Distinguishing Pure Honey from Honey Adulterated with Added Sugar Using the LC–MS/MS Method
DOI:
https://doi.org/10.15408/jkv.v12i1.50065Keywords:
Authentication, honey, LC–MS/MS, sugar adulterationAbstract
Honey adulteration through the addition of sugar such as liquid sugar, cane sugar, fructose, and glucose remains a major concern affecting honey quality and authenticity. This study aimed to differentiate pure honey, sugar-adulterated honey, and synthetic honey using a liquid chromatography tandem mass spectrometry (LC-MS/MS) fingerprinting approach. Samples included pure acacia (A), longan (K), and kapok/randu (N) honeys, their corresponding sugar-adulterated variants, and synthetic honey (S) as a negative control. The results demonstrated that LC-MS/MS fingerprints effectively distinguished pure honey from adulterated and synthetic samples. Pure honeys exhibited more complex and specific metabolite profiles, whereas sugar addition led to simplified chromatographic patterns dominated by sugar-related compounds. Fructose- and glucose-adulteration produced metabolite profiles most similar to synthetic honey, with randu honey showing the highest sensitivity to fructose addition. Fructosylated compounds such as N-(1-deoxy-1-fructosyl)phenylalanine (Fru-Phe) and isomaltol were identified as indicators of sugar addition and thermal processing. In contrast, biologically and botanically derived metabolites, including umbelliferone, indoleacrylic acid, hypoxanthine, O-glutarylcarnitine, cyclic melatonin, phenethyl tiglate, and indole-3-carboxaldehyde, reflected nectar origin and natural biological processes. The detection of phenanthrene-3,4-diol further indicated environmental influences on honey composition. Overall, integrating processing indicators with biological and botanical markers provides a comprehensive and reliable framework for honey authentication via LC-MS/MS fingerprinting.
Downloads
References
1. da Silva, P. M., Gauche, C., Gonzaga, L. V., Costa, A. C. O., & Fett, R. (2016). Honey: Chemical composition, stability and authenticity. Food Chemistry, 196, 309–323. https://doi.org/10.1016/j.foodchem.2015.09.051
2. Fakhlaei, R., Selamat, J., Khatib, A., Razis, A. F. A., Sukor, R., Ahmad, S., & Babadi, A. A. (2020). The Toxic Impact of Honey Adulteration: A Review. Foods, 9(11), 1538. https://doi.org/10.3390/foods9111538
3. Girma Tura, A., & Bersissa Seboka, D. (2020). Review on Honey Adulteration and Detection of Adulterants in Honey. International Journal of Gastroenterology, 4(1), 1. https://doi.org/10.11648/j.ijg.20200401.11
4. Sumarlin, L. O., Enita, N., Afandi, F. R., & Fathoni, A. (2021). Identification of Active Chemical Compounds of Honey from Some Regions in Indonesia. Science and Technology Indonesia, 6(2), 74–84. https://doi.org/10.26554/sti.2021.6.2.74-84
5. Kawasaki, N., Ohta, M., Hyuga, S., Hashimoto, O., & Hayakawa, T. (1999). Analysis of Carbohydrate Heterogeneity in a Glycoprotein Using Liquid Chromatography/Mass Spectrometry and Liquid Chromatography with Tandem Mass Spectrometry. Analytical Biochemistry, 269(2), 297–303. https://doi.org/10.1006/abio.1999.4026
6. Cocuron, J.-C., Ross, Z., & Alonso, A. P. (2020). Liquid Chromatography Tandem Mass Spectrometry Quantification of 13C-Labeling in Sugars. Metabolites, 10(1), 30. https://doi.org/10.3390/metabo10010030
7. Takahashi, Y., Yoshida, I., Yokozeki, T., Hirakawa, Y., & Fujita, K. (2025). Development of an LC-MS/MS Method for Quantitation of Western Honeybee (<i>Apis mellifera</i>) α-Glucosidase III as a Potential Honey Authenticity Marker. Journal of Applied Glycoscience, 72(2), 7202106. https://doi.org/10.5458/jag.7202106
