HPLC-Based Analysis of Caffeine in Different Matrices: An Appraisal of the Literature Published in 2025

Satyajit D. Sarker; Lutfun Nahar

doi:10.53941/npa.2026.100001

Abstract

Caffeine, also known as methyltheobromine, is a methylxanthine alkaloid, found in cocoa, coffee, tea, and a few other plants, e.g., Cola acuminata Schott & Endl., Ilex guayusa Loes, I. paraguariensis A. St.-Hil., I. vomitoria Sol. ex Aiton, Paullinia cupana Kunth. and P. yoco R. E. Schult. & Killip. Caffeine is widely used in various energy drinks, cosmetics, and pharmaceutical products. High-performance liquid chromatography (HPLC), inclusive of ultra-performance liquid chromatography (UPLC) or ultra-high-performance liquid chromatography (UHPLC), is the most widely used technique for caffeine analysis. This review article critically appraises the literature published in 2025 on caffeine analysis from different matrices using HPLC, revealing the popularity and usefulness of this chromatographic technique in the analysis and quantification of caffeine in different matrices. While an increase in the use of UPLC (or UHPLC) was observed, conventional HPLC using standard 5 μm columns of 150 or 250 mm length remained the instrument of choice because of its lower price than a UPLC (or UHPLC) system. Only a handful of studies reported in 2025 used an MS detector, but many of the studies used a UV or diode array detector (DAD, also known as photodiode array detector, PDA). Most of the studies published last year involved caffeine analysis in various cocoa, coffee, and tea samples.

