Cacao detection

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. 

An examination of treated cacao beans for toxic residues of Hanane showed that no Hanane was detectable chemically. Further analyses indicated that Hanane residue in no case exceeded 0.1 p.p.m., a limit tentatively set by the U. S. Food and Drug Administration (32). However, until more complete evidence has been accumulated about the use of this type of insecticide, there will remain some doubt concerning its application to cacao. 

Acetic acid occurs naturally in many plant species including Merrill flowers Telosma cordata), in which it was detected at a concentration of 2,610 ppm (Furukawa et al., 1993). In addition, acetic acid was detected in cacao seeds (1,520 to 7,100 ppm), celery, blackwood, blueberry juice (0.7 ppm), pineapples, licorice roots (2 ppm), grapes (1,500 to 2,000 ppm), onion bulbs, oats, horse chestnuts, coriander, ginseng, hot peppers, linseed (3,105 to 3,853 ppm), ambrette, and chocolate vines (Duke, 1992). 

Different authors used RP-HPLC and UV detection to monitor peptide formation during cheese ripening [174-178], providing valuable information about proteolysis. When large hydrophobic peptide need to be separated an lEC represents the best choice [179]. Nevertheless, the identification of these peptides is essential for the complete understanding of the proteolytic process. The peptides eluted from the LC column can be subjected to ESl-MS for molecular weight determination and MS/MS for amino acid sequence determination, which allow rapid peptide identification [172]. HPLC-ESl-MS and MS/MS techniques have been successfully used for peptide mass fingerprint purposes for sequence analysis of purified albumin from Theobroma cacao seeds [180,181]. 

Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometric Study of the Flavonoids of Cocoa (Jheobroma cacao) with Negative Ion Detection... 

Catechin and the proanthocyanidin prodelphinidin B3 are, respectively, the major monomeric and dimeric flavan-3-ols found in barley and malt where prodelphinidin B3 is the main contributor for the radical scavenging activity [Dvorakova et al., 2007], Proanthocyanidins have also been detected in nuts. Hazelnuts (Corylus avellana) and pecans (Carya illinoensis) are particularly rich in proanthocyanidins containing ca. 5 g kg, whereas almonds (Prunus dulcis) and pistachios (Pistachio vera) contain 1.8-2.4 mg kg 1, walnuts (Juglans spp.) ca. 0.67 g kg, roasted peanuts (Arachis hypgaea) 0.16 g kg, and cashews (Anarcardium occidentale) 0.09 g kg 1 [Crozier et al., 2006c]. Dark chocolate derived from the roasted seeds of cocoa (Theobroma cacao) is also a rich source of procyanidins [Gu et al., 2004], Monomeric flavan-3-ols and the proanthocyanidin B2, B5 dimers, and Q trimer are found in fresh cocoa beans (Fig. 1.13). Flavan-3-ols have also been detected in mint... 

Iverson, J.L. (1972) Gas-liquid chromatographic detection of palm kernel and coconut oils in cacao butter. J. Assoc. Off. Anal. Chem., 55, 1319-1322. 

Subagio, A. Sari, P. Morita, N. 2001. Simultaneous determination of ( + )-cateehin and (—)-epicatechin in cacao and its products by high-performance liquid chromatography with electrochemical detection. Phytochem. Anal. 12 271-276. 

B (System B) Treatment with IfHGl reagent generates a dark-brown zone of caffeine at R, 0.45 (T2) in extracts (1) and (3), less concentrated in (2) and (4). Theobromine at R, — 0.4 (T3) i.s delected as a grey, fast-fading zone in Mate folium (2), The concentration of theobromine in Cacao semen (4) is low, the amount of theophylline (R, — 0.6) in the extracts 1-4 is not. sufficient for detection. 

Theobromine (3,7-dimethylxanthine, 3,7-dihydro-3,7-dimethyl-lW-purine-2,6-dione). C7H,N402, Mr 180.17 formula, see under theophylline. Monoclinic, bitter tasting needles, mp. 357 °C, sublimes at 290-295 °C, soluble in hot water, alkali hydroxides, concentrated acids, moderately soluble in ammonia, poorly soluble in cold water and alcohol. With acids T. forms salts which decompose in water detection by the murexid reaction. T. is the main alkaloid of cocoa (Theobroma cacao, 1.5-3 wt.-%), from which it is obtained - especially from the husks in which it accumulates during fermentation. The typical bitter taste of cocoa is the result of interactions between T. and the pip-erazinediones formed in the roasting process. T. has diuretic, vasodilatory, and stimulating effects on cardiac muscle. The activities are weaker than those of the structurally related caffeine (a methylation product of T.) with which it co-occurs in cola nuts. For further pharmacological properties, see table under theophylline. 

Food residues containing cocoa were also found in vessels dating from the preclassical Maya period (900 BC-250 AD). The vessels were burial artefacts, which were found in the city of Colha (52 km north of Belize City). Cocoa (Theobroma cacao) is the only Central American plant which contains theobromine. Therefore, It Is the indicator substance to prove the prior existence of cocoa at those archaeological sites its detection Involves HPLC coupled mass spectrometry. [135]... 

Kim and Keeny [55] detected between 43,270 and 21,890-mg kg epicatechin in eight different cacao genotypes Tomas-Barberm et al. [56] quantified 25,650-mg kg fat-free dry matter Gotti et al. [57] reported on 15,300-5,700 mg kg It is still unclear whether environmental or genotypic factors dominate the variation. The diastereoisomer (+) catechin is lower in mature cocoa seeds 646 mg kg [56], 450-140 mg kg [57]. 

Kenyhercz TM, Kissinger PT (1978) Determination of selected acidic, neutral, and basic natural-products in cacao beans and processed cocoa. Liquid chromatography with electrochemical detection. Lloydia 41 130-139... 

Cooper, R.M., Resende, M.L.V., Flood, J., Rowan, M.G., Beale, M.H. andPotter, U. (1996) Detection and cellular localization of elemental sulphur in disease-resistant genotypes of Theobroma cacao. Nature, 319, 159-162... 

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