Theobromine and Caffeine

Theobromine (3,7-dimethylxanthine), which is 1.2% in cocoa, provides a stimulating effect, which is less than that of caffeine in coffee. Therefore, it is of physiological importance. Caffeine is also present, but in much lower amounts (average 0.2%). A cup of cocoa contains 0.1 g of theobromine and 0.01 g of caffeine. Theobromine crystallizes in the form of small rhombic prisms which sublime at 290 °C without decomposition. In cocoa beans theobromine is often weakly bound to tannins and is released by the acetic acid formed during fermentation of the beans. Part of this theobromine then diffuses into the shell. 

Cocoa fat (cocoa butter), because of its abundance and value, is the most significant ingredient of cacao beans, and is dealt with in detail elsewhere (cf. 14.3.2.2.3). 

The content of phenolic compounds is also of interest from the standpoint of chemoprevention. With 84 mg/g gallic acid equivalents (GAE) and 

Inner cotyledon tissue with pigment cells 

Constituent Polyphenol storage cell (%) Cotyle- dons (%r 

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Table 8. Variations in Theobromine and Caffeine Content of Various Chocolate Liquors...

The theobromine and caffeine contents of several finished chocolate products as determined by hplc at Hershey s laboratories are presented in... 

Table 9. Theobromine and Caffeine Content of Finished Chocolate Products ...

Mehtylxanthines are naturally occurring drugs, including theophylline, theobromine and caffeine. Methylxanthines at relatively high doses inhibit phosphodiesterases, which results in an increase in intracellular cAMP... 

Seasonal variations in the metabolic fate of adenine nucleotides prelabelled with [8—1-4C] adenine were examined in leaf disks prepared at 1-month intervals, over the course of 1 year, from the shoots of tea plants (Camellia sinensis L. cv. Yabukita) which were growing under natural field conditions by Fujimori et al.33 Incorporation of radioactivity into nucleic acids and catabolites of purine nucleotides was found throughout the experimental period, but incorporation into theobromine and caffeine was found only in the young leaves harvested from April to June. Methy-lation of xanthosine, 7-methylxanthine, and theobromine was catalyzed by gel-filtered leaf extracts from young shoots (April to June), but the reactions could not be detected in extracts from leaves in which no synthesis of caffeine was observed in vivo. By contrast, the activity of 5-phosphoribosyl-1-pyrophosphate synthetase was still found in leaves harvested in July and August. 

Typical Thin-Layer Chromatography (TLC) Systems for Theobromine and Caffeine Determinations... 

HPLC allows a quantitative determination with relatively simple extractions. In many cases, extraction only involves a heating of the commodity with water, followed by filtration and injection onto an HPLC column. In the determination of caffeine, theobromine, and theophylline in cocoa, coffee, or tea, as well as in other foods, there is scarcely a month that passes without a new paper on this assay. Kreiser and Martin provide typical conditions for analysis.28 In their studies, samples were extracted in boiling water and filtered prior to injection onto the HPLC column. The HPLC conditions used a Bondapak reversed phase column and a mobile phase of water methanol acetic acid (74 25 1) with detection at 280 nm. This method is accurate, precise, and conserves time. It has also been adopted by the AOAC as an official method for the determination of theobromine and caffeine in cocoa beans and chocolate products.29... 

Hieda et al. determined theophylline, theobromine, and caffeine in human plasma and urine by gradient capillary HPLC with frit fast atom bombardment (FAB) mass spectrometry with 7-ethyl theophylline as the internal standard.64... 

Moores, R.G. and Campbell, H.A., Determination of theobromine and caffeine in cocoa materials, Anal. Chem., 20,40,1948. 

Li, S., Berger, J., Hartland, S., UV spectrophotometric determination of theobromine and caffeine in cocoa beans, Analytics Chimica Acta, 232,409,1990. 

Kreiser, W.R. and Martin, R.A. Jr., High pressure liquid chromatographic determination of theobromine and caffeine in cocoa and chocolate products, JAOAC, 61,1424,1978. 

Hieda, Y., Kashimura, S., Hara, K., and Kageura, M., Highly sensitive and rapid determination of theophylline, theobromine and caffeine in human plasma and urine by high performance liquid chromatography frit fast atom bombardment spectrometry, J. Chromatogr., 667,241,1995... 

Although low levels of methylxanthines have been detected in the leaves and flowers of T. cacao, the primary storage location is within the seed or bean.16 The cocoa bean is the major natural source of the methylxanthine theobromine, but contains only small amounts of caffeine. Theophylline has been detected in cacao beans, but at such low concentrations that its presence generally is ignored. Together, theobromine and caffeine account for up to 99% of the alkaloid content of T. cacao beans. Alkaloid content is affected by genetic makeup, maturity of beans at harvest, and fermentation process. Analytical methodology also is partially responsible for some of the disparity in methylxanthine values since many early methods were unable to separate theobromine and caffeine. 

In live cocoa seeds, the methylxanthines are localized in polyphenolic storage cells. Bean death, which occurs 24 to 48 h after initiation of fermentation, triggers diffusion of the methylxanthines from the nib to the shell. The early studies of Humphries state that cacao cotyledons lose about 40% of their theobromine during fermentation.17 According to Knapp and Wadsworth, the loss of theobromine and caffeine becomes significant on the third day when the methylxanthines begin to diffuse into the shell.18 This migration continues until the concentration of the methylxanthines in nibs and shell are almost equal. 

