THE USE OF THEOBROMINE AS INTERNAL STANDARD IN THE RAPID HPLC ANALYSIS OF THEOPHYLLINE IN SMALL BLOOD-SERUM VOLUME

A modified method for a qualitative and quantitative analysis of theophylline in small blood serum volume (40 mul) was developed. According to this method, blood serum samples containing theobromine, as internal standard and caffeine from coffee consumption of the patients are centrifuged with acetonitrile to precepitate proteins. These serum samples are evaporated in a water bath at 45-degrees-C under stream of nitrogen to remove organic solvents. Then the samples were treated by solid - phase liquid extraction using C18 Bond Elut cartridges preconditioned with methanol and water. The Chromatographic Separation was achieved on a Lichrosorb RP-18 10 mum ODS, 250x4 mm I.D. using methanol: 0.05M ammonium acetate (42:58) at a pH 7.0. The eluted components are detected at 272 nm. The retention time is 3.03 min for theobromine and 3.76 min for theophylline. Theophylline is quantitated by comparing theophylline peak areas with that of known quantities of the internal standard. The peak areas were found to be linearly related to theophylline concentrations providing a quantitative means of assaying theophylline in biological samples. The absolute detection limit is 0.501 ng and the linearity is observed up to 120.0 ng per 20 mul injection. The proposed method was applied to the analysis of theophylline in blood samples from patients undergoing theophylline treatment without interference from caffeine, a constituent very common in human blood samples.