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 theop hylline in small blood serum volume (40 mul) was developed. According to this method, blood serum samples containing theobromine, as interna l standard and caffeine from coffee consumption of the patients are ce ntrifuged with acetonitrile to precepitate proteins. These serum sampl es are evaporated in a water bath at 45-degrees-C under stream of nitr ogen to remove organic solvents. Then the samples were treated by soli d - phase liquid extraction using C18 Bond Elut cartridges preconditio ned with methanol and water. The Chromatographic Separation was achiev ed on a Lichrosorb RP-18 10 mum ODS, 250x4 mm I.D. using methanol: 0.0 5M ammonium acetate (42:58) at a pH 7.0. The eluted components are det ected at 272 nm. The retention time is 3.03 min for theobromine and 3. 76 min for theophylline. Theophylline is quantitated by comparing theo phylline peak areas with that of known quantities of the internal stan dard. The peak areas were found to be linearly related to theophylline concentrations providing a quantitative means of assaying theophyllin e 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 pro posed method was applied to the analysis of theophylline in blood samp les from patients undergoing theophylline treatment without interferen ce from caffeine, a constituent very common in human blood samples.