Abstract: AIM: To develop an HPLC method with ultraviolet detection for simultaneous determination of theophylline and its two metabolites 1, 3-dimethyluric acid(1, 3-DMU)and 3-methylxanthine (3-MX)in human urine.METHODS: Urine samples were extracted with mixture of isopropanol and dichloromethane (2/8).The aliquot of the organic phase was removed and dried under air.The residue was dissolved in 0.2 mL of mobile phase, and 20 μL of aliquots were injected immediately into the column.The analytes were separated on a Diamonsil ODS C18, 5 μm, 150 mm ×4.6 mm I.D.column with the mobile phase consisting of 0.1 %formic acid/L and acetonitrile (95:5)at a flow rate of 1.0mL/min and the eluent was detected at 280 nm.Concentrations of theophylline and its two metabolites 1, 3-DMU and 3-MX in urine of 12 healthy subjects were determined, and accumulative amounts of excretion during 24 h urine were calculated following single dose and multidose of theophenlline.RESULTS: Linear quantitative response curve was generated over a concentration ranges of 0.312 - 40.0 μg/mL, 0.156 -20.0 μg/mL and 0.078 - 10.0 μg/mL in urine for theophylline, 3-MX and 1, 3-DMU respectively.Lowest limits of quantification for theophylline, 3-MX and 1, 3-DMU were measured to be 0.312, 0.156 and 0.078 μg/mL, respectively.Intraand inter-batch precision and accuracy were acceptable for all quality control samples, which RSD and relative standard errors were <15 %.The mean recoveries from urine were >70 %.CONCLUSION: The specific, sensitive and precise method is suitable for monitoring of theophylline and its two metabolites in urine of human.

Key words: theophylline, pharmacokinetics, HPLC, CYP1A2