Document Type : Original Article


Adama Science and Technology University, P.O. Box 1888, Adama, Ethiopia


Caffeine is a stimulant alkaloid found in aerial parts of many hot beverages, including coffee and tea. Due to its health impact, quantification of caffeine level in coffee is of paramount importance for consumers and traders, as well. Therefore, this study was designed to determine the caffeine content in coffee beverage prepared from coffee beans, pulp and leaves using high performance liquid chromatography coupled with a reverse phase C8 column and UV-detector. In this study, caffeine sample was extracted from coffee beans, pulp and leaves with boiled distilled water followed by solvent partition with chloroform. The extracted caffeine samples were analyzed alongside caffeine standard solutions over the concentration range of 5-25 μg/mL. For quantitative purposes, the standard caffeine gave an equation of Y=1270560x + 986903 (R2 = 0.998) and the retention time of 1.84 ± 0.01 min. In parallel to the standard caffeine solution, peak area of caffeine contents in 20 μL extracted caffeine samples of 2.5 g coffee powder in 100 mL of distilled water coffee beverage were registered. By using the peak area, caffeine concentration in injected sample and its concentration in the total sample solution was calculated. The percentage masses of caffeine (w/w%) in the original coffee samples were 1.30 ± 0.11% for beans, 0.90 ± 0.11% for pulp and 0.65 ± 0.10% for leaves. These results of caffeine contents in coffee beans in Wollega zone show high caffeine contents when compared with previously reported coffee beans (1.01 ± 0.04-1.19 ± 0.02%) of other parts of Ethiopia using UV/Vis. spectrophotometric technique.

Graphical Abstract

Determination of caffeine content in Wollega Zones, Ethiopian coffee bean, pulp and leaves by high performance liquid chromatography


