Solid-phase extraction for determination of caffeine and its dimethylxanthine metabolites in rat urine using high performance liquid chromatography and its application

A solid-phase extraction (SPE) method for use with high performance liquid chromatography to measure caffeine (CF) and its metabolites in rat urine was developed and validated. Oasis HLB cartridges were used and washed with water, 1% acetic acid: 3% methanol-water (0.5:99.5 v/v), and 1% ammonium hydroxide: 3% methanol-water (0.5:99.5 v/v), respectively, and eluted with methanol:acetronitrile (80:20 v/v). Method validation showed good linearity (r>0.9990) over all calibration ranges for CF and its metabolites. The method was precise (1.0–17.6%RSD) and accurate (-13.8–(+) 16.0%DEV). The SPE method resulted in a high recovery for all analytes (72.9-98.9%). The lower limits of quantification were 0.1 µg/ml (PX), 0.25 µg/ml (CF and theophylline), and 0.5 µg/ml (theobromine and 1,3,7-trimethyluric acid). This method was successfully applied to determine the urinary profiles of CF and its metabolites, and the caffeine metabolic ratios in rats receiving a single oral dose of CF