The fate of caffeine in man and a method for its estimation in biological material.

CHE~IICAL METHOD. The estimation of cafeine. Older methods for the determination of car7eine in biological material involve extraction into chloroform, followed by assay of the drug by its nitrogen content (Kunz, 1935) or by a color-producing murexide reaction (Tanaka and Ohkubo, 1937). These methods are insensitive and inaccurate. Recently, Ishler et al. (1948) described a sensitive method, using ultraviolet spectrophotometry, for the estimation of caffeine in coffee preparations. This method was applied by Fisher et at. (1949) to the estimation of caffeine in urine. In this method caffeine is extracted from the biological material with chloroform, which also extracts other methyl xanthines and interfering normal constituents of biological materials. In the following procedure, caffeine is isolated from biological material at pH 7-8 by extraction into benzene. The compound is then returned to an aqueous phase by shaking with 5 N HCl and its absorption in the ultraviolet is measured at 273 rn+ The relativel? nonpolar solvent benzene was chosen as the solvent least likely to extract metabolic products of caffeine and normal constituents of urine according to principles previous described (Brodie ef al., 1947). The ext,racticn is augmented by saturating the aqueous phase with sodium chloride. A small amount of interfering blank material in urine and tissues which is also extracted into benzene is almost entirely removed by shaking the solvent extract with dilute XaOH. Since caffeine is basic it can be extracted from the benzene phase into 5 X HCl. This effectively separates it from methytated xanthines such as theobromine and theophylline. Procedure for plasma. Pipet 1 to 5 ml. of plasma into a 60 ml. glass-stoppered bottle containing 40 ml. of benzene* and about 3 gm. of sodium chloride. &ske for 20 minutes. Centrifuge the bottle and pipet 30 ml. of the benzene phase int,o a 60 ml. glass-stoppered bottle’ containing4 ml. of 5 N HCl. Sh&e for 5 minutes, transfer 3 ml. of the acid phase to a quarhz cuvct. Determine the optical density at 273 mp in a Beckman DU spectrophotometer.’