Abera, B., 2014. Medicinal plants used in traditional medicine by Oromo people, Ghimbi District, Southwest Ethiopia. J. Ethnobiol Ethnomed. 10(1), 40.
Abbood, A., Aldiab, D., 2017. HPLC determination of caffeine in some beverages and pharmaceutical dosage forms available in Syrian market. J. Chem. Pharm. Sci. 10(3), 1174-1179.
Aibinu, I., Adenipekun, T., Adelowotan, T., Ogunsanya, T., Odugbemi, T., 2007. Evaluation of the antimicrobial properties of different parts of Citrus aurantifolia (lime fruit) as used locally. Afr. J. Tradit. Complement. Altern. Med. 4(2), 185.
Akhtar, S., 2013. Evaluation of cardiovascular effects of Citrus aurantifolia (Linn.) fruit. Available at SSRN:
Ali, M.M., Eisa, M., Taha, M.I., Zakaria, B.A., Elbashir, A.A., 2012. Determination of caffeine in some Sudanese beverages by high performance liquid chromatography. Pak. J. Nutr. 11(4), 336.
Alliance, C., Chan, S.C., 2013. Chemical Test for Caffeine. Hong Kong Chemistry Olympiad for Secondary Schools (2013-14), Memorial College.
Amare, M., Admassie, S., 2012. Polymer modified glassy carbon electrode for the electrochemical determination of caffeine in coffee. Talanta 93, 122-128.
American Public Health Association (APHA), 1999. Standard Methods for the Examination of Water and waste Water. Washington DC. 20th Ed.
Aresta, A., Palmisano, F., Zambonin, C.G., 2005. Simultaneous determination of caffeine, theobromine, theophylline, paraxanthine and nicotine in human milk by liquid chromatography with diode array UV detection. Food Chem. 93(1), 177-181.
Anonynous, 1985. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, American Water Works Association, Water Environment Federation, Washington.
Belay, A., Ture, K., Redi, M., Asfaw, A., 2008. Measurement of caffeine in coffee beans with UV/Vis spectrometer. Food Chem. 108(1), 310-315.
 Balemie, K., Kelbessa, E., Asfaw, Z., 2004. Indigenous medicinal plant utilization, management and threats in Fentalle area, Eastern Shewa, Ethiopia. E. J. Biol. Sci. 3(1), 37-58.
Belitz, H.-D., Grosch, W., Schieberle, P., 2009. Coffee, tea, cocoa. Food Chem. 938-970.
Beyene, B., Tolosa, T., Rufael, T., Hailu, B., Teklue, T., 2015. Foot and mouth disease in selected districts of western Ethiopia: seroprevalence and associated risk factors. Rev. Sci. Tech. Off. Int. Epiz 34(3), 2.
Bote, A.D., Struik, P.C., 2011. Effects of shade on growth, production and quality of coffee (Coffea arabica) in Ethiopia. J. Hortic. For. 3(11), 336-341.
Carrillo, J.A., Benitez, J., 2000. Clinically significant pharmacokinetic interactions between dietary caffeine and medications. Clin. Pharmacokinet. 39(2), 127-153.
Castro, J., Pregibon, T., Chumanov, K., Marcus, R.K., 2010. Determination of catechins and caffeine in proposed green tea standard reference materials by liquid chromatography-particle beam/electron ionization mass spectrometry (LC-PB/EIMS). Talanta 82(5), 1687-1695.
Chen, Q.-c., Mou, S.-f., Hou, X.-p., Ni, Z.-m., 1998. Simultaneous determination of caffeine, theobromine and theophylline in foods and pharmaceutical preparations by using ion chromatography. Anal. Chim. Acta 371(2-3), 287-296.
Chou, K.H., Bell, L., 2007. Caffeine content of prepackaged national‐brand and private‐label carbonated beverages. J. Food Sci. 72(6), C337-C342.
Clifford, M., Ramirez-Martinez, J., 1991. Tannins in wet-processed coffee beans and coffee pulp. Food Chem. 40(2), 191-200.
Demissie, E.G., Woyessa, G.W., Abebe, A., 2016. UV/Vis spectrometer determination of caffeine in green coffee beans from Hararghe, Ethiopia, using beer-lambert's law and integrated absorption coefficient techniques. Sci. Study Res. Chem. Chem. Eng. Biotechnol. Food Ind. 17(2), 109.
Feyissa, T.F., Hailemariam, T.B., Regassa, T., Kergano, N.K., 2017. Ethnobotanical study of ethnoveterinary plants in Kelem Wollega Zone, Oromia Region, Ethiopia. J. Med. Plants Res. 11(16), 307-317.
Fujioka, K., Shibamoto, T., 2008. Chlorogenic acid and caffeine contents in various commercial brewed coffees. Food Chem. 106(1), 217-221.
Grujić-Letić, N., Rakić, B., Šefer, E., Milanović, M., Nikšić, M., Vujić, I., Milić, N., 2016. Quantitative determination of caffeine in different matrices. Maced. Pharm. Bull. 62(1) 77-84.
Khare, C., 2007. Indian Medicinal Plants: An Illustrated Dictionary Springer-Verlag. Berlin pg, 699-700.
