An isocratic high-performance liquid chromatographic system for simultaneous determination of theophylline and its major metabolites in human urine.

A simple and highly selective isocratic high-performance liquid chromatography method is presented for the simultaneous determination of theophylline and its major metabolites in human urine using beta-hydroxyethyl theophylline as an internal standard. The method utilizes direct injection of diluted urine samples followed by separation and quantitation by reversed-phase isocratic elution and ultraviolet detection. The assay is accurate and reproducible with a sensitivity of 1 microgram ml-1 for theophylline and 0.5 micrograms ml-1 for its metabolites. The assay was employed for the analysis of theophylline and its major metabolites in urine following the oral administration of theophylline to four healthy volunteers.