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.     

Caffeine daily intake from dietary sources in Brazil

A survey on the potential intake of caffeine was carried out in Campinas, SP, Brazil, in the summer of 1993. The survey was based on a representative sample of 600 individuals, 9-80 years old, who were asked about their habitual usage of coffee, tea, chocolate products and carbonated beverages. Caffeine levels in the products were determined by high performance liquid chromatography with a UV-visible detector at 254 nm. Individual daily intakes (mg/kg b.w.) of caffeine were calculated from the consumption data generated by the survey and the caffeine content of the analysed products. Of all those interviewed, 81% consumed soft drinks regularly, 75% coffee, 65% chocolate products and 37% tea. Of the analysed products, coffee showed the highest amount of caffeine. The average and median potential daily intake of caffeine by the studied population were, respectively, 2.74 and 1.85mg/kg b.w. Coffee, tea, chocolate products and carbonated beverages accounted for median individual daily intakes of 1.90, 0.32, 0.19, and 0.19mg/kg b.w., respectively. These data show that coffee is the most important vehicle for caffeine intake within the studied population.     

Determination of the caffeine contents of various food items within the Austrian market and validation of a caffeine assessment tool (CAT)

The caffeine content of 124 products, including coffee, coffee-based beverages, energy drinks, tea, colas, yoghurt and chocolate, were determined using RP-HPLC with UV detection after solid-phase extraction. Highest concentrations of caffeine were found for coffee prepared from pads (755?mg?l^?1) and regular filtered coffee (659?mg?l^?1). The total caffeine content of coffee and chocolate-based beverages was between 15?mg?l^?1 in chocolate milk and 448?mg?l^?1 in canned ice coffee. For energy drinks the caffeine content varied in a range from 266 to 340?mg?l^?1. Caffeine concentrations in tea and ice teas were between 13 and 183?mg?l^?1. Coffee-flavoured yoghurts ranged from 33 to 48?mg?kg^?1. The caffeine concentration in chocolate and chocolate bars was between 17?mg?kg^?1 in whole milk chocolate and 551?mg?kg^?1 in a chocolate with coffee filling. A caffeine assessment tool was developed and validated by a 3-day dietary record (r ^2?=?0.817, p?<?0.01) using these analytical data and caffeine saliva concentrations (r ^2?=?0.427, p?<?0.01).     

Energy drink consumption and impact on caffeine risk

The impact of caffeine from energy drinks occurs against a background exposure from naturally occurring caffeine (coffee, tea, cocoa and foods containing these ingredients) and caffeinated beverages (kola-type soft drinks). Background caffeine exposure, excluding energy drinks, was assessed for six New Zealand population groups aged 15 years and over (n = 4503) by combining concentration data for 53 caffeine-containing foods with consumption information from the 2008/09 New Zealand Adult Nutrition Survey (ANS). Caffeine exposure for those who consumed energy drinks (n = 138) was similarly assessed, with inclusion of energy drinks. Forty-seven energy drink products were identified on the New Zealand market in 2010. Product volumes ranged from 30 to 600 ml per unit, resulting in exposures of 10–300 mg caffeine per retail unit consumed. A small percentage, 3.1%, of New Zealanders reported consuming energy drinks, with most energy drink consumers (110/138) drinking one serving per 24 h. The maximum number of energy drinks consumed per 24 h was 14 (total caffeine of 390 mg). A high degree of brand loyalty was evident. Since only a minor proportion of New Zealanders reported consuming energy drinks, a greater number of New Zealanders exceeded a potentially adverse effect level (AEL) of 3 mg kg^?1 bw day^?1 for caffeine from caffeine-containing foods than from energy drinks. Energy drink consumption is not a risk at a population level because of the low prevalence of consumption. At an individual level, however, teenagers, adults (20–64 years) and females (16–44 years) were more likely to exceed the AEL by consuming energy drinks in combination with caffeine-containing foods.     

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.     

