Test solution— Finely powder not fewer than 5Tablets,and transfer an accurately weighed portion of the powder,equivalent to about 40mg of bisoprolol fumarate,to a 50-mLflask.Add about 20mLof a mixture of dichloromethane and methanol (70:30),shake for 30minutes,centrifuge,and use the clear solution.

Application volume: 20µL.

Developing solvent system— Prepare a mixture of dichloromethane,methanol,and ammonia TS(70:10:0.8).

Procedure— Proceed as directed in the chapter,except to develop the chromatogram until the solvent front has moved about two-thirds of the length of the plate and to dry the plate in a current of cold air.

Medium: water;900mL.

Apparatus 2: 75rpm.

Time: 20minutes.

Diluent— Prepare a mixture of methanol,water,triethylamine,and phosphoric acid (160:35:5:2.5).

Mobile phase— Mix 20mLof triethylamine with 1000mLof water and 680mLof methanol,adjust with phosphoric acid to a pHof 4.0±0.1,and mix.

Standard stock solution— Quantitatively dissolve an accurately weighed quantity of USP Bisoprolol Fumarate RSin water to obtain a solution having a known concentration of about twice the concentration of bisoprolol fumarate in the Test solution.

Standard solution— Dilute an accurately measured volume of Standard stock solutionwith an equal accurately measured volume of Diluent.

Test solution— Withdraw a portion of the solution under test,filter,dilute with an equal volume of Diluent,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 227-nm detector and a 3.9-mm ×30-cm column that contains packing L7.The flow rate is about 1mLper minute.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the peak areas for bisoprolol.Calculate the quantity,in mg,of bisoprolol fumarate [(C18H31NO4)2·C4H4O4]dissolved.

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

Diluent— Prepare a mixture of water and acetonitrile (65:35).

Mobile phase— Prepare a mixture of water and acetonitrile (65:35).Add 5mLof heptafluorobutyric acid,5mLof diethylamine,and 2.5mLof formic acid.Mix,filter,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

System suitability solution— Prepare a solution in Diluenthaving a concentration of 0.5mg of propranolol hydrochloride per mLand 1.0mg of bisoprolol fumarate per mL.

Standard preparation— Quantitatively dissolve an accurately weighed quantity of USP Bisoprolol Fumarate RSin Diluentto obtain a solution having a known concentration of about 1mg 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 bisoprolol fumarate,to a 25-mLvolumetric flask.Add 10mLof Diluent,and sonicate for 10minutes.Cool,dilute with Diluentto volume,and mix.Centrifuge for 20minutes,and use the clear supernatant.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 273-nm detector and a 4.6-mm ×12.5-cm column that contains packing L7.The flow rate is about 1mLper minute.Chromatograph the System suitability solution,and record the peak areas as directed for Procedure:the resolution,R,between bisoprolol and propranolol is not less than 7.0.Chromatograph the Standard preparation,and record the peak areas as directed for Procedure:the tailing factor for the analyte peak is not more than 2.0;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in mg,of bisoprolol fumarate [(C18H31NO4)2·C4H4O4]in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Bisoprolol Fumarate RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.