High performance liquid chromatographic determination of methylxanthines in canine serum, gastric and pancreatic juices

A convenient high performance liquid chromatographic method for the determination of methylxanthines in biological samples is described. Separation was achieved by reversed phase chromatography using a mobile phase consisting of tetrahydrofuran + methanol + 0.01M potassium dihydrogen phosphate, pH 3.5 (1:20:79, v/v/v), on a 7 μm C18 column and a C18 Lichrosorb precolumn at a flow rate of 0.8 mL/min. Levels varying from 0.25 – 16 mg/L could be detected by UV at 280 nm. In this range, standard curves were established for 4 methylxanthines: theobromine, paraxanthine, theophylline and caffeine in 4 media: mobile phase, serum, gastric and pancreatic juices, and were found to be linear (r ≥ 0.9975). Overall characteristics of the method were determined as: percent recovery (89.54%), accuracy (≥99.4%) and reproducibility (≥95%). Retention times ranged from 4.21 ± 0.01 (1‐methyluric acid) to 10.8 ± 0.03 min (caffeine). Animal experiments (5 and 10 mg/kg boluses) were used to determine caffeine half life in dog's blood (310 ± 46 and 453 ± 59 min, respectively) and its secretion into pentagastrin stimulated gastric juice (mean concentrations 2.51 and 6.04 mg/L; mean outputs 351 and 1206 μg/2.25 h; both statistically different at p < 0.001 level).