Caffeine intake and its sources: A review of national representative studies

Aim of this review is to summarize current daily caffeine intake of children, adolescents, and adults, and trends in caffeine intake over the past decade. A literature search was conducted (1997–2015) which yielded 18 reports on nationally representative studies, describing caffeine consumption of over 275,000 children, adolescents and adults. The data revealed that mean total daily caffeine intake in children, adolescents, and adults is below caffeine intake recommendations such as those stated by Health Canada (2.5 mg/kg bw/day for children and adolescents, and 400 mg/day for adults) and the European Food Safety Authority, EFSA (3 mg/kg bw/day for children and adolescents, and 400 mg/day for adults). Total daily caffeine intake has remained stable in the last 10–15 years, and coffee, tea and soft drinks are the most important caffeine sources. Across all age groups, energy drinks contribute little to total caffeine intake. The highest potential for reducing daily caffeine intake is by limiting coffee consumption, and in some countries and age groups, by reducing tea and soft drink consumption.     

CAFFEINE CONTENT IN COLAS FROM NEW YORK STATE AND ONTARIO

Thirty-nine canned soft drinks obtained from New York (NY) State and from Ontario in 1986 were analyzed for caffeine. The major brands of cola drinks from either location differed little in caffeine concentration, with mean values of 92.5 μg/mL for NY State and 86.5 μg/mL for Ontario. However, different can volumes contributed toward the difference in caffeine content per can of cola beverages obtained in NY State and Ontario. The mean caffeine values for the "regular colas" were 34.3 and 21.5 mg/can (concentration range: 2.3–133.4 μg/mL and 0.1–104.9 μg/mL) for the NY State and Ontario products, respectively. The range of caffeine content for all products was from 0.8 to 50.8 mg/can for the NY State colas and from 0.03 to 29.4 mg/can for the Ontario products. Comparison with earlier reports (for USA from 1979:47.3 mg/can; for Ontario from 1976: 40 mg/can) indicated a general decrease in the amount of caffeine in all types of cola beverages in the last 7 years.     

Effects of caffeine on human health

Caffeine is probably the most frequently ingested pharmacologically active substance in the world. It is found in common beverages (coffee, tea, soft drinks), in products containing cocoa or chocolate, and in medications. Because of its wide consumption at different levels by most segments of the population, the public and the scientific community have expressed interest in the potential for caffeine to produce adverse effects on human health. The possibility that caffeine ingestion adversely affects human health was investigated based on reviews of (primarily) published human studies obtained through a comprehensive literature search. Based on the data reviewed, it is concluded that for the healthy adult population, moderate daily caffeine intake at a dose level up to 400 mg day^-1 (equivalent to 6 mg kg^-1 body weight day^-1 in a 65-kg person) is not associated with adverse effects such as general toxicity, cardiovascular effects, effects on bone status and calcium balance (with consumption of adequate calcium), changes in adult behaviour, increased incidence of cancer and effects on male fertility. The data also show that reproductive-aged women and children are 'at risk' subgroups who may require specific advice on moderating their caffeine intake. Based on available evidence, it is suggested that reproductive-aged women should consume ≤ 300 mg caffeine per day (equivalent to 4.6 mg kg^-1 bw day^-1 for a 65-kg person) while children should consume ≤ 2.5 mg kg^-1 bw day^-1.     

Evaluation of aluminium concentrations in samples of chocolate and beverages by electrothermal atomic absorption spectrometry

Samples of chocolate, cocoa, tea infusions, soft drinks and fruit juice have been examined by, electrothermal atomic absorption spectrometry (ETA-AAS) for the presence of aluminium (Al). Fruit juices and chocolate were analysed after an adequate sample preparation; the other products were evaluated directly. Sampling was performed in duplicate for 248 independent samples. The mean Al concentration in chocolate was 9.2 +/- 7.5 mg kg(-1), and individual values were correlated with the per cent of cocoa in samples (Y = 0.63 + 0.27X, r = 0.78, p < 0.0001). Al concentration in commercial tea infusions ranged from 0.9 to 3.3 mg l(-1) (mean = 1.80 +/- 65 mg l(-1), whereas in laboratory-prepared samples it was 2.7 +/- 0.93 mg l(-1). In soft drinks, the concentrations of Al were lower, ranging from 9.1 to 179 microg l(-1); the highest values were observed in samples of orange squash (mean = 114 +/- 56 microg l(-1)). Apricot juice showed the highest Al level (mean = 602 +/- 190 microg l(-1)), being statistically, different from that of pear (mean = 259 +/- 102 microg l(-1)), but not different from that of peach juice (mean = 486 +/- 269 microg kg(-1)). Toxicologically, the amount of Al deriving from the consumption of these products is far below the acceptable daily intake of 1 mg kg(-1) body weight indicated by the FAO/WHO, and it is a verv low percentage of the normal Al dietary intake.     