Gebeyehu, B.T., Bikila, S.L., 2015. Determination of caffeine content and antioxidant activity of coffee. Am. J. Appl. Chem. 3, 69-76.
Gole, T.W., 2003. Vegetation of the Yayu forest in SW Ethiopia: impacts of human use and implications for in situ conservation of wild Coffea arabica L. populations. Cuvillier.
Gowrisankar, D., Abbulu, K., Bala Souri, O., Sujana, K., 2010. Validation and calibration of analytical instruments. J. Biomed. Sci. Res. 2(2), 89-99.
Illy, E., 2013. The complexity of coffee. Sci. Am. 86-91.
Islam, M., Rahman, M., Abedin, M., 2002. Isolation of caffeine from commercially available available tea and tea waste. Jahangirnagar Uni. J. Sci. 25, 9.
Juliano, L.M., Griffiths, R.R., 2004. A critical review of caffeine withdrawal: empirical validation of symptoms and signs, incidence, severity and associated features. Psychopharmacology 176(1), 1-29.
Komes, D., Horzic, D., Belscak, A., Kovacevic Ganic, K., Bljak, A., 2009. Determination of caffeine content in tea and maté tea by using different methods. Czech J. Food Sci. 27, 213-216.
Martın, M.J., Pablos, F., González, A., 1998. Characterization of green coffee varieties according to their metal content. Anal. Chim. Acta. 358(2), 177-183.
Minamisawa, M., Yoshida, S., Takai, N., 2004. Determination of biologically active substances in roasted coffees using a diode-array HPLC system. Anal. Sci. 20(2), 325-328.
Mohammadhosseini, M., Sarker, S.D., Akbarzadeh, A., 2017. Chemical composition of the essential oils and extracts of Achillea species and their biological activities: A review. J. Ethnopharmacol. 199, 257-315.
Ng, T., Liu, F., Wang, Z., 2000. Antioxidative activity of natural products from plants. Life Sci. 66(8), 709-723.
Norton, T.R., Lazev, A.B., Sullivan, M.J., 2011. The “buzz” on caffeine: Patterns of caffeine use in a convenience sample of college students. J. Caffeine Res. 1(1), 35-40.
Oestreich-Janzen, S., 2013. Chemistry of coffee. CAFEA GmbH, Hamburg, Germany.
Perrone, D., Donangelo, C.M., Farah, A., 2008. Fast simultaneous analysis of caffeine, trigonelline, nicotinic acid and sucrose in coffee by liquid chromatography-mass spectrometry. Food Chem. 110(4), 1030-1035.
Ranheim, T., Halvorsen, B., 2005. Coffee consumption and human health-beneficial or detrimental?-Mechanisms for effects of coffee consumption on different risk factors for cardiovascular disease and type 2 diabetes mellitus. Mol. Nutr. Food Res. 49(3), 274-284.
Reuter, J., Merfort, I., Schempp, C.M., 2010. Botanicals in dermatology. Am. J. Clin. Dermato. 11(4), 247-267.
Ribeiro, A., Estanqueiro, M., Oliveira, M., Sousa Lobo, J., 2015. Main benefits and applicability of plant extracts in skin care products. Cosmetics 2(2), 48-65.
Sarker, S.D., Nahar, L., 2018. Phytochemicals and phyto-extracts in cosmetics. Trends Phytochem. Res. 2(4) 185-186.
Schenker, S., Heinemann, C., Huber, M., Pompizzi, R., Perren, R., Escher, R., 2002. Impact of roasting conditions on the formation of aroma compounds in coffee beans. J. Food Sci. 67(1), 60-66.
Shaker, S.A., Farina, Y., Mahmmod, S., 2010. Synthesis and characterization of mixed ligand complexes of caffeine, adenine and thiocyanate with some transition metal ions. Sains. Malays. 39(6), 957-962.
Tamene, B., 2000. A floristic analysis and ethnobotanical study of the semi-wetland of Cheffa area South Wello. Unpublished MSc. Thesis, Addis Ababa University, Addis Ababa, Ethiopia.
Wansi, J.D., Sewald, N., Nahar, L., Martin, C., Sarker, S.D., 2018. Bioactive essential oils from the Cameroonian rain forest: A review - Part I. Trends Phytochem. Res. 2(4), 187-234.
Wansi, J.D., Sewald, N., Nahar, L., Martin, C., Sarker, S.D., 2019. Bioactive essential oils from the Cameroonian rain forest: A review - Part II. Trends Phytochem. Res. 3(1), 3-52.
Weldegebreal, B., Redi-Abshiro, M., Chandravanshi, B.S., 2017. Development of new analytical methods for the determination of caffeine content in aqueous solution of green coffee beans. Chem. Cent. J. 11(1), 126.
Wondimkun, Z.T., Jebessa, A.G., Molloro, L.H., Haile, T., 2016. The determination of caffeine level of wolaita zone, Ethiopia coffee using UV-visible spectrophotometer. Am. J. Appl. Chem. 4(2), 59-63.
Yigzaw, D., Labuschagne, M., Osthoff, G., Herselman, L., 2007. Variation for green bean caffeine, chlorogenic acids, sucrose and trigonelline contents among Ethiopian Arabica coffee accessions. SINET: Ethiop. J. Sci. 30, 77-82.