int. j. sec. metabolite International Journal of Secondary Metabolite 2148-6905 İzzet KARA 10.21448/ijsm.1682941 Natural Products and Bioactive Compounds Doğal Ürünler ve Biyoaktif Bileşikler Extraction and determination of caffeine content in ground coffee commercially available on Bosnian market Extraction and determination of caffeine content in ground coffee commercially available on Bosnian market https://orcid.org/0000-0001-6093-7127 Dzudzevic-cancar Hurija University of Sarajevo-Faculty of Pharmacy https://orcid.org/0000-0003-1920-7645 Dedić Alema University of Sarajevo-Faculty of Pharmacy https://orcid.org/0000-0001-8447-936X Alispahic Amra University of Sarajevo-Faculty of Pharmacy https://orcid.org/0000-0002-7432-9349 Boškailo Emina Faculty of Social Sciences Dr. Milenko Brkić, Herzegovina University https://orcid.org/0000-0003-4936-1846 Yavuz Musa ISPARTA UYGULAMALI BİLİMLER ÜNİVERSİTESİ, ZİRAAT FAKÜLTESİ 03 09 2026 Advanced Online Publication 04 25 2025 11 22 2025 Copyright © 2014, International Journal of Secondary Metabolite 2014 International Journal of Secondary Metabolite

Caffeine (1,3,7-trimethylxanthine) is an alkaloid that acts as a stimulant on the central nervous system. Caffeine consumption brings a momentary increase in awareness and alertness in humans, as well as in experimental animals. The refluxing method and the regular coffee preparation method were used to extract caffeine from several coffee varieties that were sold in Bosnia. TLC and melting point analysis were performed before and after purification using vacuum sublimation. Chemical characterization of samples by FTIR (Fourier Transform Infrared Spectroscopy) analysis was performed by comparing with caffeine standard spectra. The percentage of extracted impure caffeine was higher in coffee samples obtained by the ordinary brewing method compared to reflux. After purification of crude extracts, all refluxed samples yielded more caffeine than those prepared by conventional brewing. In developed TLC chromatograms, Rf values of samples and standard match the literary Rf values of caffeine. Unprocessed caffeine samples showed a melting point from 185 to 216°C, corresponding to the range of 180 to 220°C, the melting point of crude caffeine. After vacuum sublimation, the melting point of all samples was between 235 and 237 °C, which corresponds to the literary values (235-239 °C for the “Merck” caffeine standard). Comparing the FTIR spectra of samples and standard, characteristic caffeine signals were observed confirming successful caffeine isolation. The caffeine content in the analyzed coffee samples from the Bosnia and Herzegovina market complies with FDA (Food and Drug Administration) regulations on the daily amount of caffeine intake per cup of coffee.

Caffeine (1,3,7-trimethylxanthine) is an alkaloid that acts as a stimulant on the central nervous system. Caffeine consumption brings a momentary increase in awareness and alertness in humans, as well as in experimental animals. The refluxing method and the regular coffee preparation method were used to extract caffeine from several coffee varieties that were sold in Bosnia. TLC and melting point analysis were performed before and after purification using vacuum sublimation. Chemical characterization of samples by FTIR (Fourier Transform Infrared Spectroscopy) analysis was performed by comparing with caffeine standard spectra. The percentage of extracted impure caffeine was higher in coffee samples obtained by the ordinary brewing method compared to reflux. After purification of crude extracts, all refluxed samples yielded more caffeine than those prepared by conventional brewing. In developed TLC chromatograms, Rf values of samples and standard match the literary Rf values of caffeine. Unprocessed caffeine samples showed a melting point from 185 to 216°C, corresponding to the range of 180 to 220°C, the melting point of crude caffeine. After vacuum sublimation, the melting point of all samples was between 235 and 237 °C, which corresponds to the literary values (235-239 °C for the “Merck” caffeine standard). Comparing the FTIR spectra of samples and standard, characteristic caffeine signals were observed confirming successful caffeine isolation. The caffeine content in the analyzed coffee samples from the Bosnia and Herzegovina market complies with FDA (Food and Drug Administration) regulations on the daily amount of caffeine intake per cup of coffee.