References

1.
Kupka, T.; Makieieva, N.; Jewginski, M.; et al. Caffeine—Legal natural stimulant with open research perspective: Spectroscopic and theoretical characterisation. Molecules 2024, 29, 4382.
2.
Kim, Y.-S.; Choi, Y.-E.; Sano, H. Plant vaccination: Stimulation of defense system by caffeine production in planta. Plant Signal Behav. 2010, 5, 489–493.
3.
Waldvogel, S.R. Caffeine—A drug with a surprise. Angew. Chem. 2003, 42, 604–605.
4.
Reddy, V.S.; Shiva, S.; Manikantan, S.; et al. Pharmacology of caffeine and its effects on the human body. Eur. J. Med. Chem. Rep. 2024, 10, 100138.
5.
Sarker, S.D.; Nahar, L. Natural Products Isolation, 3rd ed.; Humana Press-Springer-Verlag: Totowa, NJ, USA, 2012.
6.
Nahar, L.; Sarker, S.D. Advances in applications of high-performance liquid chromatography in the analysis of herbal products. In Evidence-Based Validation of Herbal Medicine: Translational Research on Botanicals; Mukherjee, P., Ed.; Elsevier: Cambridge, MA, USA, 2022; pp. 431–461.
7.
Bragagnolo, F.S.; Strieder, M.M.; Sanches, V.L.; et al. An updated gradient PLE-SPE×HPLC-PDA system for the extraction, concentration, fractionation, and analysis of valuable compounds from cocoa bean shells. Food Chem. 2025, 494, 146198.
8.
Nascimento, L.L.; dos Santos, P.N.A.; Granja, H.S.; et al. Mixture design and Doehlert matrix for optimization of energized dispersive guided extraction (EDGE) of theobromine and caffeine from cocoa bean shells. Foods 2025, 14, 740.
9.
Mercogliano, F.; Bani, C.; Tretola, M.; et al. Preliminary assessment of alkaloid content in cocoa (Theobroma cacao L.) hulls for safe consumption as a feed ingredient. Toxins 2025, 17, 441.
10.
Rodriguez-Gonzalez, B.E.; Amador-Espejo, G.G.; Penagos-Velez, L.; et al. Chemical composition and volatile profile of cocoa liquors from Colombian Criollo cacao. J. Food Sci. 2025, 90, e70143.
11.
Muriqi, S.; Cervenka, L.; Ceslova, L.; et al. Physicochemical, antioxidant and mineral composition of Cascara beverage prepared by cold brewing. Food Technol. Biotechnol. 2025, 63, 46–56.
12.
Duke, B.W.; Mokonon, M.A.; Mekonin, N.D.; et al. Effects of roasting degree and grinding size on caffeine content and sensorial quality of coffee. J. Food Qual. 2025, 2025, 2405668.
13.
Al-Zoubi, S.M.; Awwad, S.H.; Issa, R.A.; et al. Quantification of bioactive compounds in Coffea arabica and their impact with omega-3 supplementation on non-high-density lipoprotein levels in hyperlipidemia-induced rats. Baghdad Sci. J. 2025, 22, 5022.
14.
Sijko-Szpanska, M.; Mystkowska, I.; Smitrowicz, A. Influence of brewing methods on the bioactive and mineral composition of coffee beverages. Molecules 2025, 30, 4080.
15.
DePaula, J.; Partelli, F.L.; Batista, A.M.; et al. Major bioactive compounds in seeds, husks, and leaves of selected genotypes of Coffea canephora cv. Conilon from three consecutive crops. Plants 2025, 14, 1040.
16.
DePaula, J.; Cunha, S.G.; Partelli, F.L.; et al. Major bioactive compounds, volatile and sensory profiles of Coffea canephora flowers and infusions for waste management in coffee production. Foods 2025, 14, 911.
17.
Park, J.B.; Peters, R. Compositional analysis of coffee containing javamide I/II and investigation of health effects in rats fed a high fat diet. Sci. Rep. 2025, 15, 28806.
18.
Palacios-Ruiz, V.; Suarez-Quiroz, M.; Figueroa-Hernandez, C.; et al. Influence of the use of yeast in the fermentation of coffee pulp on its aromatic and sensory quality. Biotecnia 2025, 27, e2244.
19.
Rendon-Mera, A.M.; Vega-Oliveros, C.; Ordonez-Lozano, S.; et al. Multivariate analysis of FT-IR, bioactive compounds, and antioxidant activity in coffee pulp (Coffea arabica var. Catimor): Combined effects of ripening stage and drying method. Food Chem. X 2025, 29, 102828.
20.
Soontontaweesub, S.; Lertsuwan, R.; Pruksatrakul, T.; et al. Multisolvent metabolite profiling of coffee waste by UHPLC-HRMS/MS and molecular networking. Comput. Struc. Biotechnol. J. 2025, 27, 5116–5128.
21.
Hajicek, J.; Hoca, G.; Varady, M.; et al. Comparative analysis of chemical composition and antioxidant activity in conventional, civet, and elephant coffees: Is there a definitive authentication marker of elephant coffee? Beverages 2025, 11, 79.
22.