Effects of extraction solvents on concentration and antioxidant activity of black and black mate tea polyphenols determined by ferrous tartrate and Folin–Ciocalteu methods

Effect of the use of water and different organic solvents such as acetone, N,N-dimethylformamide (DMF), ethanol or methanol at various concentrations on the total polyphenol content and antioxidant activity was studied for the black tea and mate tea. Polyphenol contents of extracts were determined using ferrous tartrate (method # 1) and Folin–Ciocalteu (method # 2) assays. For black tea, 50% DMF extract showed the highest polyphenol content of 131.9 mg/g and 99.8 mg GAE/g by method # 1 and method # 2, respectively. For mate tea, 50% acetone showed the highest polyphenol content of 132.5 mg/g and 120.4 mg GAE/g by method # 1 and method # 2, respectively. Fifty percent ethanol extract from mate tea and 50% acetone from black tea had the greatest antioxidant activity. The results showed that solvent with different polarity had significant effect on polyphenol content and antioxidant activity. A high correlation between polyphenol content and antioxidant activity of tea extracts was observed.     

茶黄素和咖啡碱浓度对茶乳酪形成的影响

以茶黄素和咖啡碱的相互作用来模拟茶乳酪的形成体系,通过分析其透光率、沉淀量、茶黄素和咖啡碱质量浓度的变化,研究茶黄素和咖啡碱对茶乳酪形成的影响。结果表明:随茶黄素和咖啡碱质量浓度的增加,其聚合后原溶液和贮藏液的透光率降低,贮藏液的沉淀量增加;并且当固定茶黄素和咖啡碱二者中任一物质的质量浓度,随另外一个物质质量浓度的增加,上述变化趋势显著。灭菌不影响整体的变化趋势,除此之外通过高效液相色谱分析咖啡碱和茶黄素各单体质量浓度的变化发现,咖啡碱和茶黄素-3,3’-没食子酸酯是影响模拟体系中茶乳酪形成的关键因子。基于减少茶乳酪生成,提高红茶饮料的感官效果,并同时尽可能多地保留茶黄素质量浓度,应当选择茶黄素质量浓度低于100mg/L、咖啡碱质量浓度低于200mg/L的红茶原料来制作红茶饮料。      

Caffeine levels in beverages from Argentina's market: application to caffeine dietary intake assessment

The caffeine content of different beverages from Argentina's market was measured. Several brands of coffees, teas, mates, chocolate milks, soft and energy drinks were analysed by high-performance liquid chromatography (HPLC) with ultraviolet detection. The highest concentration level was found in short coffee (1.38 mg ml^?1) and the highest amount per serving was found in instant coffee (95 mg per serving). A consumption study was also carried out among 471 people from 2 to 93 years of age to evaluate caffeine total dietary intake by age and to identify the sources of caffeine intake. The mean caffeine intake among adults was 288 mg day^?1 and mate was the main contributor to that intake. The mean caffeine intake among children of 10 years of age and under was 35 mg day^?1 and soft drinks were the major contributors to that intake. Children between 11 and 15 years old and teenagers (between 16 and 20 years) had caffeine mean intakes of 120 and 240 mg day^?1, respectively, and mate was the major contributor to those intakes. Drinking mate is a deep-rooted habit among Argentine people and it might be the reason for their elevated caffeine mean daily intake.     

Tea caffeine: Metabolism, functions, and reduction strategies

Tea is a product made up from topmost part of the plant Camellia sinensis. This part includes bud, first leaf (next to bud), second leaf, and stem (spanning from bud to second leaf). Tea is the second most consumed beverage in the world, well ahead of coffee, beer, wine, and carbonated soft drinks. Clinical studies have demonstrated that one of the harmful effects of tea over consumption, at least in sensitive peoples is due to its caffeine content. In view of this, major points discussed in this article are the following: i) a brief overview on tea and its biochemical composition, ii) health effects of tea drink, iii) caffeine metabolism and its functions, iv) possible strategies for caffeine reduction in tea, and v) feasibility of tea improvement through biotechnological approaches.     

