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Journal of Food and Nutrition Research, 2018, Vol. 6, No. 8, 513-517 Available online at http://pubs.sciepub.com/jfnr/6/8/5 ©Science and Education Publishing DOI:10.12691/jfnr-6-8-5

The Content in Beverages Commercially Distributed in Indonesia

Sukrasno*, Irena Aria Rivera, Komar Ruslan Wirasutisna

School of Pharmacy, Institut Teknologi Bandung, Bandung, Indonesia *Corresponding author: [email protected] Received June 25, 2018; Revised August 25, 2018; Accepted August 31, 2018 Abstract Coffee beverages are widely consumed by Indonesian, however, some of them encounter the adverse effect after drinking coffee, such as difficulty to sleep, arrhythmia and pain in the stomach. This work was aimed at determining the caffeine content of coffee beverages that are commercialized in Indonesia, covering coffee powders, coffee instants and brewed . Samples were collected from the market and coffee shops. The caffeine from the samples were extracted followed by fractionation using liquid-liquid extraction with ethyl acetate. Caffeine presence in the ethyl acetate fraction was then separated on TLC and the quantity analyzed using TLC scanner. Twenty coffee samples were analyzed and the caffeine contents were ranging from 13 mg to 159 mg per-serve. Caffeine in coffee instants were approximately 50 mg per-serve, while some products of coffee powders and brewed coffees higher than 100 mg per-serve. The level of caffeine in the coffee beverage distributed in Indonesia is still below the maximal recommended level in various countries. Keywords: caffeine contents, coffee products, Indonesian market Cite This Article: Sukrasno, Irena Aria Rivera, and Komar Ruslan Wirasutisna, “The Caffeine Content in Coffee Beverages Commercially Distributed in Indonesia.” Journal of Food and Nutrition Research, vol. 6, no. 8 (2018): 513-517. doi: 10.12691/jfnr-6-8-5.

caffeine also evaporates directly from a solid into vapor that can be recovered through condensation to form 1. Introduction caffeine crystals. There are at least two kinds of coffee beans, Drinking coffee has been a traditional habit of the arabica L. and Coffea robusta, now named as Coffea people throughout the world. Mostly, a coffee drink is canephora Pierre ex A. Froehner, the former contains less prepared by roasting coffee beans followed by grinding caffeine, but more expensive than the latter. The caffeine and the addition of hot water into a cup containing coffee content of C. arabica beans is approximately 1 %, while powder. There are some variations in serving coffee, with in C. robusta 2 % [4]. The conventional coffee brew is or without , with or without milk. Currently coffee usually prepared by adding two table spoons of ground can be served directly from the coffee powder, instant roasted beans with hot water. The intensity of coffees that have been mixed with other ingredients or roasting influences the caffeine content of the bean. The even using dried coffee extract, using coffee machine that level of roasting includes light, medium and heavy can serve different style of coffee drink such as , roasting with light roasting contain the highest caffeine , , and served using a special recipe in content [5]. coffee shops. A number of side effects may be encountered during Roasting coffee is designed to degrade chlorogenic acid drinking coffee such as insomnia, increase heart rate or that was claimed to induce pain in the gastric. Roasting atrial fibrillation, gastric pain, increase urinary frequency, temperature is ranging from 230°C to 250°C with roasting anxiety and increase blood pressure [2]. The side effect of time from 12 to 21 minutes. Light roasting eliminates coffee brew from certain brands was so obvious and approximately 50 % chlorogenic acid content while after consistent, but not shown by the other brands. One of the heavy roasting only traces amounts of chlorogenic possible side effects is the level of caffeine present in the acid can be detected [1]. Nowadays chlorogenic acid is coffee brew in addition to the individual sensitivity. becoming popular as a health food or food supplement, Indeed, certain individuals are very sensitive toward especially to prevent obesity [2]. Chlorogenic acid coffee brew or caffeine containing beverage or drug and demonstrated a number of biological activities such as do not drink coffee at all. Accordingly, we are interested hypoglycemia, anti hyperlipidemias, antibacterial activities, to evaluate the caffeine content in various coffee products antioxidant and anticarcinogenic activities [3]. During including conventional coffee powder, and roasting not only chlorogenic acid degraded but part of available in Indonesian market.