Abou El-Ezz, R.F.; Meselhy, K.; Emara, S.; et al. A new segmented gradient hplc-dad and spectra index plots for measuring caffeine and chlorogenic acid in dark and green coffees. ACS Omega 2025, 10, 50896–50903.
23.
da Silva, W.B.; Rocha, L.M.; Moura, L.D.G.; et al. Optimization of methodology for simultaneous quantification of trigonelline, 5-caffeoylquinic acid, and caffeine in green and roasted coffee extracts by HPLC. ACS Omega 2025, 10, 40304–40312.
24.
Rusinek, R.; Dobrzanski, B., Jr.; Gawrysiak-Witilska, M.; et al. Effect of climate, growing region, country of origin, and post-harvest processing on the content of chlorogenic acids (CGAs) and aromatic compounds in roasted coffee beans. Sci. Rep. 2025, 15, 30117.
25.
Pompei, F.; Montillo, G.; Lepore, E.; et al. More than water: Water-based binary deep eutectic solvents (aquoDESs) for the effective ultrasound-assisted extraction of bioactive compounds from spent coffee grounds. Food Bioprocess. Technol. 2025, 19, 72.
26.
Fernandes, F.; Freitas, M.; Pinho, C.; et al. Upcycling coffee waste into sustainable nano zerovalent iron for environmental contaminant remediation: Characterization, applicability and cytotoxicity. Nanomaterials 2025, 15, 1788.
27.
Orpong, P.; Sriwiang, W.; Chookaew, S.; et al. Comprehensive analysis of elemental profiles, caffeine and chlorogenic acid contents, antioxidant activities, and isotope ratios in Thai single-origin green coffee beans. Meas. Food 2025, 20, 100265.
28.
He, W.; Chen, X.M.; Sun, J.; et al. Unveiling key aroma compounds formation during the roasting process of Yunnan Arabica coffee using molecular sensory science approaches. Food Chem. 2025, 494, 145853.
29.
Konwar, T.; Baruah, I.; Chaudhary, M.K.; et al. Evaluation of genetic diversity of tea [Camellia sinensis (L.) Kuntze] germplasm of Assam based on characterization of flavour-related metabolites. Genet. Resour. Crop Evol. 2025, 72, 7061–7077.
30.
Muthulingam, P.; Popovich, D.G.; Punyasiri, P.A.N.; et al. Development of a validated efficient HPLC-DAD analysis for assessing polyphenol transformation during black tea processing. J. Food Compos. Anal. 2025, 148, 108330.
31.
Yang, H.Y.; Li, Y.K.; Xie, T.; et al. Red-light withering increases the aroma content and diversity, and blue-light withering improves the fresh sweetness of black tea. J. Food Sci. Technol. 2025. https://doi.org/10.1007/s13197-025-06408-7.
32.
Lin, X.Y.; Huang, W.; Qiu, Z.H.; et al. Impact of fresh leaf elements on flavor components and aroma quality in ancient Dancong tea gardens across varying altitudes. Plants 2025, 14, 1339.
33.
Yu, J.; Wu, Y.J.; Zeng, C.X.; et al. Phytochemicals from fractioned dark tea water extract enhance the digestive enzyme inhibition, antioxidant capacities and glucose-lipid balance. Food Res. Int. 2025, 204, 115957.
34.
Chen, J.; Li, Z.Q.; Zhang, S.Y.; et al. The key quality components in Fenggang green tea with different zinc content and their correlations with zinc. Food Res. Int. 2025, 208, 116195.
35.
Kirci, D.; Zengin, G.; Karadag, A.E.; et al. A multi-analytical exploration of flavored black teas: Profiling volatile compounds, caffeine content, and biological effects. Food Sci. Nutri. 2025, 13, e70585.
36.
Parvez, S.; Wani, I.A. Comparative study of composition and antioxidant properties of green tea extracts from different commercial grades. ACS Food Sci. Technol. 2025, 5, 1678–1686.
37.
Govindan, D.; Kumar, S.S.; Ananda, D.H.; et al. Nutritional, bioactive potentials and antioxidant activities of processed green tea residue extracts against Hep-G2 and MCF-7 cell lines. J. Food Sci. Technol. 2025. https://doi.org/10.1007/s13197-025-06382-0.
38.
Zan, J.Z.; Chen, W.X.; Yuan, H.B.; et al. Evaluation of key taste components in Huangjin green tea based on electronic tongue technology. Food Res. Int. 2025, 201, 115569.
39.
Wu, Z.B.; Liao, W.W.; Zhao, H.B.; et al. Differences in the quality components of Wuyi rock tea and Huizhou rock tea. Foods 2025, 14, 4.
40.
Cai, M.; Peng, Z.; Xu, P.; et al. Comprehensive analysis of the flavor and color characteristics of light-fermented sour tea mediated by Aspergillus niger RAF106. Food Chem. 2025, 481, 143866.
41.
Shan, X.J.; Niu, L.C.; Zhang, Q.T.; et al. Quantitative non-volatile sensometabolome of Longjing tea and discrimination of taste quality by sensory analysis, large-scale quantitative metabolomics and machine learning. Food Chem. 2025, 485, 144496.
42.
