Prepeak of trolox caused by theophylline and its application to the determination of theophylline in rat plasma

A novel electrochemical detection coupled with high-performance liquid chromatography (HPLC) was developed for determining theophylline in plasma samples. On a voltammogram of acetonitrile containing trolox (TrOH), theophylline and LiClO4, a new oxidation peak (termed a prepeak) appeared at a potential less positive than that for the oxidation peak of TrOH without theophylline. Since the prepeak current linearly increased with the theophylline concentration, the electrochemical detection was carried out by measuring the prepeak current height at a constant potential in a flow-type thin-layer cell. HPLC using a silica gel column and a mobile phase of acetonitrile was conducted for the separation of the theophylline. The eluate from the column was mixed with a solution of 1.5 mmol L−1 TrOH and 20 mmol L−1 LiClO4 in acetonitrile. The chromatographic peak height for theophylline at a detection potential of +1.1 V vs. Ag/AgCl was found to be linearly related to the concentrations injected, ranging from 2.0 to 100 μmol L−1 (r > 0.999). The detection limit of the concentration of theophylline was 0.55 μmol L−1. The present method, which required only 10 μL of plasma sample, was applied to the determination of theophylline in rat plasma. The concentration–time profile of theophylline in rat plasma was monitored to determine pharmacokinetic parameters.