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2. Materials and Methods water each followed by filtration and the filtrates combined. Coffee brew was transferred to a 500 ml flask, Coffee samples consisted coffee powder, coffee instant adjust the volume with hot water and mixed well, and coffee brews that were bought from the market and subsequently 50 ml was taken as a sample for coffee shops. Caffeine present in the sample was extracted fractionation. The sample was transferred to a separatory and then analyzed by TLC scanner (Camag). funnel and extracted three times with 25 ml ethyl acetate respectively. Combined ethyl acetate fraction was evaporated to dryness and redissolved in 5 ml ethyl 2.1. Caffeine Standard Solution acetate for further analysis. Dilution was conducted if the Caffeine marker 25 mg was dissolved in 20 ml ethyl size of caffeine spot was too big compared to the acetate then transferred into 25 ml measuring flask and the calibration experiment data. volume adjusted to 25 ml. This solution contained 1,000 ppm caffeine. Into 5 ml measuring flask were transferred 2.7. TLC Densitometry 125, 250, 500, 750 and 1,000 µl caffeine solution and the final volume adjusted to 5 ml to make a caffeine solution Ten microlitre of sample was applied on TLC plate at 25, 50, 100, and 200 µg/ml respectively. coated with SiGel 60 GF254 using CAMAG linomat 5 and developed using 5 % acetic acid in ethyl acetate. The AUC was measured using CAMAG TLC Scanner 3 at 2.2. TLC of Caffeine 275 nm. Each sample was measured twice and the average TLC of caffeine was conducted on Silica plate calculated. 20x20 cm, thickness 100 µm, particle size 10-12µm (E Merck) with solvent system 5% acetic acid in ethyl acetate. 3. Results 3.1. Method Validation 2.3. Maximum Wavelength Measurement Calibration curve and equation was made by measuring Caffeine standard solution 10 µl was applied on TLC the density of caffeine spot after TLC of caffeine at plate using CAMAG linomat 5 and developed using the various concentrations. The TLC densitogram of caffeine developing solvent and the AUC measured at 200-400 nm. after being developed with 5 % acetic acid in ethyl The densitogram of the spot that give maximum AUC was acetate is as shown in Fig 1. The AUC of caffeine spot at then used as the wavelength for AUC measurement. various concentrations is as shown in Table 1. The calibration equation was Y= 40,629 X + 1,844.5, with 2 2.4. Calibration Curve R = 0.995. Acceptable value for regression coefficient is 0.995 [6]. The limit of detection (LOD) was calculated Each of known concentration caffeine was applied on based on the regression equation and it was found to be TLC plate using CAMAG linomat 5 and developed using 0.556 ppm, while the limit of quantification (LOQ) was the TLC solvent. Subsequently the AUC of caffeine peak 1.854 ppm. was measured at 275 nm. The regression curve was then prepared by using concentration and AUC data, the calibration equation and the regression coefficient calculated. Linearity, limit of detection and limit of quantification was calculated from the calibration curve experiment

2.5. Precision and Accuracy Test Caffeine solution was prepared by dissolving 10; 15 and 20 mg in 100 ml ethyl acetate that has been used to extract roasted corns. As much as 10µl sample was applied on TLC plate SiGel 60 GF254 using Camag Linomat 5, and developed using 5% acetic acid in ethyl acetate. The AUC of caffeine spots was measured using Camag TLC Scanner 3 at 275 nm. Measurement was conducted in three replicates. Precision was measured by Figure 1. Densitogram of caffeine marker calculating the variation coefficient, while accuracy by Table 1. AUC of caffeine spots at various concentrations calculating the recovery. The acceptable variation coefficient according to UNODC is ≤ 2% and for the No Caffeine (ppm) AUC recovery between 95 % - 105 % [6]. 1 25 2,607.9 2 50 4,176.8 2.6. Sample Preparation 3 100 5,979.0 One gram of coffee powder was mixed with 25 ml 4 150 7,776.8 water and boiled for 10 minutes, filtered through filter 5 200 10.012.6 paper, the residue was then reextracted twice with 10 ml

