The determination and the urinary excretion of caffeine in animals.

Caffeine, its physiological actions, and its metabolic fate have been of great interest to the medical profession for many years because of the therapeutic value of the drug as a respiratory and circulatory stimulant, and its wide-spread consumption in beverages. Despite this, a suitable method for the detection and quantitative determination of small amounts of caffeine in body fluids has not been available. It is the purpose of this paper to present such a technique, along with certain observations concerning the excretion of caffeine by the dog and horse. Chronologically, caffeine determination has passed from the stage of ultimate analysis of extractive residues early in this century (1, 2), through calorimetric procedures in the 1930’s (3), to the use of ultraviolet spectrophotometry (4). The former are not applicable to urine and blood because of the impracticability of obtaining caffeine-containing extracts of sufficient purity for routine carbon and nitrogen analyses to yield significant results. The calorimetric procedure, with use of the murexide reaction, as described by Tanaka and Ohkubo (3), has, in our experience, failed to yield sufficient precision and specificity to allow its use in analysis of caffeine-containing urines. Ishler, Finucane, and Borker (4) have reported the use of the ultraviolet absorption spectrum of caffeine for its determination in coffees and crude caffeine preparations. This laboratory has been studying the ultraviolet absorption spectra of various methylated xanthines during the past year and has developed a technique based on these phenomena which allows detection of caffeine in amounts as small as 2.5 y per cc. of urine. Ishler et al. have described the ultraviolet absorption spectrum of caffeine and found that it conforms to the Beer-Lambert law. We also confirm these findings and have obtained a molecular extinction of 10,800 (log lo/l = 0.635 at 273 rnp for 12.5 y per cc.). Fig. 1 shows the spectrum of pure caffeine.