Automated high-performance liquid chromatographic assay for monitoring caffeine and its metabolites in biological fluids of monkeys consuming caffeine.

A recently reported high-performance liquid chromatographic procedure, using a 5 micron C18 reversed-phase column to separate and quantitate caffeine and seven of its metabolites was modified for use with an automatic sampler to allow the continuous analysis of a large number of samples of various biological fluids obtained from monkeys consuming caffeine. The sensitivity for most metabolites was in the range of 0.1-0.3 microgram/ml from a 0.1 ml sample. The repeatability of the method regarding within-day variations was excellent and the absolute retention time for eight standards differed by less than +/- 0.03 min. Excellent repeatability in the day-to-day assay, with almost quantitative recoveries, was found for most of the analyzed compounds in various biological fluids. The standard deviation for the quantitation of all standards was in a range of 0.41-2.01 micrograms/ml, with the standard error less than 0.02. Using this method an analytical chemist could process between 40 and 60 samples of biological fluids in 24 h. The main metabolite of caffeine in the plasma of the monkey was theophylline, while theophylline and 1,3-dimethyluric acid were the major metabolites in urine. A close correlation was observed for the pattern of metabolites found in plasma and milk.