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

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

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

Mobile phase— Mix 200 mL of methanol, 960 mg of sodium 1-pentanesulfonate, and sufficient water to make 1 L. Adjust with glacial acetic acid to a pH of 2.9 ± 0.1, filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

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

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

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

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

Chromatographic system (see Chromatography 621)—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 1 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.65 for theobromine and 1.0 for 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 10 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 theophylline (C7H8N4O2) in the portion of Aminophylline taken by the formula: in which C is the concentration, in mg per mL, of USP Theophylline RS in the Standard preparation; and rU and rS are the theophylline peak responses obtained from the Assay preparation and the Standard preparation, respectively.