 Coffee Caffeine Extraction TLC FTIR Coffee Caffeine Extraction TLC FTIR Agafonova, E.V., Moshchenskii, Y.V., & Tkachenko, M.L. (2012). Determining the main thermodynamic parameters of caffeine melting by means of DSC. Russian Journal of Physical Chemistry A, 86, 1035-1037. https://doi.org/10.1134/S0036024412060027 Amirkia, V., & Heinrich, M. (2014). Alkaloids as drug leads – A predictive structural and biodiversity-based analysis. Phytochemistry Letters, 10, 48-53. https://doi.org/10.1016/j.phytol.2014.06.015 Barone, J.J., & Roberts, H. (1984). Human Consumption of Caffeine. In: Dews, P.B. (eds) Caffeine. Springer, 453 Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-69823-1_4 Cappelletti, S., Piacentino, D., Sani, G., & Aromatario, M. (2015). Caffeine: cognitive and physical performance enhancer or psychoactive drug? Current Neuropharmacology, 13(1), 71 88. https://doi.org/10.2174/1570159X13666141210215655 Dabrowska-Molenda, M., Szwedzik, K. & Zabłudowska, Z. (2019). Analysis of caffeine content in selected types of coffee. Post. Tech. Przetw. Spoz., 2, 68–71. de Paula, J., & Farah, A. (2019). Caffeine Consumption through Coffee: Content in the Beverage, Metabolism, Health Benefits and Risks. Beverages, 5(2), 37. https://doi.org/10.3390/beverages5020037 Derry, C.J., Derry, S. &Moore, R.A. (2014) Caffeine as an analgesic adjuvant for acute pain in adults. Cochrane Database of Systematic Reviews, 12, CD009281. https://doi.org/10.1002/14651858.CD009281.pub3 Džudžević-Čančar, H., & Salihović, M. (2016). Praktikum iz Organske hemije I, za studente farmaceutskog fakulteta. Interna skripta, Farmaceutski fakultet u Sarajevu. Ejuh, G.W., Ndjaka, J.M.B., Tchangnwa, N.F., Ndukum, P.L., Fonkem, C., Tadjouteu Assatse, Y., & Yossa Kamsi, R.A. (2020). Determination of the structural, electronic, optoelectronic and thermodynamic properties of the methylxanthine molecules theophylline and theobromine. Optical and Quantum Electronics, 52, 498. https://doi.org/10.1007/s11082-020-02617-w European Food Safety Authority-EFSA. (2015) Panel on Dietetic Products, Nutrition and Allergies. Scientific opinion on the safety of caffeine. EFSA J., 13, 4102. https://doi.org/10.2903/j.efsa.2015.4102 Farah, A., & Ferreira dos Santos, T. (2015). Chapter 1 - The Coffee Plant and Beans: An Introduction. Editor(s): Preedy, V.R, Coffee in Health and Disease Prevention. Academic Press. Fisone, G., Borgkvist, A., & Usiello, A. (2004). Caffeine as a psychomotor stimulant: mechanism of action. Cellular and Molecular Life Sciences, 61(7-8), 857-872. https://doi.org/10.1007/s00018-003-3269-3 Furushima, Y., Schick, C., & Toda, A. (2018). Crystallization, recrystallization, and melting of polymer crystals on heating and cooling examined with fast scanning calorimetry. Polymer Crystallization, 1(1-2), e10005. https://doi.org/10.1002/pcr2.10005 Jarosz, M., Wierzejska, R. & Siuba, M. (2012). Maternal caffeine intake and its effect on pregnancy outcomes. European Journal of Obstetrics and Gynecology and Reproductive Biology, 160(2), 156 160. https://doi.org/10.1016/j.ejogrb.2011.11.021 Kole, J., & Barnhill, A. (2013). Caffeine Content Labeling: A Missed Opportunity for Promoting Personal and Public Health. Journal of Caffeine Research, 3(3), 108 113. https://doi.org/10.1089/jcr.2013.0017 Kovačević, N. (2004). Osnovi farmakognozije. Srpska školska knjiga, Beograd, 113-116. Ludwig, I.A., Clifford, M.N., Lean, M.E.J., Ashihara, H., & Crozier, A. (2014). Coffee: biochemistry and potential impact on health. Food & Function, 5(8), 1695 1717. https://doi.org/10.1039/c4fo00042k Messina, G., Zannella, C., Monda, V., Dato, A., Liccardo, D., De Blasio, S., Valenzano, A., Moscatelli, F., Messina, A. & Cibelli, G. (2015). The