Determination of theobromine,theophylline and caffeine in cocoa samples by a high-performance liquid chromatographic method with on-line sample cleanup in a switching-column system

A simple reversed-phase high-performance liquid chromatographic method was developed for the determination of theobromine, theophylline and caffeine in cocoa samples. In the sample cleanup step, the procedure involves an on-line solid-phase extraction of analytes from cocoa samples into a home-made dry-packed pre-column with ODS-C₁₈ using a column-switching system. The separation was performed on a C₁₈ Nova-Pak column (150 mm × 3.9 mm, 4 μm) using a mobile phase consisting of a solution of 20% of methanol in water under isocratic conditions, at a flow-rate of 1.4 ml/min. The validation method revealed quantitative recoveries (>95.0%) with a coefficients of variation <3.2% and it also provided a good precision for data validation. The overlap of sample cleanup, analysis and recondition of the precolumn increases the sample throughput to 8 samples/h. Furthermore, the proposed method was successfully applied to analysis of cocoa samples “Trinitario”, “Forastero” and “Criollo” grown in different seasons of the year and fermented for 3 and 7 days, respectively. The results showed a slight reduction in the theobromine and caffeine content according to the fermentation times. In the same way, the theobromine/caffeine ratio was assessed, with the purpose of establishing a correlation with the genotype of the studied samples.     

Theobromine and caffeine content of the cocoa bean during its growth

Changes in the concentrations of caffeine and theobromine in the cocoa pod during its growth were determined. Theobromine and caffeine concentrations increased markedly after about 12 weeks from flowering to the time of harvesting. During the stage when the bean was only a mucilaginous mass, the quantity of caffeine and theobromine was negligible. Caffeine and theobromine levels in the shell, once this is formed, increased rapidly until harvest.     

Caffeine and Theobromine Levels in Cocoa and Carob Products

Seventy-nine cocoa or chocolate products and eighteen carob products were analyzed by HPLC for caffeine and theobromine content. After extraction into boiling water, the methylxanthines were identified and quantified with the use of a reverse phase column. Mean theobromine and caffeine levels respectively, were 0.695 mg/g and 0.071 mg/g in cocoa cereals; 1.47 mg/g and 0.152 mg/g in chocolate bakery products; 1.95 mg/g and 0.138 mg/g in chocolate toppings; 2.66 mg/g and 0.208 mg/g in cocoa beverages; 0.621 mg/g and 0.032 mg/g in chocolate ice creams; 0.226 mg/g and 0.011 mg/g in chocolate milks; 74.8 mg/serving and 6.5 mg/serving in chocolate puddings. Theobromine and caffeine levels in carob products ranged from 0-0.504 mg/g and 0-0.067 mg/g, respectively.     

Comparison of polyphenol,methylxanthines and antioxidant activity in Theobroma cacao beans from different cocoa-growing areas in Colombia

The aims of the current work were to study the influence of geographical areas on polyphenol and methylxanthine contents and the antioxidant activity in cocoa beans from different cocoa-growing areas of Colombia, and to evaluate the possibility of establishing a classification based on the geographical areas of the cocoa-growing regions. Eighteen cocoa farms located in eleven cocoa-growing areas were analyzed. The statistical analysis showed significant differences (p < 0.05) in total polyphenol content (TPC), flavan-3-ol, epicatechin, catechin, caffeine and theobromine contents as well as the theobromine/caffeine ratio and the antioxidant capacity between some of the different sampled farms, showing a significant effect of the cocoa-producing region on these parameters. Generally, a proportional relationship has been proposed to exist between the polyphenol content with changes in altitude of plant crops, despite this, our results suggest that the lower the altitude, the more polyphenols, flavan-3-ols and epicatechin are produced by the cocoa plant. Results of the principal component analysis (PCA) indicate that caffeine content and the theobromine/caffeine ratio could serve as parameters to establish a cocoa bean classification according to the geographical area of the cocoa-growing regions.     