Chocolate liquor is the solid or semiplastic food prepared by finely grinding the nib of the cacao bean. It is commonly called baking chocolate, unsweetened chocolate, or bitter chocolate and, in Europe, is frequently referred to as chocolate mass or cocoa paste. Chocolate liquor is essentially the starting point from which all chocolate products are produced. Table 5 lists the theobromine and caffeine content of 22 various chocolate liquor samples determined by high pressure liquid chromatography (HPLC). The liquors averaged 1.22% theobromine and 0.214% caffeine.27- 28 The ratio of theobromine to caffeine ranged from 2.5 1 to 23.0 1. 

According to Schutz, it is well known that cocoa contains about 2.5% by weight of theobromine and caffeine.29 Although the presence of theophylline has been demonstrated, its concentration is so low that it is generally ignored. In a summary of the literature values for 27 commercial cocoa powders and cocoa masses, Hadorn found that the mean total alkaloid content was 3.36 0.48%.23 Theobromine and caffeine content of several cocoa powders is shown in Table 6. HPLC analyses by DeVries et... 

Cocoa butter is the fat obtained from subjecting chocolate liquor to pressure. Since the alkaloids are sparingly soluble in fat, only trace amounts of theobromine and caffeine in cocoa butter have been reported. The theobromine and caffeine content of four cocoa butter samples averaged 0.008% and 0.038%, respectively.33... 

The combination of ground cocoa beans and sugar produces a very hard substance with an unpleasant mouthfeel. However, the addition of extra cocoa butter results in a product that melts easily in the mouth. Additionally, the production of cocoa butter results in a lower fat cocoa powder which can be used more readily in both beverages and foods. As the amounts of cocoa butter and other fats, milk solids, sugar, and other ingredients increase, the amount of theobromine and caffeine in the final product decreases. 

Bunker and McWilliams found that two beverages prepared from instant cocoa (with Dutch process cocoa) contained 10 to 17 mg caffeine per cup.37 Table 7 lists the theobromine and caffeine concentration of hot cocoa and chocolate milk prepared from instant mixes. Zoumas et al. analyzed five commercial hot cocoa mixes and reported an average of 65 mg per serving of theobromine and 4 mg per serving of caffeine.28 Similar results were reported by Blauch and Tarka.36... 

Chocolate milk samples prepared from sweetened cocoa powders averaged 58 mg per serving of theobromine and 5 mg per serving of caffeine.28 Analysis of a "home-style" recipe resulted in higher methylxanthine values — 94 mg theobromine and 10 mg caffeine per serving. However, the authors noted that this recipe also had a stronger chocolate flavor. The lower values reported by Zoumas et al. and Blauch and Tarka compared to others was attributed to the inability of older methods to separate theobromine and caffeine, and the lack of precision and accuracy of the older methods. A compendium of theobromine and caffeine values reported for chocolate beverages from both published and unpublished studies has been compiled in Table 8. 

It is necessary to determine the methylxanthine content of chocolate foods, as well as beverages, in order to obtain an accurate assessment of the total amount of theobromine and caffeine that is ingested via the diet. This area of analysis has received little attention, and only scant published data exist on the methylxanthine content of chocolate foods. 

Theobromine and Caffeine Concentration in Hot Cocoa and Chocolate Milk Prepared From Instant Mixes... 

Most chocolate is consumed in the form of chocolate confectionery. Sweet chocolate is produced from chocolate liquor with the addition of sugar and cocoa butter. Sometimes called dark chocolate, sweet chocolate must contain at least 15% chocolate liquor, but may contain as much as 50%. Semisweet or bittersweet chocolate consists of a minimum of 35% chocolate liquor. The chocolate liquor content results in sweet and semisweet chocolate containing the highest amount of theobromine and caffeine per serving of any type of chocolate confectionery (Table 9). Within brands of sweet chocolate, there is wide variation in the methylxanthine... 

Chocolate liquor is a semifinished product commonly called "baking" or unsweetened chocolate. The average theobromine and caffeine content of liquors has been reported at 1.2% and 0.21%, respectively. Cocoa powder, which is prepared after partial removal of the cocoa butter, contains about 1.9 to 2.7% theobromine and 0.16 to 0.26% caffeine. Milk chocolate averages 0.168% theobromine and 0.022% caffeine, thus providing about 65 mg of theobromine and less than 10 mg of caffeine in a 40-g serving. The methylxanthine content of chocolate foods has received only slight attention in the literature, yet is necessary in order to obtain an accurate assessment of the total amount of theobromine and caffeine that is ingested via the diet. 

Timbie, D., Sechrist, L., Keeney, P., Application of high pressure liquid chromatography to the study of variables affecting theobromine and caffeine concentrations in cocoa beans, J. Food Sci., 43, 560, 1978. 

Fig. 12. Mechanism for formation of allantoins from the methylated uric acid-4,5-diol derived from theobromine and caffeine. Molar amounts of products are those formed in 1 M HOAc...

In the case of the methylated xanthines, particularly theophylline, theobromine and caffeine, the preponderance of data on the metabolism of these compounds in man suggests that a methylated uric acid is the principal product. However, the data presented earlier proposes at best a 77 per cent accounting of the methylated xanthine administered. The question can be raised as to whether the final products observed upon electrochemical oxidation of these compounds aids these studies. Very recently studies of metabolism of caffeine have revealed that 3,6,8-trimethylallantoin is a metabolite of caffeine 48>. This methylated allantoin is, of course, a major product observed electrochemically. The mechanism developed for the electrochemical oxidation seems to nicely rationalize the observed products and electrochemical behavior. The mechanism of biological oxidation could well be very similar, although insufficient work has yet been performed to come to any definite conclusions. There is however, one major difference between the electrochemical and biological reactions which is concerned with the fact that in the former situation no demethylation occurs whereas in the latter systems considerable demethylation appears to take place. 

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