A: Dissolve about 500mg in 20mLof water,add,with constant stirring,1mLof 3Nhydrochloric acid,filter (retain the filtrate),wash the precipitate with small portions of cold water,and dry at 105for 1hour:the precipitate of theophylline so obtained melts between 270and 274.

B: To about 10mg of the dried precipitate obtained in Identificationtest A,contained in a porcelain dish,add 1mLof hydrochloric acid and 100mg of potassium chlorate,evaporate on a steam bath to dryness,and invert the dish over a vessel containing a few drops of 6Nammonium hydroxide:the residue acquires a purple color,which is destroyed by solutions of fixed alkalies.

C: To the filtrate obtained in Identificationtest Aadd 0.5mLof benzenesulfonyl chloride and 5mLof 1Nsodium hydroxide to render alkaline,shake by mechanical means for 10minutes,add 5mLof 3Nhydrochloric acid to acidify,chill,collect the precipitated disulfonamide of ethylenediamine,wash with water,recrystallize from water,and dry at 105for 1hour:the dried precipitate melts between 164and 171.

Mobile phase— Mix 200mLof methanol,960mg of sodium 1-pentanesulfonate,and sufficient water to make 1L.Adjust with glacial acetic acid to a pHof 2.9±0.1,filter,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Diluent: a mixture of water and methanol (4:1).

Standard preparation— Dissolve an accurately weighed quantity of USP Theophylline RSin Diluent,and dilute quantitatively,and stepwise if necessary,with Diluentto obtain a solution having a known concentration of about 0.08mg per mL.

Resolution solution— Dissolve a suitable quantity of theobromine in the Standard preparationto obtain a solution containing about 0.08mg per mL.Transfer 20.0mLof this solution to a 25-mLvolumetric flask,dilute with Diluentto volume,and mix.

Assay preparation— Transfer about 24mg of Aminophylline,accurately weighed,to a 250-mLvolumetric flask,dissolve in and dilute with Diluentto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm ×15-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.65for theobromine and 1.0for theophylline;the tailing factor for the theophylline peak is not more than 2.0;and the resolution,R,between theobromine and theophylline is not less than 3.0.Chromatograph the Standard preparation,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 10µL)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 theophylline (C7H8N4O2)in the portion of Aminophylline taken by the formula: in which Cis the concentration,in mg per mL,of USP Theophylline RSin the Standard preparation;and rUand rSare the theophylline peak responses obtained from the Assay preparationand the Standard preparation,respectively.