Determination of theophylline and its metabolites in human urine and plasma by high-performance liquid chromatography.

A method for the quantitation of theophylline (13DMX) and the three metabolites, 1-methyluric acid (1MU), 3-methylxanthine (3MX) and 1,3-dimethyluric acid (13DMU) in human plasma and urine has been developed. The method is based on a simple one-step liquid-liquid extraction with ethylacetate-2-propanol followed by isocratic, reversed-phase high-performance liquid chromatography with UV detection (detection wavelength: 273 nm). The overall mean recoveries ranged from 86 to 95% for the four compounds. The detection limit was 1 microM for 1MU, 3MX and 13DMU and 2 microM for 13DMX in urine, and 0.1 microM for 1MU, 3MX and 13DMU and 0.2 microM for 13DMX in plasma. The intra-day and inter-day coefficient of variation was < 6% and < 9%, respectively, and the accuracy was within +/- 10% in both urine and plasma. The simple but sensitive method is highly suitable for the development of theophylline as a probe drug for assessing CYP1A2 activity in man.