Caffeine determination in rat plasma. A comparative study of micromethods

Confronted with the need for a sensitive (1 mg/1), specific and reproducible (less than 5%) method for the determination of caffeine in small plasma samples (10-50 microliter) of small laboratory animals, two existing methods, radioactive labelling and gas chromatography, were adapted. The first step, common to both methods, is chloroform extraction, followed by either gas-chromatographic analysis or radioactivity measurement. These methods were compared by using a common internal standard, labelled caffeine, measured before and after the extraction step. The initial requirements were fulfilled and the correlation of the results proved to be excellent. The methods can be extended to animals other than the rat, and to organs or biological fluids other than plasma. The very small amount of plasma needed for the determination allows pharmacokinetic studies to be conducted in small laboratory animals. Further, by combining the two methods it is possible to perform rather complex investigations, such as evaluating the extent of caffeine metabolism and, at the same time, determining levels in the case of chromic administration.