus 2: 75rpm.

Times: 20minutes for bisoprolol fumarate;30minutes for hydrochlorothiazide.

Triethylamine solution— Mix 2mLof triethylamine with 1000mLof water,and adjust with phosphoric acid to a pHof 3.0.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and Triethylamine solution(4:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard stock solution 1— Quantitatively dissolve an accurately weighed quantity of USP Bisoprolol Fumarate RSin Mediumto obtain a solution having a known concentration of about 0.5mg per mL.

Standard stock solution 2— Transfer about 30mg of USP Hydrochlorothiazide RS,accurately weighed,to a 50-mLvolumetric flask,dissolve in 5mLof methanol,dilute with Mediumto volume,and mix.

Standard solution— Dilute accurately measured volumes of Standard stock solution 1and Standard stock solution 2with Mediumto obtain a solution having known concentrations of bisoprolol fumarate and hydrochlorothiazide corresponding to those of the solution under test.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a UVdetector capable of measuring peak responses at 227nm and 272nm,simultaneously,and a 3.9-mm ×15-cm column that contains packing L11.The flow rate is about 1.5mLper minute.Chromatograph the Standard solution,and record the peak areas as directed for Procedure:the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard solutionand the filtered portions of the solution under test into the chromatograph,record the chromatograms,and measure the peak areas for bisoprolol at 227nm and for hydrochlorothiazide at 272nm.Calculate the quantities,in mg,of bisoprolol fumarate (C18H31NO4)2·C4H4O4and hydrochlorothiazide (C7H8ClN3O4S2)dissolved.

Tolerances— Not less than 80%(Q)of the labeled amount of (C18H31NO4)2·C4H4O4is dissolved in 20minutes and not less than 80%(Q)of the labeled amount of C7H8ClN3O4S2is dissolved in 30minutes.

Diluent,Solution A,Solution B,Mobile phase,and System suitability solution— Proceed as directed in the Assay.

Standard solution— Dissolve an accurately weighed quantity of USP Hydrochlorothiazide RSin Diluent,and quantitatively dilute with Diluent,if necessary,to obtain a solution having a known concentration of about 2µg per mL.

Test stock solution— Proceed as directed for Assay stock preparationin the Assay.

Test solution— Quantitatively dilute an accurately measured volume of the Test stock solutionwith Diluentto obtain a solution having a concentration of about 100µg of bisoprolol fumarate per mL.

Chromatographic system (see Chromatography á621ñ)— Prepare as directed in the Assay,but use a 260-nm detector.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the resolution,R,between chlorothiazide and hydrochlorothiazide is not less than 1.5.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the tailing factor is not more than 1.3;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for all the peaks.Calculate the percentage of each impurity in the portion of Tablets taken by the formula: in which Fis the response factor,equal to 1.2for the peak with a relative retention time of 0.69and 1.4for the peak with a relative retention time of 1.2,both retention times relative to that of the hydrochlorothiazide peak;WBand WHare the labeled quantities,in mg,of bisoprolol fumarate and hydrochlorothiazide,respectively,in each Tablet;CSis the concentration,in mg per mL,of USP Hydrochlorothiazide RSin the Standard solution;CBis the concentration,in mg per mL,of bisoprolol fumarate in the Test solution;riis the peak response of each of the two impurities obtained from the Test solution;and rSis the response for the hydrochlorothiazide peak obtained from the Standard solution:not more than 1.0%for the impurity with a relative retention time of 0.69is found;and not more than 2.0%for the impurity with a relative retention time of 1.2is found.

Diluent— Mix 10mLof 1Mdibutylammonium phosphate with 1000mLof water.

Solution A— Filter and degas a portion of the Diluent.

Solution B— Prepare a mixture of acetonitrile and water (3:2).Add 10mLof 1Mdibutylammonium phosphate per liter,stir vigorously for 2minutes,filter,and degas.

Mobile phase— Use variable mixtures of Solution Aand Solution Bas directed for Chromatographic system.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

System suitability solution— Prepare a solution of USP Chlorothiazide RSand USP Hydrochlorothiazide RSin Diluentcontaining 40µg of each per mL.

Standard preparation— Dissolve suitable quantities of USP Bisoprolol Fumarate RSand USP Hydrochlorothiazide RSin Diluentto obtain a solution having known concentrations of about 100µg of each per mL.Stir by mechanical means for 1hour.

Assay stock preparation— Weigh 10Tablets,and transfer to a 100-mLvolumetric flask.Add about 50mLof Diluent,sonicate for 10minutes,and cool.Dilute with Diluentto volume,stir by mechanical means for 1hour,and centrifuge.

Bisoprolol fumarate assay preparation— Quantitatively transfer a portion of the Assay stock preparationto a 50-mLvolumetric flask,and dilute with Diluentto volume to obtain a solution having a concentration of about 100µg of bisoprolol fumarate per mL.

Hydrochlorothiazide assay preparation— Quantitatively transfer a portion of the Assay stock preparationto a 50-mLvolumetric flask,and dilute with Diluentto volume to obtain a solution having a concentration of about 62.5µg of hydrochlorothiazide per mL.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with 225-nm detector and an 8-mm ×10-cm column that contains packing L11.The flow rate is about 3mLper minute.The chromatograph is programmed as follows. Chromatograph the System suitability solution,and record the peak areas as directed for Procedure:the resolution,R,between chlorothiazide and hydrochlorothiazide is not less than 1.5.Chromatograph the Standard preparation,and record the peak areas as directed for Procedure:the tailing factor for the hydrochlorothiazide peak is not more than 1.3;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparation,Bisoprolol fumarate assay preparation,and Hydrochlorothiazide assay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantities,in mg,of bisoprolol fumarate (C18H31NO4)2·C4H4O4and hydrochlorothiazide (C7H8ClN3O4S2)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Bisoprolol Fumarate RSor USP Hydrochlorothiazide RSin the Standard preparation,as appropriate;Vis the volume of the Assay stock preparationused to prepare the Bisoprolol fumarate assay preparationor the Hydrochlorothiazide assay preparation;rUis the peak area obtained from the Bisoprolol fumarate assay preparationor the Hydrochlorothiazide assay preparation,as appropriate;and rSis the corresponding peak area obtained from the Standard preparation.