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The results of accuracy and precision tests were as coffee with the lowest caffeine content (13.04 mg) and shown in Table 2 and Table 3. The accuracy was ranging Good Day Cappuccino was the highest caffeine content from 96.2 to 100.4 % for samples between 100 to 200 (62.64 mg). Nescafe although the dosage is only 2 g, it ppm, while the precission that is indicated by the contained relatively high caffeine (50.2 mg), this product coefficient variation was ranging from 0.2 to 1.9 % may contain coffee extract only and may need sweetener, creamer or milk on serving. Table 2. Experiment for Accuracy Testing Actual Measured Average Recovery amount AUC quantity Recovery (%) (ppm) (ppm) (%) 10,080.7 202.7 101.4 200 9,815.9 196.2 98.1 100.4 10,099.9 203.2 101.6 7,694.6 144.0 96.9 150 7,675.4 143.5 95.7 96.2 7,755.6 145.5 97,0 5,854.8 98.7 98.7 100 5,869.0 99.1 99.1 99.3 5,908.0 100.0 100.0

Table 3. Precission testing Actual amount Measured Average Stdev CV (%) (ppm) quantity (ppm) 202.7 200 196.2 200.7 3.9 1.9 203.2 144.0 150 143.5 143.7 0.3 0,2 145.5 98.7 100 99.1 99.3 0.7 0.7 100.0

3.2. Densitogram of Caffeine in Coffee Extract The spot of caffeine was detected using UV at 275 nm and the densitorgam of the extract from coffee powder, instant coffee and coffee brews are as shown in Figure 2. Caffeine appeared as a single major spot and other spots only appeared in the origin and some minor spots near the front of the development. Coffee powder is mostly prepared by grinding roasted coffee beans and sold as bulk material and packed in a package with certain weight such as 100 g, 250 g or Figure 2. Densitogram of caffeine extracted from the coffee powder (a), 500 g. On serving, two tablespoons of coffee powder instant coffee (b) and brewed coffee (c). (approximately 12.84 g) are transferred to a glass or a cup then hot water is added. Some coffee lovers added sugar, Table 4. Caffeine in coffee powder creamer or milk when they drink coffee. Five samples No Brand Caffeine (mg) /serve were evaluated for their caffeine content, and mostly 1 Kapal api 42,12 contain relatively high caffeine. Kapal Api contained the least caffeine, 42.12 mg per-serve compared to other 2 Aroma Arabica 70,93 products. Two products, Aroma Robusta and Kopi bubuk 3 Excelso Arabika 78,74 senang contain very high caffeine content, 128.04 mg and 4 Kopi bubuk senang 123,39 123.39 mg per-serve respectively. Instant coffee is a that is presented as 5 Kopi Aroma Robusta 128,04 a package, usually in a sachet and use for once serving. For serving, usually a sachet of instant coffee is added Some coffee drinks are served in various styles such as with one glass or one cup of hot water. The main cappuccino, espresso, original, latte, etc. with special ingredient of instant coffee is mostly extract only or added branding. The size of glass for serving is also varied, such with other ingredients that make the product ready to be as, large, medium or small sizes. serves brewed consumed after the addition of hot water. Seven instant in four cup sizes short (8 FL Oz), tall (12 FL Oz), grande coffee products were analyzed in these experiments and (16 FL Oz) and venti (20 FL Oz) with caffeine the results were as shown in Table 2. Torabika was instant content and size 180 mg, 260 mg, 330 mg and 415 mg