Li, L.; Zhao, Y.; Li, Y.; et al. Effects of storage time on the quality and microbial community of ripe Pu-erh tea. Food Chem. X 2026, 33, 103389.
43.
Kalisz, O.; Catani, M.; Bocian, S. Greener and whiter analytical procedure for theobromine and caffeine determination in tea using dimethyl carbonate as an extraction solvent and mobile phase constituent in reversed-phase liquid chromatography. ACS Omega 2025, 10, 12432–12440.
44.
Ayakdas, G.; Agagunduz, D. Determination of L-theanine and caffeine contents in tea infusions with different fermentation degrees and brewing conditions using the chromatographic method. Foods 2025, 14, 2313.
45.
Chen, T.L.; Wu, H.R.; Luo, J.W.; et al. Phytochemical characterization of low-caffeine and high-theobromine wild tea Camellia yungkiangensis var. yuanbaoshanica reveal its special compositions. Food Chem. 2025, 496, 146685.
46.
Abdullah, A.F.; Piro, S.J.; Ali, N.H.; et al. Cucurbita pepo seed extract and green-synthesized silver nanoparticles: Antioxidant, phytochemical, and antibacterial properties. J. Mol. Eng. Mater. 2025, 2025, 2550027.
47.
Fernandez, M.A.; Ochoa-Ocampo, M.; Garzon, Y.; et al. Exploring variability in the methylxanthine content within Ilex guayusa Loes: Impact of soil conditions, age, and sunlight exposure. ACS Agric. Sci. Technol. 2025, 5, 1034–1046.
48.
Lener, T.; Sticha, M.; Bauerova, M.; et al. Historical analgesic tablets revisited: Development of a versatile RP-HPLC method for the stability study of single- and multi-component pharmaceutical preparations. Monatshefte Fur Chem. 2025, 156, 1013–1025.
49.
Al-Khateeb, L.A.; Dahas, F.A. Development of an eco-friendly liquid chromatography method for the separation and trace determination of caffeine in beverages, pharmaceuticals, and environmental water samples utilizing high-temperature and superheated water: A greenness assessment. J. Anal. Chem. 2025, 80, 693–706.
50.
Wang, J.; Hu, Y.; Chang, Y.; et al. Transcriptomic insights into caffeine degradation pathways in Desarmillaria tabescens. Microorganisms 2025, 13, 2720.
51.
Sun, X.; Chang, Q.-Z.; Wang, C.; et al. Determination of caffeine and taurine in electronic cigarette oil by high-performance liquid chromatography-tandem mass spectrometry. Chin. J. Chromatog. 2025, 43, 1374–1379.
52.
Copsey, S.; Menacho, B.; Rafoo, L.; et al. Improving the performance of HPLC–MS using radial flow splitting columns: The analysis of caffeine in energy drinks. Anal. Bioanal. Chem. 2025, 417, 3927–3933.
53.
Unal, N.; Dinc, E. A novel UPLC method development for quantifying active and inactive ingredients in a commercial energy drink product using chemometric optimization methodology. Food Anal. Meth. 2025, 18, 1255–1268.
54.
Karlsen, C.E.; Cravino, J.A.; Soliven, A.; et al. Quantification of caffeine in energy and cola drinks via rapid high-performance liquid chromatography assays with ultraviolet diode array detection. Beverages 2025, 11, 39.
55.
Jankulovska, M.S.; Stojchevska, S.; Velkoska-Markovska, L. Development and validation of an RP-HPLC method for the determination of caffeine and preservatives (sodium benzoate and potassium sorbate) in sports and energy drinks. J. Food Measure. Character. 2025, 19, 5448–5458.
56.
Wahba, I.A.; Hassan, S.A.; Fayed, A.S.; et al. A new chromatographic approach for the simultaneous determination of tramadol, ibuprofen, and caffeine in a novel fixed-dose combination tablet: An integrated framework to analytical sustainability and multimodal analgesia. BMC Chem. 2026, 20, 13.
57.
Nishi, H.; Ueda, M.; Kimoto, A.; Kawabata, K. Elution behavior of eight tea catechins and caffeine in HPLC separation with core-shell type reversed-phase columns and application to determination of these contents in green tea drinks containing large amount of catechin. Bunseki Kagaku 2025, 74, 29–36.
58.
Szynkaruk, G.; Puchalska, M.; Kerner, J.; et al. Stability of caffeine citrate intravenous dilutions in sodium chloride and glucose solutions. Eur. J. Hosp. Pharm. 2025. https://doi.org/10.1136/ejhpharm-2025-004706.
59.
Portillo-Castillo, O.J.; Cobos-Cervantes, P.; Álvarez-Román, R.; et al. Analytical method development and validation with greenness assessment of HPLC-DAD method to determine methylxanthines. Acta Chim. Slov. 2025, 72, 180–194.
60.
