Assay
Buffer solution
Transfer 2.72 g of sodium acetate trihydrate to a 2000-mL volumetric flask, add about 200 mL of water, and shake until dissolution is complete. Add 10.0 mL of glacial acetic acid, dilute with water to volume, and mix.
Mobile phase
Transfer 70.0 mL of acetonitrile to a 1000-mL volumetric flask, dilute with
Buffer solution to volume, and mix. Degas, and filter before using. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Internal standard solution
Transfer about 50 mg of theobromine, accurately weighed, to a 100-mL volumetric flask, dissolve in 10.0 mL of 6 N ammonium hydroxide, dilute with Mobile phase to volume, and mix.
Standard preparation
Dissolve an accurately weighed quantity of
USP Theophylline RS in
Mobile phase, and dilute quantitatively, and stepwise if necessary, with
Mobile phase to obtain a solution having a known concentration of about 1 mg per mL. Transfer 10.0 mL of this solution to a 100-mL volumetric flask, add 20.0 mL of
Internal standard solution, dilute with
Mobile phase to volume, and mix to obtain a solution having a known concentration of about 0.1 mg of
USP Theophylline RS per mL.
Assay preparation
Transfer about 100 mg of Theophylline, accurately weighed, to a 100-mL volumetric flask, add about 50 mL of Mobile phase, shake by mechanical means until solution is complete, dilute with Mobile phase to volume, and mix. Transfer 10.0 mL of this solution to a second 100-mL volumetric flask, add 20.0 mL of Internal standard solution, dilute with Mobile phase to volume, and mix.
Chromatographic system
(see
Chromatography 621)The liquid chromatograph is equipped with a 280-nm detector and a 4-mm × 30-cm column that contains packing L1. The flow rate is about 1.0 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the resolution,
R, between the theophylline and theobromine peaks is not less than 2.0, the tailing factor for the theophylline peak is not more than 2.0, and the relative standard deviation for replicate injections is not more than 1.5%.
Procedure
Separately inject equal volumes (between 10 µL and 25 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, and measure the peak responses for the major peaks. The retention time of theophylline relative to that of theobromine is about 1.6. Calculate the quantity, in mg, of C
7H
8N
4O
2 in the portion of Theophylline taken by the formula:
1000C(RU / RS),
in which
C is the concentration, in mg per mL, of
USP Theophylline RS in the
Standard preparation, and
RU and
RS are the response ratios of the theophylline peak to the internal standard peak obtained from the
Assay preparation and the
Standard preparation, respectively.