HPLC Determination of Caffeine and Theobromine in Coffee, Tea, and Instant Hot Cocoa Mixes

Popular brands of instant and ground coffee, instant and bag tea, and instant hot cocoa mixes were prepared according to manufacturer's directions and analyzed for caffeine and/or theobromine content. After extraction into boiling water, the methylxanthines were separated by high performance liquid chromatography using a reverse phase C₁₈ column and a mobile phase of acetonitrile and water (8:92, v/v). Caffeine content ranged from 32.4–35.0 mg/cup in instant tea, 30.2–67.4 mg/cup in bag tea, 1.0–7.8 mg/cup in instant hot cocoa mixes, 46.7–67.6 mg/cup in instant coffee, and 93.0–163.5 mg/cup in ground coffee. Theobromine concentrations ranged from 1.4–2.3 mg/cup in instant tea, 1.2–4.4 mg/cup in bag tea, and 39.5–79.5 mg/cup in instant hot cocoa mixes.     

Determination of purine alkaloids and trigonelline in instant coffee and other beverages using high performance liquid chromatography

A simple, rapid high performance liquid chromatography method is reported for the simultaneous analysis of trigonelline and caffeine (1,3,7-trimethylxanthine) in coffee, and theobromine (3,7-dimethylxanthine) and caffeine in samples of cocoa, drinking chocolate, tea and cola. Chromatography was carried out on Spherisorb-ODS columns with gradient elution. The eluate was monitored at 272 nm with an ultraviolet spectrophotometer and quantification was achieved with external standards. Results for different brands of instant coffees, one decaffeinated coffee, and for samples of cocoa, drinking chocolate, tea and cola are reported.     

Cocoa Phytochemicals: Recent Advances in Molecular Mechanisms on Health

Recent reports on cocoa are appealing in that a food commonly consumed for pure pleasure might also bring tangible benefits for human health. Cocoa consumption is correlated with reduced health risks of cardiovascular diseases, hypertension, atherosclerosis, and cancer, and the health-promoting effects of cocoa are mediated by cocoa-driven phytochemicals. Cocoa is rich in procyanidins, theobromine, (?)-epicatechin, catechins, and caffeine. Among the phytochemicals present in consumed cocoa, theobromine is most available in human plasma, followed by caffeine, (?)-epicatechin, catechin, and procyanidins. It has been reported that cocoa phytochemicals specifically modulate or interact with specific molecular targets linked to the pathogenesis of chronic human diseases, including cardiovascular diseases, cancer, neurodegenerative diseases, obesity, diabetes, and skin aging. This review summarizes comprehensive recent findings on the beneficial actions of cocoa-driven phytochemicals in molecular mechanisms of human health.     

Biologically active substances in grated cocoa and cocoa butter

In the article results of comparative analysis of grated cocoa and cocoa butter samples are presented. The investigation was done by modern instrumental methods such as HPLC, GC, UV- VIS-spectroscopy, and also with application of titrimetric and grarimetric methods. In the analyzed samples contents of total phenolics changes in an interval 1,0-3,2%, including monomeric proantocyanidins 0,6-1,35%; pyrroloquinoline quinine (PQQ) 0,34-0,76 microg/g; phenyl ethylamine from 2,79 to 14,97 microg/g, tyramine from 9,56 to 71,68 microg/g, dopamine from 5,3 to 25,85 microg/g; theobromine from 3,3 to 8%, caffeine from 0,49 to 0,70%; among the amino acids at the greatest quantities were presented glutaminic and asparaginic acids, arginin and leucin; three main fatty acids were determined - palmitinic (31+/-2% rel.), oleinic (35+/-2% rel.) and stearinic (35+/-2% rel.); the main phytosterins were sytosterin (up to 192 mg%) and obtusifoliol (up to 198,5 mg%).     

