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

Application volume: 20 µL.

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; 900 mL.

Apparatus 2: 75 rpm.

Time: 20 minutes.

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

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

Standard stock solution— Quantitatively dissolve an accurately weighed quantity of USP Bisoprolol Fumarate RS in 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 solution with 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 621)— 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 1 mL per 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 50 µL) of the Standard solution and the Test solution into 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·C4H4O4 is dissolved in 20 minutes.

(Official January 1, 2007)

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

Mobile phase— To a 1-L portion of Diluent add 5 mL of heptafluorobutyric acid, 5 mL of diethylamine, and 2.5 mL of formic acid. Mix, filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

System suitability solution— Prepare a solution in Diluent having a concentration of 0.5 mg of propranolol hydrochloride per mL and 1.0 mg of bisoprolol fumarate per mL.

Standard preparation— Quantitatively dissolve an accurately weighed quantity of USP Bisoprolol Fumarate RS in Diluent to obtain a solution having a known concentration of about 1 mg per mL.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 25 mg of bisoprolol fumarate, to a 25-mL volumetric flask. Add 10 mL of Diluent, and sonicate for 10 minutes. Cool, dilute with Diluent to volume, and mix. Centrifuge for 20 minutes, and use the clear supernatant.

Chromatographic system (see Chromatography 621)— 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 1 mL per 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 10 µL) of the Standard preparation and the Assay preparation into 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 C is the concentration, in mg per mL, of USP Bisoprolol Fumarate RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.