High-performance liquid chromatographic assay for theophylline and its major metabolites in human urine.

A highly selective automated high-performance liquid chromatographic (HPLC) method has been developed for the assay of theophylline and its major metabolites in urine. The method involves direct injection of urine on to a reversed-phase column, followed by gradient elution and ultraviolet detection. Quantitation is achieved by the peak-height ratio method with reference to an internal standard, 8-chlorotheophylline. The assay is accurate and reproducible, with a sensitivity of 1 microgram/ml in urine. In order to confirm its accuracy, theophylline and its major metabolites in urine were quantified after HPLC separation by both peak-height ratio of ultraviolet absorbance and liquid scintillation spectrometry after oral administration of [14C]theophylline to a human volunteer. The assay was also applied to the analysis of theophylline and its major metabolites in urine following the oral administration of theophylline to three healthy volunteers.