Caffeine Content of Retail Market Coffee in Portugal

How much caffeine does one ingest when drinking a simple cup of coffee in Portugal? The study presented herein tried to answer this question through the assessment of caffeine content of commercially available espresso coffee samples, both caffeinated and decaffeinated, using a high-performance liquid chromatography assay. Caffeine was rapidly separated from the sample matrix using a RP-18 column (250?×?4 mm i.d., 5 μm). The flow rate was 1.0 mL/min and the mobile phase consisted of water acidified with 5% of orthophosphoric acid/methanol (35:65, v/v). Caffeine was detected directly at 273 nm. The assay was validated for linearity, lower limit of quantification and limit of detection, precision, accuracy, and stability. Seventeen different brands of caffeinated coffee and six of decaffeinated coffee were analyzed. As for capsule coffee, eight caffeinated and two decaffeinated blends were analyzed. The caffeine content of caffeinated coffee varied from 53.8?±?5.9 to 141.3?±?5.3 mg/cup, whereas for caffeinated capsule coffee caffeine concentrations ranged from 45.0?±?5.3 to 60.8?±?6.2 mg/cup. As for decaffeinated coffee, caffeine concentrations ranged from 0.96?±?0.04 to 3.9?±?0.1 mg/cup and for decaffeinated capsule coffee from 0.93?±?0.04 to 1.2?±?0.1 mg/cup.     

Optimized method for simultaneous determination of catechin, gallic acid, and methylxanthine compounds in chocolate using RP-HPLC

The simultaneous determination of catechins, gallic acid, and three methylxanthine compounds was determined in chocolate extract by reversed-phase HPLC. An isocratic HPLC procedure using Hypersil-ODS column with methanol three distilled water-formic acid (48.75:200.5:0.75, v/v/v) as a mobile phase was used and the HPLC profiles of 11 chocolate products were obtained. (-)- Epicatechin, (-)-epigallocatechin gallate, levels were very high whereas (+)-catechin, (-)-epigallocatechin and (-)-epicatechin gallate were lower values. Gallic acid was predominant compounds with extremely high levels in all chocolate samples. The content of three methylxanthine, theobromine, theophylline and caffeine was quantified with this procedure. Caffein amounts were low in all samples, while theobromine levels were high in milk and dark chocolates. Theophylline was at trace levels in some chocolates and could not be detected in some samples. This method was reliable, reproducible and objective for quantitative identification of flavanols and alkaloid compounds in the chocolate industry.     

Survey of caffeine levels in retail beverages in Portugal.

The caffeine content of 85 retail beverage samples purchased from local supermarkets between 1995 and 2004 was determined. The potential intake of caffeine through the consumption of these beverages (but excluding coffee) was estimated for students of the University of Coimbra, Portugal. The caffeine content of the beverages ranged from 47.5 to 282.5 mg l(-1) for teas, from 20.1 to 47.2 mg l(-1) for tea extracts samples, and from 80.7 to 168.7 mg l(-1) for cola soft drinks. Caffeine was not completely absent from caffeine-free colas, and energy drinks had a far greater caffeine content than regular drinks, ranging from 21 to 2175 mg l(-1). Soft drinks were consumed by 72% of the individuals, although 14% of the survey participants did not drink any of the different types of the beverages studied. Contrary to expectations for this age group, no consumptions of energy drinks was reported. Daily caffeine intake was estimated to range from 4.7 to 200 mg day(-1), but with only 5% reporting a daily intake around 200 mg caffeine. Cola-type beverages were an important dietary source of caffeine for the population studied. Statistical differences in the caffeine intake between the male and female populations were found, with p = 0.014, being higher for the male population. Of the beverages studied, cola-type drinks showed statistical differences for the male population, p = 0.03, and tea showed statistical differences for female population p = 0.013, respectively.     

