Determination of theophylline metabolites in human liver microsomes by high-performance liquid chromatography

A method for the quantitation of three metabolites of theophylline, 1-methylxanthine (1 MX), 3-methylxanthine (3MX), and 1,3-dimethyluric acid (13DMU) in human liver microsomes has been developed. The method is based on a simple one-step extraction followed by isocratic, reversed-phase high-performance liquid chromatography with uv detection (detection wavelength: 273 nm). The detection limit was 0.03 nmol.mg-1.h-1, which corresponds to 10 pmol per sample for all three metabolites. Linear standard curves were obtained for all three compounds within a concentration range of 0.6-6.0 nmol.mg-1.h-1 for 3MX and 1MX and 2.4-24.0 nmol.mg-1.h-1 for 13DMU. The absolute recoveries ranged from 61 to 80%, 68 to 74%, and 74 to 85% for 1MX, 3MX, and 13DMU, respectively, within the concentration range of the standard curve. The reproducibility and repeatability showed a coefficient of variation < 12% at five concentrations within the standard curve range. The accuracy for all three metabolites was within +/- 5% at three concentrations, except for 1MX in the lowest concentration (12%). The simple but sensitive method developed is highly suitable as a probe for cytochrome P4501A2 (CYP1A2) and probably also CYP2E1 function in human liver microsomes.