A: Infrared Absorption 197M.

B: A solution (10 mg per mL) responds to the tests for Sulfate 191.

Test solution: 10 mg per mL, in methanol.

Organic phase— Prepare a mixture of methanol and acetonitrile (610:390). Make adjustments if necessary (see System Suitability under Chromatography 621).

Aqueous phase— Dissolve 11 g of sodium 1-heptanesulfonate in 1000 mL of water, add 5.0 mL of triethylamine, adjust with phosphoric acid to a pH of 2.70 ± 0.05, and filter through a filter having a porosity of 0.5 µm or finer. Make adjustments if necessary (see System Suitability under Chromatography 621).

Solvent mixture— Prepare a mixture of Organic phase and Aqueous phase (600:400).

Test solution— Transfer about 50 mg of Penbutolol Sulfate to a 25-mL volumetric flask, dissolve in and dilute with Solvent mixture to volume, and mix.

Diluted test solution— Transfer 1.0 mL of the Test solution to a 100-mL volumetric flask, dilute with Solvent mixture to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 271-nm detector, a preinjection guard column that contains packing L4, and a 4.6-mm × 25-cm analytical column that contains packing L1 and is maintained at a constant temperature between ambient and 40, and is programmed to provide variable mixtures of Organic phase and Aqueous phase. Before each injection, the system is equilibrated with a mobile phase consisting of a mixture of 60% Organic phase and 40% Aqueous phase. After each injection, this composition of the mobile phase is maintained for 15 minutes, then the proportion of Organic phase is increased linearly over the next 20 minutes so that the mobile phase consists of 80% Organic phase and 20% Aqueous phase. The proportion of Organic phase is then decreased to 60% over 1 minute. The flow rate is about 1 mL per minute. Chromatograph the Diluted test solution, and record the peak responses as directed for Procedure: the resolution, R, between the penbutolol peak and any impurity peak is not less than 1.5, and the tailing factor is not more than 2, when calculated by the formula: in which W0.1 is the width of the peak at 10% of peak height.

Procedure— [note—Use peak areas where peak responses are indicated.] Separately inject equal volumes (about 20 µL) of the Solvent mixture, the Test solution, and the Diluted test solution into the chromatograph, and measure the peak responses for all the peaks. Calculate the percentage of each individual impurity in the Penbutolol Sulfate taken by the formula: in which ri is the peak response for an individual impurity in the chromatogram of the Test solution, and rD is the penbutolol peak response obtained from the Diluted test solution: not more than 1.2% of any impurity is found.

Organic phase— Prepare a mixture of methanol and acetonitrile (610:390). Make adjustments if necessary (see System Suitability under Chromatography 621).

Aqueous phase— Dissolve 11 g of sodium 1-heptanesulfonate in 1000 mL of water, add 5.0 mL of triethylamine, adjust with phosphoric acid to a pH of 2.70 ± 0.05, and filter through a filter having a porosity of 0.5 µm or finer. Make adjustments if necessary (see System Suitability under Chromatography 621).

Mobile phase— Prepare a mixture of Organic phase and Aqueous phase (650:350). Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Prepare a solution of 3,4-dimethylbenzophenone in Mobile phase containing about 0.01 mg per mL.

Standard preparation— Transfer about 24 mg of USP Penbutolol Sulfate RS, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with Internal standard solution to volume, and mix.

Assay preparation— Transfer about 24 mg of Penbutolol Sulfate, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with Internal standard solution to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 271-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 0.9 for penbutolol and 1.0 for 3,4-dimethylbenzophenone, and the resolution, R, between the penbutolol peak and the 3,4-dimethylbenzophenone peak is not less than 1.5, and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the areas of the responses for the major peaks. Calculate the quantity, in mg, of (C18H29NO2)2·H2SO4 in the portion of Penbutolol Sulfate taken by the formula: in which C is the concentration, in mg per mL, of USP Penbutolol Sulfate RS in the Standard preparation, and RU and RS are the ratios of the penbutolol peak response to the internal standard peak response obtained from the Assay preparation and the Standard preparation, respectively.