Effects of cocoa extract containing polyphenols and methylxanthines on biochemical parameters of obese‐diabetic rats

BACKGROUND: Previous studies have indicated that cocoa extract possesses hypoglycaemic and hypocholesterolaemic properties in streptozotocin‐induced diabetic rats. However, there has been limited research on the effects of cocoa extract on obese‐diabetic (Ob‐db) rats that mimic human diabetes syndrome. Hence this study was initiated to determine the effect of cocoa extract containing polyphenols and methylxanthines on several biochemical parameters, namely glucose level, insulin sensitivity and lipid profiles of Ob‐db rats. RESULTS: Intake of cocoa extract supplemented with polyphenols (2.17 mg epicatechin, 1.52 mg catechin, 0.25 mg dimer and 0.13 mg trimer g^?1 cocoa extract) and methylxanthines (3.55 mg caffeine and 2.22 mg theobromine g^?1 cocoa extract) for 4 weeks significantly (P < 0.05) reduced the plasma total cholesterol, triglycerides and low‐density lipoprotein cholesterol of obese‐diabetic rats (Ob‐db + cocoa) compared with non‐supplemented animals (Ob‐db). Short‐term (acute) supplementation of cocoa extract significantly (P < 0.05) reduced the plasma glucose level at 60 and 90 min compared with untreated rats as assessed by the oral glucose tolerance test. However, no significant differences were observed in plasma glucose level, insulin level and insulin sensitivity after chronic (4 weeks) cocoa extract supplementation. CONCLUSION: The results of this study suggest that cocoa extract possesses hypocholesterolaemic properties and can exert a transient glucose‐lowering effect but not long‐term glucose control. Copyright © 2008 Society of Chemical Industry     

Relationship between bioactive compounds and sensory properties of dark chocolate produced from Brazilian hybrid cocoa

This work evaluated the influence of bioactive compounds on the sensory profile and acceptability of dark chocolate produced from Brazilian hybrid cocoa. The results showed that higher contents of catechin, epicatechin, caffeine, total phenolic compounds and flavonoids contributed to higher intensity of bitterness, cocoa flavour, acid taste, and astringency, and negatively affected the acceptance of chocolate samples from the TSH1188, PH16, and CEPEC2002 varieties. Other varieties SR162 and BN34 showed lower contents of caffeine and phenolic compounds, and higher concentration of theobromine, revealed notes of sweet/caramel and fruity flavour, presented a higher acceptance. This research showed the great potential of the ‘Catongo’ (SR162) and BN34 hybrids from southern Bahia (Brazil) for the production of fine cocoa. The chemical and sensory evaluations may be a strategic tool to help cocoa producers in deciding which genetic varieties should be marketed as fine cocoa, adding value to the product and highlighting promising cocoa varieties.     

Partial removal of various food components from aqueous solution using crosslinked polymers of cyclodextrins with epichlorohydrin

Cyclodextrin (CD), its derivatives and dextrin were polymerised with epichlorohydrin (ECH) to form crosslinked polymers gels. The effects of various types of such gels in removing natural food components—naringin, caffeine and theobromine—from aqueous solution were investigated. The capacity of the gel to remove natural products was highest for naringin, intermediate for vanillin and lowest for caffeine and theobromine. The influence of gel structure on removal capacity was studied by varying the molar ratio of CD, or its derivatives, to ECH. Aqueous sodium hydroxide (0·5 M ) effectively regenerated a β-CD–ECH gel for repeated binding of naringin. About 97% of relative removal efficieny was retained after seven regenerations in batch operation. A column procedure was also effective for naringin removal.     