Chauhan, N.; Kumar, S.; Rana, S.S.; et al. Development of an AQbD-driven HPLC method for the simultaneous estimation of caffeine and misoprostol in nanoparticle formulations. Anal. Meth. 2026, 18, 389–400.
61.
Alqarni, M.; Nagub, I.A.; Arida, H. Assessment of the greenness of hplc methods employing common detectors for analysis of caffeine in pharmaceutical dosage forms. J. Chem. 2025, 2025, 6420273.
62.
Jan, A.; Ahmad, F.; Raza, N.; et al. Development and validation of a rapid RP-HPLC method for the simultaneous quantification of paracetamol, caffeine, and chlorpheniramine maleate in pharmaceutical tablets. ChemistrySelect 2025, 10, e04531.
63.
Uddin, M.J.; Mohaimenul, M.; Aktaruzzaman, M.; et al. Isolation and quantification of caffeine in marketed tea and carbonated beverage products in Bangladesh. Toxicol. Anal. Et Clin. 2025, 37, 159–166.
64.
Kegele, C.S.; Mariani, B.; Polonini, H.C. Personalized compounded hair loss treatments: A compatibility assessment of the TrichoConcept vehicle line. Int. J. Pharm. Compd. 2025, 29, 239–253.
65.
Knott, H.; Rivas, C.; Barnes, J.; et al. Caffeine disclosure in weight loss teas and its public health implications: An investigation using high-performance liquid chromatography. J. Chem. Edu. 2025, 102, 4603–4610.
66.
Sridhar, D.; Parimalarenganayaki, S. Evaluation of sources, spatial and temporal distribution, ecological and health risk associated with CAF (Caffeine) and DEET (N,N-diethyl-meta-toluamide) contamination in the urban groundwater parts of Vellore city, Tamilnadu, India. Environ. Geochem. Health 2025, 47, 44.
67.
Sanchez-Herrera, A.L.; Leal-Bautista, R.M.; Alvarado-Flores, J. Presence of caffeine in the Karst Lake Punta Laguna of the Yucatán Peninsula and its environmental risk. Rev. Int. Contam. Ambient. 2025, 41, 655–665.
68.
Koziarska, M.; Strzebonska, M.; Szalinska, E. Development and validation of a green/blue UHPLC-MS/MS method for trace pharmaceutical monitoring. Sci. Rep. 2025, 15, 30902.
69.
Al Awawdeh, S.; Shafie, N.H.; Ishak, A.H.; et al. Analytical method development for polyphenol detection in rat plasma using green tea-loaded chitosan-iron oxide nanoparticles. BioNanoScience 2026, 16, 69.
70.
Chen, X.D.; Guo, Q.W.; Li, J.; et al. The association between urinary caffeine and caffeine metabolites and diabetic retinopathy in individuals with diabetes: NHANES 2009–2014. Sci. Rep. 2025, 15, 15827.
71.
Baharum, Z.; Akim, A.M.; Hin, T.Y.Y.; et al. Theobroma cacao: Review of the extraction, isolation, and bioassay of its potential anti-cancer compounds. Trop. Life Sci. Res. 2016, 27, 21–42.
72.
Sarker, S.D.; Nahar, L. Computational Phytochemistry, 2nd ed.; Elsevier: Amsterdam, The Netherlands, 2025.
73.
Zhao, N.; Liu, Z.; Yu, T.; et al. Spent coffee grounds: Present and future of environmentally friendly applications on industries-A review. Trends Food Sci. Technol. 2024, 143, 104312.
74.
Campos-Vega, R.; Loarca-Pina, G.; Vergara-Castaneda, H.A.; et al. Spent coffee grounds: A review on current research and future prospects. Trends Food Sci. Technol. 2015, 45, 24–36.
75.
Ma, J.; Li, J.; He, H.; et al. Characterization of sensory properties of Yunnan coffee. Curr. Res. Food Sci. 2022, 5, 1205–1215.
76.
Boros, K.; Jedlinkzki, N.; Csupor, D. Theanine and caffeine content of infusions prepared from commercial tea samples. Pharmacog. Mag. 2016, 12, 75–79.
77.
Gramza-Michalowska, A. Caffeine in tea Camellia sinensis—Content, absorption, benefits and risks of consumption. J. Nutri. Health Aging 2014, 18, 143–149.
78.
Jia, W.; Rajani, C.; Lv, A.; et al. Pu-erh tea: A review of a healthful brew. J. Trad. Chin. Med. Sci. 2022, 9, 95–99.
79.
Derry, C.J.; Derry, S.; Moore, R.A. Caffeine as an analgesic adjuvant for acute pain in adults. Cochrane Database Syst. Rev. 2014. https://doi.org/10.1002/14651858.CD009281.pub3.
80.
Reissig, C.J.; Strain, E.C.; Griffiths, R.R.; et al. Caffeinated energy drinks—A growing problem. Drug Alcohol. Depend. 2009, 99, 1–10.
81.
Costantino, A.; Maiese, A.; Lazzari, J.; et al. The dark side of energy drinks: A comprehensive review of their impact on the human body. Nutrients 2023, 15, 3922.
82.
Alsunni, A.A. Energy drink consumption: Beneficial and adverse health effects. Int. J. Health Sci. 2015, 9, 468–474.

Scilight Press

Author Information

Abstract

Keywords

References

About Scilight

Journals

Publishing Policies

Contact Us