Standard solution— Transfer an accurately weighed quantity of about 20 mg of USP 8-Bromotheophylline RS to a 100-mL volumetric flask, add 25 mL of water, 50 mL of methanol, and a small amount of dilute ammonium hydroxide. Swirl the flask to effect solution. Dilute the contents of the flask with methanol to volume, and mix.

Test solution— Transfer an accurately weighed quantity of about 25 mg of Pamabrom to a 100-mL volumetric flask, add 25 mL of water, and swirl to dissolve. Dilute the contents of the flask with methanol to volume, and mix.

Diluting solution, Mobile phase, and Chromatographic system— Proceed as directed in the Assay for 8-bromotheophylline.

Standard solution— Dissolve an accurately weighed quantity of USP Theophylline RS in Diluting solution, add a few drops of ammonium hydroxide, sonicating if necessary, to obtain a solution having a known concentration of about 1 mg of USP Theophylline RS per mL. Dilute a volume of this solution quantitatively, and stepwise if necessary, with Diluting solution to obtain a solution having a known concentration of about 5 µg per mL.

Test solution— Transfer an accurately weighed quantity of about 200 mg of Pamabrom to a 200-mL volumetric flask. Add about 50 mL of Diluting solution, and sonicate for 5 minutes. Cool to room temperature, dilute with Diluting solution to volume, and mix.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the percentage of theophylline in the portion of Pamabrom taken by the formula: in which C is the concentration, in µg per mL, of USP Theophylline RS in the Standard solution, W is the weight, in mg, of Pamabrom taken, and rU and rS are the peak responses of theophylline obtained from the Test solution and the Standard solution, respectively: not more than 0.5% is found.

Diluting solution— Prepare a mixture of water and methanol (70:30).

Mobile phase— Prepare a filtered and degassed mixture of water, methanol, and glacial acetic acid (69:30:1), filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Dissolve an accurately weighed quantity of caffeine in Diluting solution, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a concentration of about 125 µg of caffeine per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP 8-Bromotheophylline RS in Diluting solution, add a few drops of ammonium hydroxide, sonicating if necessary, to obtain a solution having a known concentration of about 0.75 mg of USP 8-Bromotheophylline RS per mL. Transfer 5.0 mL of this solution to a 100-mL volumetric flask, add 10.0 mL of Internal standard solution, dilute with Mobile phase to volume, mix, and filter.

Assay preparation— Transfer an accurately weighed quantity of about 200 mg of Pamabrom to a 200-mL volumetric flask, add about 50 mL of Diluting solution and two drops of ammonium hydroxide, and sonicate for 5 minutes. [note—If a hazy solution is present after 5 minutes of sonication, add 1 additional drop of ammonium hydroxide.] Cool, dilute with Diluting solution to volume, and mix. Transfer 5.0 mL of this solution to a 100-mL volumetric flask, add 10.0 mL of Internal standard solution, dilute with Mobile phase to volume, mix, and filter.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm × 15-cm column that contains packing L1. The flow rate is about 1.5 mL per minute. Chromatograph 20 µL of the Standard preparation, and record the peak responses as directed under Procedure: the relative retention times are about 0.6 for caffeine and 1.0 for 8-bromotheophylline, the resolution, R, between caffeine and 8-bromotheophylline is not less than 2.0, 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 responses for the major peaks. Calculate the quantity, in mg, of 8-bromotheophylline (C7H7BrN4O2) in the portion of Pamabrom taken by the formula: in which C is the concentration, in mg per mL, of USP 8-Bromotheophylline RS in the Standard preparation, and RU and RS are the peak response ratios of the 8-bromotheophylline peak and the internal standard peak obtained from the Assay preparation and the Standard preparation, respectively.