The simultaneous determination of theophylline, theobromine and caffeine in plasma by high performance liquid chromatography

A high performance liquid chromatographic procedure for the simultaneous micro-scale determination of theophylline, theobromine and caffeine in plasma is described. After a single dichloromethane extraction of 0.05 - 0.2 ml of acidified plasma, the evaporated residue is chromatographed on a reverse-phase gC-18) column. With a mobile phase of acetate buffer (pH 4) - acetonitrile (88:12) at a flow-rate of 2.0 ml/min., the three methylxanthines are separated within six minutes. Detection at 276-280 nm enables quantitation of 0.1 mg/l of drug in a 0.1 ml sample. The method is reproducible, correlates well with EMIT for plasma theophylline, and is applicable to the routine monitoring of both paediatric and adult patients as well as to metabolic studies.