A simple liquid-chromatographic method applied to determine caffeine in plasma and tissues.

In this simple, reliable assay for quantifying caffeine in plasma and tissues, methylxanthines are first partly purified from plasma and acid extracts of tissue by passage through solid-phase columns. The ease of this extraction method permits a relatively large number of samples to be processed daily. Quantification is by reversed-phase high-pressure liquid chromatography (mobile phase: acetic acid/acetonitrile/water, 2/6/92 by vol). Caffeine is eluted in 20 min. The reliability of this method allows its automation. This method has been adapted to measure caffeine in brain and kidney extracts and in as little as 10 microL of plasma. After 10 days of oral administration of caffeine (1 g/L, in drinking water) to six rats, the mean (+/- SEM) concentrations of caffeine in plasma, brain, and kidney were 18.6 +/- 6.0 micrograms/mL, 16.2 +/- 1.5 micrograms/g, and 18.9 +/- 2.0 micrograms/g, respectively. Correlations were linear between concentrations of caffeine in plasma and brain (r = 0.86) and between concentrations in plasma and kidney (r = 0.91). This method should be useful in studying the effects and mechanisms of actions of methylxanthines.