beneficial effects of coffee in human nutrition. Biology and Medicine, 7(4), 1-5. https://doi.org/10.4172/0974-8369.1000240 Nawrot, P., Jordan, S., Eastwood, J., Rotstein, J., Hugenholtz, A., & Feeley, M. (2003). Effects of caffeine on human health. Food Additives & Contaminants, 20(1), 1 30. https://doi.org/10.1080/0265203021000007840 Nikolić, T. (2010). Izolacija alkaloida iz prirodnih spojeva [Master thesis, Josip Juraj Strossmayer University of Osijek]. https://urn.nsk.hr/urn:nbn:hr:182:150250 Oestreich-Janzen, S. (2016). Caffeine: Characterization and properties. In B. Caballero, P. M. Finglas, & F. Toldrá (Eds.), Encyclopedia of food and health (pp. 556 572). Elsevier. https://doi.org/10.1016/B978-0-12-384947-2.00098-2 Ogawa, N., & Ueki, H. (2007). Clinical importance of caffeine dependence and abuse. Psychiatry Clinical Neurosciences, 61(3), 263-268. https://doi.org/10.1111/j.1440-1819.2007.01652.x Pradeep, S., Rameshaiah, G.N., & Ashoka, H. (2015). Caffeine extraction and characterization. Int. J. Curr. Res. Rev., 7(9), 16-19. https://ijcrr.com/abstract.php?article_id=555 Prance, G., & Nesbitt, M. (2005). The Cultural History of Plants. Routledge. 176 177, https://doi.org/10.4324/9780203020906 Reyes, C.M. & Cornelis, M.C. (2018). Caffeine in the diet: Country-level consumption and guidelines. Nutrients, 10, 1772. https://doi.org/10.3390/nu10111772 Rosenfeld, L.S., Mihalov, J.J., Carlson, S.J., & Mattia, A. (2014). Regulatory status of caffeine in the United States. Nutrition Reviews, 72, 23-33. https://doi.org/10.1111/nure.12136 Roy, A. (2017). A review on the alkaloids an important therapeutic compound from plants. Int. J. Plant Biotechnol., 3(2), 1-9. https://doi.org/10.37628/ijpb.v3i2.199 Saimaiti, A., Zhou, D.D., Li, J., Xiong, R.G., Gan, R.Y., Huang, S.Y., Shang, A., Zhao, C.N., Li, H.Y. & Li, H.B. (2023). Dietary sources, health benefits, and risks of caffeine. Critical Reviews in Food Science and Nutrition, 63, 9648–9666. https://doi.org/10.1080/10408398.2022.2074362 Sanda, B., Ganea, M., Moisa, C., & Caraban, A. (2015). Method for quantitative determination caffeine from coffee. Ecotox. Zooteh. Ind. Alim., 14(B), 39 44. https://api.semanticscholar.org/CorpusID:40680512 Sherma, J. (2006). Thin-Layer Chromatography. Encyclopedia of Analytical Chemistry. John Wiley & Sons. https://doi.org/10.1002/9780470027318.a5918 Sivrikaya, S. (2020). A deep eutectic solvent based liquid phase microextraction for the determination of caffeine in Turkish coffee samples by HPLC-UV. Food Additives & Contaminants: Part A, 37(3), 488-495. https://doi.org/10.1080/19440049.2020.1711972 Tavares, C., & Sakata, R.K. (2012). Caffeine in the treatment of pain. Brazilian Journal of Anesthesiology, 62(3), 387-401. https://doi.org/10.1016/S0034-7094(12)70139-3 Tucakov, J. (1964) Farmakognozija. Zavod za izdavanje udžbenika Socijalističke Republike Srbije. Beograd, 92-94. Wierzejska, R.E. & Gielecinska, I. (2024). Evaluation of the Caffeine Content in Servings of Popular Coffees in Terms of Its Safe Intake-CanWe Drink 3-5 Cups of Coffee per Day, as Experts Advise? Nutrients, 16(15), 2385. https://doi.org/10.3390/nu16152385 Yu, N.Y., Bieder, A., Raman, A., Mileti, E., Katayama, S., Einarsdottir, E., Fredholm, B.B., Falk, A., Tapia-Páez, I., Daub, C.O., & Kere, J. (2017). Acute doses of caffeine shift nervous system cell expression profiles toward promotion of neuronal projection growth. Scientific Reports, 7, 11458. https://doi.org/10.1038/s41598-017-11574-6 Ziegler, J., & Facchini, P.J. (2008). Alkaloid biosynthesis: metabolism and trafficking. Annual Review of Plant Biology, 59, 735-769. https://doi.org/10.1146/annurev.arplant.59.032607.092730