可可碱、茶碱和咖啡碱的快速测定及其色谱保留行为

建立基于钛胶反相色谱固定相快速测定食品中可可碱、茶碱和咖啡碱的高效液相色谱（high performance liquid chromatography,HPLC）法。结果表明,可可碱、茶碱和咖啡碱的标准曲线线性良好（R2＞0.999 0）,线性范围分别为5～500、5～1 000 mg/mL和6～1 200 mg/mL,检出限分别为0.04、0.08 mg/mL和0.03 mg/mL,样品加标回收率分别为99.0%～102.0%、96.7%～106.7%和99.4%～102.3%,相对标准偏差（n=9）小于0.23%。3 种生物碱在HPLC上的保留行为由焓变ΔH0驱动,保留顺序与实验结果相符,依次为可可碱、茶碱和咖啡碱。建立方法操作简单、分析速度快,精密度和准确度均可满足测定要求。用于食品中可可碱、茶碱和咖啡碱的同时测定,结果满意。     

Chemical characterization and antioxidant properties of a new coffee blend with cocoa,coffee silverskin and green coffee minimally processed

The search for new technologies and ingredients with interesting characteristics and potential for incorporation into functional foods emerges in parallel with the demand for alternative sustainable and economically viable blends. Pursuing these aims, the formulation of a new coffee blend with 94% roasted coffee powder (Coffea canephora cv. Robusta and Coffea arabica, 70/30, w/w), 3% cocoa powder, 2% coffee silverskin and 1% golden coffee (green coffee minimally processed) was developed. The influence of the ingredients in the blend was compared with two other commercial coffee blends (in capsule and in a sealed package with a one-way degassing valve), being characterized the formulation, the physicochemical parameters, as its innovation. It is concluded that the developed coffee blend shows an enriched content of bioactive compounds (chlorogenic acids, trigonelline, theobromine and caffeine), displays an important antioxidant capacity and was favorably appreciated due to its sensory characteristics. Moreover, the addition of skin by-product becomes an additional valorization and the processing of green coffee and cocoa was minimized by adding innovation and an optimized extraction.     

Biodegradation of caffeine: Formation of theophylline and theobromine from caffeine in mature Coffea arabica fruits

The level of theophylline in mature and ripened fruit is 20–50 times that in the immature green fruit of Coffea arabica L. Biodegradation of caffeine occurs in the mature, ripened coffee fruits through theophylline and theobromine as the first biodegradation products. It is now clear that theophylline is associated primarily with caffeine biodegradation, whereas theobromine is involved in both biosynthesis and biodegradation of caffeine.     

High‐performance liquid chromatography procedure for the determination of flavor enhancers in consumer chocolate products and artificial flavors

BACKGROUND: A number of individual high‐performance liquid chromatography (HPLC) procedures exist for the analysis of maltol, theobromine, ethyl maltol, catechin, vanillic acid, caffeine, vanillin, epicatechin, and ethyl vanillin. A single procedure utilizing simple sample preparation and less sophisticated equipment would be advantageous for the analysis of different sample types containing these compounds. RESULTS: An HPLC procedure has been developed using water as the extract for consumer products and artificial flavors. A methanol–water gradient was used to elute the compounds of interest using a reverse‐phase column. Absorbance detection using a wavelength of 273 nm was used to monitor the eluent. Recoveries for these compounds ranged from 88% to 104%. CONCLUSIONS: Results obtained for theobromine, catechin, caffeine, and epicatechin in Standard Reference Material 2380 Baking Chocolate compare well with those found in its certificate of analysis verifying that the procedure is valid. Vanillic acid and ethyl vanillic acid were found as oxidation products of vanillin and ethyl vanillin in both standards and some consumer products. Copyright © 2008 Society of Chemical Industry     

Comparative study of cytotoxic and cytoprotective activities of cocoa products affected by their cocoa solids content and bioactive composition