超高效液相串联质谱检测茶叶及茶饮料中咖啡因含量

建立超高效液相串联质谱检测茶叶及茶饮料中咖啡因含量的方法。茶叶及茶饮料经前处理后进样,以0.1%甲酸水溶液-乙腈(80:20,v/v)为流动相等度洗脱,流速为0.3 mL/min,经C₁₈色谱柱分离,柱温25 ℃;质谱采用电喷雾离子源正离子模式、多反应检测模式检测,脱溶剂气(N₂)温度500 ℃、流速700 L/Hr,毛细管电压3.5 kV,锥孔电压55 V,碰撞气为高纯氩气。在上述分析条件下,本法咖啡因的检出限为2.0 μg/L;在2.0~1000 μg/L浓度范围内的线性关系良好,相关系数r为0.9969;加标回收率达90.2%~103.9%;相对标准偏差在1.00%~3.00%(n=6)。本方法简便、快速,选择性、重现性好、灵敏度高,适用于茶叶及茶饮料中咖啡因的定量分析,结果准确可靠,对于复杂样品中咖啡因的检测有明显优势。     

Determination of the decomposition products of Usal in model systems and determination of dioxopiperazine in soft drinks by HPLC

A HPLC method for the determination of Usal (Aspartame hydrochloride, L-aspartyl-L-phenylalanine methyl ester hydrochloride) and its decomposition products was elaborated. Aspartic acid, phenylalanine, phenylalanine methyl ester, aspartyl-phenylalanine, phenylalanyl-aspartic acid, 5-benzyl-3,6-dioxo-2-piperazineacetic acid (DOP) and Usal were separated on Separon SI C-18. The mobile phase was: 0.5 M NaH2PO4 (pH 2.1) and methanol (85:15 v/v). The detection was carried out at 200 nm. The method for DOP determination was tested by the analysis of 10 types of soft drinks to which DOP was added. In two newly developed sorts of soft drinks sweetened with Usal the formation of DOP was followed during storage. The DOP increment after 34 days of storage reached 0.7 and 6 mg/l at 7 and 20 degrees C, resp. The method is also suitable for DOP determination in the sweetener itself.     

ANALYSIS OF COFFEE, TEA AND ARTIFICIALLY FLAVORED DRINKS PREPARED FROM MINERALIZED WATERS

ABSTRACT— Coffee, black tea and artificially flavored and artificially sweetened orange and grape soft drinks were prepared from solutions of 8 minerals each at 750 ppm. Additionally, coffee and tea were brewed, using 6 natural drinking waters which ranged from 42–1,725 ppm of total dissolved solids. Beverages made from solutions containing carbonates were the least desirable, having flat, insipid characteristics. Although distilled water resulted in an acceptable soft drink, coffee and tea prepared from it were excessively sour and astringent, respectively. The recommended formula of 53.3 g of coffee per liter of water was considered too strong and "burnt' for the panel of trained judges, so that the experiments were repeated at a lower concentration of 47 g coffee/liter water. Large differences in the direction and magnitude of the visual characteristics of the coffee and tea brewed from the various waters also were observed in the marked changes in luminous transreflectance, purity and shifts in dominant wavelengths measured by thin-layer reflectometry.     

茶叶中茶多酚和生物碱的测定及聚类和线性判别分析

目的：建立一种反相高效液相色谱方法,测定绿茶、乌龙茶、红茶、白茶和普洱茶中儿茶素[(+)-catechin,C]、表儿茶素[(-)-epicatechin,EC]、表儿茶素没食子酸脂[(-)-epicatechin gallate,ECG]、表没食子儿茶素[(-)-epigallocatechin,EGC]、表没食子儿茶素没食子酸脂[(-)-epigallocatechin gallate,EGCG]、没食子酸(gallic acid,GA)、咖啡因(caffeine,CAF)、可可碱(theobromine,THEO)的水平。以这8 种组分为指标对茶叶进行聚类分析和线性判别分析,建立区分绿茶、红茶和乌龙茶的方法。方法：茶叶提取后采用HPLC 法测定儿茶素和生物碱含量,色谱柱为C18 柱,流动相由甲醇(A)、2% 的乙酸(B)等度洗脱,A、B 相的体积比为25:75,流速为1mL/min,柱温30℃。采用PDA 检测器在278nm 检测,参考波长为210nm。采用SPSS 14.0 对实验数据进行了聚类分析和线性判别分析。结果：在选择的分析条件下,样品中的8 种组分获得了理想分离, 加标回收率在8 7%～ 112.8%之间,并采用外标法对8 种组分进行定量。以这8 种组分的含量为指标,聚类分析和线性判别分析能对39 种茶叶样品进较好的区分。