Journal of Food and Nutrition Research 516 respectively. We were also interested in analyzing their Black, Kopi Aroma Arabica and Excelso Arabica high, caffeine contents. Among 8 brands tested, Good Day Starbucks Freshly Brewed Coffee, Calais America, Kopi White Vanilla contained the least caffeine content. Six Bubuk Senang and Kopi Aroma Robusta very high. out of eight brands contained relatively low caffeine The maximum allowable caffeine intake is 400 mg [7] content, less than 30 mg of caffeine for each serve. Three therefore, if someone drinks coffee twice a day, the total of them were relatively having high caffeine content, more consumption of caffeine is still less than 400 mg. than 90 mg per-serve. Calais America was coffee brew However, drinking more than three times a day need to with the highest caffeine content in the samples tested. It consider the total caffeine that will be taken every day and contained 159.23 mg caffeine for each cup. some of the side effect might be encountered. The highest caffeine content found in Indonesia for one Table 5. Instant Coffee time drink is still much lower compared to that of reported Weight/sachet Caffeine/sachet No Brand by McCusker et al [8]. One specialty of coffee brewed (g) (mg) contained caffeine up to 253 mg. The result of repeated 1 Torabika 25 13.04 assays on one specialty in 6 consecutive days varied, 2 ABC 31 18.78 ranging from 259.2 to 564.4 mg per-cup. Heckman [9] 3 Good Day Original 20 34.69 compiled that caffeine range for 8 Oz instant coffee 27-73 mg, plain and brewed coffee 102-200 mg while for 4 Luwak Original 20 40.13 espresso 240-720 mg. 5 Good Day Cappucino 20 62.64 It is very important for consummers to know the 6 Indocaffee 20 39.10 caffeine level in the coffeee beverage to anticipate the 7 Nescafe 2 50.2 physiological responses that may occur after drinking coffee, especially when taking new brand of coffee. In Table 6. Caffeine in commercially brewed coffee addition, the time and the number of cups of coffee should be considered to maintain the sleeping quality. Although No Code Caffeine (mg) /cup caffeine intake up to 400 mg per-day do not raise safety 1 Good Day White Vanilla 11.81 concerns, the unwanted physiological effects may arise, 2 Kopi ABC EXO 20.85 such as anxiety, insomnia, stomachache, atrial fibrilation, 5 J. Co Cappucino 20.96 increase blood pressure and urination. The maximum 3 Kopiko 78 C 27.22 amount of caffeine consumption has not been regulated 6 Starbucks Americano 29.21 yet. In general, the recommended maximal daily intake in a number of countries was 300 mg for pregnant woman 4 Nescafe Black 93.64 and 400-500 mg for adults [7,9]. 7 Starbucks Freshly Brewed Coffee 121.65 Cardiac arrhythmias are some times reported by 8 Calais Americano 159.23 individual drinking coffee, however this side effect only occur on certain individuals who are sensitive to caffeine. These individuals may susceptible to the small 4. Discussion electrophysiological changes induce by caffeine [11]. Coffee promotes gastro-oesophagal reflux, but is not Caffeine in coffee powder, coffee instant and coffee associated with dyspepsia and the activity is not caused by brew can be easily determined by using TLC densitometry. caffeine [12]. People with non-ulcer dyspepsia were more Extraction was performed by boiling in water similar to likely to experience dyspeptic symptoms after drinking that of brewing coffee. Caffeine has high solubility in hot coffee [13,14]. Caffeine intake will affect sleep quality, by water (66 g in 100 m1), low solubility in cold water (2 g reducing bed time and this effect is related to the caffeine in 100 ml) and soluble in ethyl acetate. Preliminary liquid- consumption. The profound effect was observed at dose liquid extraction using ethyl acetate is needed to fractionate approximately 200 mg while population who took caffeine caffeine from other compounds. Based on LOD and LOQ at 100 mg maintained their good sleep quality [15] analysis, caffeine can be detected at 0.556 ppm and quantified at 1.854 ppm. In this experiment the spot of caffeine was detected using UV light at 275 nm without 5. Conclusion visualization as that of performed by Palacios et al. [10]. Based on LOQ and LOD calculation and considering the Caffeine from caffee bean and coffee brews can be application volume was 5 µl, under this system caffeine quantified by simple and rapid methods using TLC can be detected at approximately 5 ng and quantified at Scanner with acceptable accuracy and precission. Caffeine 20 ng. content of coffee powder, coffee instant and coffee Caffeine content in coffee drink in Indonesia varied brewed commercially available in Indonesian market was from 11.81 mg/cup to 159.23 mg/cup. If caffeine content still below the allowable maximum level. is classified by considering less than 25 mg/cup is low, 25-50 mg/cup moderate, > 50-100 mg/cup high and above 100 mg/cup very high; Good Day White Vanilla, Kopi References ABC Exo, Torabika, ABC and Jco Cappucino were low; Kopiko 78 C, Starbuck Americano, Good Day Original, [1] Moon, J.K., Yoo, H.S., Shibamoto, T., “Role roasting conditions in the level of chlorogenic acid content in coffee beans: correlation Luwak White Coffee Original, Indocaffee and Kapal Api with coffee acidity”, J. Agric Food Chem, 57(12): 5365-5369, Jun moderate; Nescafe Black, Good Day Capuccino, Nescafe 2009.