Polyphenolic profile and antioxidant properties of water extracts of milk, semisweet and dark chocolates, as well as cocoa liquor, were determined and examined for potential biological activity. Non-fat cocoa solids (NFCS), phenolic compounds content and the antioxidant capacity of cocoa product extracts were determined using UV/Vis spectrophotometric methods and HPLC analysis. The increase in NFCS was consistent with the increase in polyphenol content and antioxidant properties. Methylxanthines, theobromine and caffeine constituted the most abundant bioactive compounds, followed by flavan-3-ols epicatechin and procyanidin B2. Cytotoxic and antioxidative/prooxidative effects of cocoa product extracts were determined on human laryngeal carcinoma cell line (HEp2). Cocoa liquor containing the highest NFCS exhibited the lowest HEp2 cell viability, while milk chocolate characterized by the lowest NFCS exhibited no cytotoxic effect. Experiments revealed a strong relationship between the type of product/concentration/time of exposure and antioxidant/prooxidant character of cocoa products. Lower concentrations of semisweet, dark chocolate and cocoa liquor induced an increase in ROS formation, while the higher concentrations resulted in a decrease in ROS formation when compared with control (growth medium). Principal component analysis of the obtained results revealed specific grouping of samples (milk chocolate and cocoa liquor), while the observed dispersions indicated that the outcome of cytotoxic and cytoprotective activities of cocoa products are greatly affected by their concentration.     

Quality of Malaysian cocoa butter during storage

Malaysian pure prime pressed and deodorised cocoa butters were obtained from local cocoa grinders and analysed for quality deterioration. Approximately 5 kg of each sample was packed in corrugated paper boxes lined with low‐density polyethylene plastic material of thickness 73.2 µm. The water vapour transmission rate and gas transmission rate (for oxygen) of the plastic material were 3.21 g m^?2 day^?1 (37.8 °C, 90 ± 2% RH) and 71.57 cm³ m^?2 day^?1 (23.0 °C, 0% RH) respectively. The butters were stored at 15 ± 4 °C and 70 ± 4% RH for up to 24 months. Samples were taken every 2 months and analysed for free fatty acid content, saponification value, iodine value, refractive index and peroxide value. The findings indicated that the free fatty acid content, iodine value and peroxide value of both types of cocoa butter increased with storage duration. The other parameters showed no significant changes. However, the free fatty acid content and peroxide value after 24 months of storage were still below the limits specified in Malaysia. Therefore we conclude that both types of Malaysian cocoa butter are still in good condition after 24 months of storage, with minimal quality deterioration. Copyright © 2004 Society of Chemical Industry     

液相色谱法测定饮料中咖啡因、可可碱和茶碱

采用高效液相色谱法同时测定饮料中的咖啡因、可可碱和茶碱.饮料样品经超声提取后,用高效液相色谱分离,二极管阵列检测器检测,建立标准工作曲线,以外标法定量.实验精密度为0.58%～1.85%,添加回收率范围为80.2%-96.4%.此方法快速,简便,准确,适用于饮料中咖啡因、可可碱和茶碱的测定.     

高效液相色谱法测定咖啡因的不确定度评定

目的建立高效液相色谱法测定咖啡因的不确定度的评估方法。方法试样中的咖啡因用甲醇:水:磷酸=100:400:0.5(V:V:V)混合溶液进行提取,在波长280 nm下,经流动相洗脱,色谱系统分离,采用外标法测定咖啡因的含量,然后依据JJF 1059.1-2012《测量不确定度评定与表示》,对标准物质、仪器重复测量、精密度和样品称量等因素引入的不确定度进行分析和计算。结果当试样中咖啡因含量为7.50%时,在95%的置信区间下,其扩展不确定度为±0.13%(k=2)。实验过程中的不确定度主要来源于仪器测量、标准物质及人员操作。结论该不确定度评定适用于高效液相色谱法测定咖啡因含量的过程分析,对检测结果准确度的控制具有指导意义。     

11—OBSERVATIONS ON THE CHARACTERISTICS OF AUSTRALIAN GREASY WOOL PART I: STAPLE LENGTH IN MERINO FLEECE WOOLS

The average staple length of merino fleece wool is 84 mm. Subjective classification of diameter–style groupings from one selling centre into ‘long’, ‘medium’, and ‘short’ produces differences of 7–8 mm between adjacent length categories. Sale lots within these length categories vary by ± 14 mm (95% confidence interval), so there is considerable overlap even between ‘long’ and ‘short’ wools. Individual staples within a sale lot vary by ± 25 mm about the mean (95% confidence interval). These estimates vary significantly with diameter and style category and among selling centres.