Standard solution— Transfer an accurately weighed quantity of about 20mg of USP8-Bromotheophylline RSto a 100-mLvolumetric flask,add 25mLof water,50mLof 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 25mg of Pamabrom to a 100-mLvolumetric flask,add 25mLof 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 RSin Diluting solution,add a few drops of ammonium hydroxide,sonicating if necessary,to obtain a solution having a known concentration of about 1mg of USP Theophylline RSper mL.Dilute a volume of this solution quantitatively,and stepwise if necessary,with Diluting solutionto obtain a solution having a known concentration of about 5µg per mL.

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

Procedure— Separately inject equal volumes (about 20µL)of the Standard solutionand the Test solutioninto 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 Cis the concentration,in µg per mL,of USP Theophylline RSin the Standard solution,Wis the weight,in mg,of Pamabrom taken,and rUand rSare the peak responses of theophylline obtained from the Test solutionand 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 Suitabilityunder Chromatography á621ñ).

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 125µg of caffeine per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP8-Bromotheophylline RSin Diluting solution,add a few drops of ammonium hydroxide,sonicating if necessary,to obtain a solution having a known concentration of about 0.75mg of USP8-Bromotheophylline RSper mL.Transfer 5.0mLof this solution to a 100-mLvolumetric flask,add 10.0mLof Internal standard solution,dilute with Mobile phaseto volume,mix,and filter.

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

Chromatographic system (see Chromatography á621ñ)—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 1.5mLper minute.Chromatograph 20µLof the Standard preparation,and record the peak responses as directed under Procedure:the relative retention times are about 0.6for caffeine and 1.0for 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 20µL)of the Standard preparationand the Assay preparationinto 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 Cis the concentration,in mg per mL,of USP8-Bromotheophylline RSin the Standard preparation,and RUand RSare the peak response ratios of the 8-bromotheophylline peak and the internal standard peak obtained from the Assay preparationand the Standard preparation,respectively.