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[2] McLellan,T.M., Cadwell, J.A, Lieberman, H.R., “A review of [9] Heckman, M.A., Well, J., De Mejia, E.G., “Caffeine caffeines’s effects on cognitive, physical and occupational (1,3,7-trimethylxanthine) in Foods: A Comprehensive Review on performance”, Neurosci Biobehav Rev, 71: 294-312, December Consumption, Functionality, Safety, and Regulatory Matters”, J 2016. Food Science, 75: R77-R87, April 2010. [3] Meng, S., Cao, J., Feng, Q., Peng, J., Hu, Y., “Roles of [10] Palacios, C., Salatini, M.L.F., Salatino, A, “TLC Procedure for chlorogenic acid on regulating glucose and lipid metabolism: a Determination of Approximate Contents of Caffeine in Food and review”, Evidence Based Complimentary and Alternative Beverages”, World Journal of Chemical Education, 5: 148-152, Medicine, 2013: 801457, Aug 2013 Aug 2017. [4] Belay, A., Ture, K., Redi, M., Asfaw, A., “Measurement of [11] Pelchovitz, D.J., Goldberger, J.J., “Caffeine and Cardiac caffeine in coffee beans with UV/Vis spectrometer”, Food Arrythmias: A Review of the Evidence”, The American Journal of Chemistry, 108(10): 310-315. May 2008. Medicine, 124: 284-289, Aug 2011. [5] Hecimovic, I., Cvitanovic, A.B., Horzic, D., Komes, D., [12] Boekema, P.J., Samson, M., van Berge Henegouwen, G.P., Smout, “Comparative study of and caffeine in different A.J., “Coffee and gastrointestinal function: facts and fiction. A coffee varieties affected by the degree of roasting”, Food review”, Scand J Gastroenterol Suppl., 230: 35-9. 1999. Chemistry, 129(3): 991-1000, Dec 2011. [13] Elta, G.H., Behler, E.M., Colturi, T.J., “Comparison of coffee [6] United Nation Office on Drugs and Crime, “Guidance for the intake and coffee-induced symptoms in patients duodenal ulcer, Validation of Analytical Methodology and Calibration of nonulcer dyspepsia and normal control”, Am J Gastroenterol, Equipment used for Testing of Illicit Drugs in Seized Materials 85(10): 1339-1342, Oct 1990. and Biological Specimens”, United Nations, New York, 2009, [14] Shimamoto, T., Yamamichi, N., Kodashima, S., Takahashi, Y., 15-38. Fujishiro, M., Oka, M., Mitsushima, T., Koike, K., “No [7] European Food Safety Authority, “Scientific opinion on the safety Association of Coffee Consumption with Gastric Ulcer, Duodenal of caffeine”, EFSA Journal, 13(5), 4102, May 2015. Ulcer, Reflux Esophagitis and Non-Erosive Reflux Disease: A [8] McCusker, R.R., Goldberger, B.A., Cone, E.J., “Caffeine content Cross-Sectional Study of 8,013 Healthy Subjects in Japan”, PLoS of soeciality coffees”, Journal of Analytical Toxicology, 27: ONE, 8(6): e65996, Jun 2013. 520-522, Oct 2003. [15] Watson, E.J., Coates, A.M., Kohler, M., Banks, S.; “Caffeine consumption and sleep quality in Australian adults”, Nutrients, 8: 479. Aug 2016.