8. Dewi Pobo Kitchen. (2021, May 10). Begini cara buat madu sendiri [Video]. YouTube. https://www.youtube.com/watch?v=74wYVnAHTDU
9. Maharani, T., Sukandar, D., & Hermanto, S. (2016). Karakterisasi Senyawa Hasil Isolasi dari Ekstrak Etil Asetat Daun Namnam (Cynometra Cauliflora L.) yang Memiliki Aktivitas Antibakteri. Jurnal Kimia VALENSI, 2(1), 55–62. https://doi.org/10.15408/jkv.v2i1.3084
10. Al-Hetlani, E., D’Cruz, B., Hayssam, M., Mandekar, B., & Amin, M. O. (2026). Evaluating Honey Adulteration Through Physicochemical Characterization and Liquid Chromatography–Mass Spectrometry-Based Sugar Profiling. Foods, 15(6), 1038. https://doi.org/10.3390/foods15061038
11. Sumarlin, L. O., Amilia, N., Muawanah, A., Hasanah, N. U., & Hajar, H. (2022). Characteristics and Antibacterial Activity of Apis and Trigona Honey Types against Escherichia coli and Staphylococcus Aureus on Various Heating. Jurnal Kimia Valensi, 8(2), 251–262. https://doi.org/10.15408/jkv.v8i2.27241
12. Yan, S., Wang, X., Wu, Y., Wang, K., Shan, J., & Xue, X. (2022). A metabolomics approach revealed an Amadori compound distinguishes artificially heated and naturally matured acacia honey. Food Chemistry, 385, 132631. https://doi.org/10.1016/j.foodchem.2022.132631
13. Dettmer, K., Aronov, P. A., & Hammock, B. D. (2007). Mass spectrometry‐based metabolomics. Mass Spectrometry Reviews, 26(1), 51–78. https://doi.org/10.1002/mas.20108
14. Want, E. J., Wilson, I. D., Gika, H., Theodoridis, G., Plumb, R. S., Shockcor, J., Holmes, E., & Nicholson, J. K. (2010). Global metabolic profiling procedures for urine using UPLC–MS. Nature Protocols, 5(6), 1005–1018. https://doi.org/10.1038/nprot.2010.50
15. Niessen, W. M. A. (2006). Liquid Chromatography-Mass Spectrometry. CRC Press. https://doi.org/10.1201/9781420014549
16. Gross, J. H. (2017). Mass Spectrometry. Springer International Publishing. https://doi.org/10.1007/978-3-319-54398-7
17. Morales, F. J. (2008). Hydroxymethylfurfural (HMF) and Related Compounds. In Process‐Induced Food Toxicants (pp. 135–174). Wiley. https://doi.org/10.1002/9780470430101.ch2e
18. Nursten, H. E. (2005). The Maillard Reaction: Chemistry, Biochemistry and Implications. https://api.semanticscholar.org/CorpusID:83876408
19. Murkovic, M., & Bornik, M. (2007). Formation of 5‐hydroxymethyl‐2‐furfural (HMF) and 5‐hydroxymethyl‐2‐furoic acid during roasting of coffee. Molecular Nutrition & Food Research, 51(4), 390–394. https://doi.org/10.1002/mnfr.200600251
20. Yaylayan, V. A. (1997). Classification of the Maillard reaction: A conceptual approach. Trends in Food Science & Technology, 8(1), 13–18. https://doi.org/10.1016/S0924-2244(96)20013-5
21. van Boekel, M. A. J. S. (2006). Formation of flavour compounds in the Maillard reaction. Biotechnology Advances, 24(2), 230–233. https://doi.org/10.1016/j.biotechadv.2005.11.004
22. Chaipoot, S., Wiriyacharee, P., Pathomrungsiyounggul, P., Kanthakat, G., Somjai, C., Boonyapranai, K., Srinuanpan, S., Wongwatcharayothin, W., & Phongphisutthinant, R. (2025). Antioxidant Activity and Chemical Alterations of Honeybee Brood Bio-Peptides Interacting with Honey Under Moist-Dried Thermal Aging. Antioxidants, 14(3), 254. https://doi.org/10.3390/antiox14030254
23. Bonsignore, G., Martinotti, S., & Ranzato, E. (2024). Honey Bioactive Molecules: There Is a World Beyond the Sugars. BioTech, 13(4), 47. https://doi.org/10.3390/biotech13040047
24. Leoni, V., Panseri, S., Giupponi, L., Pavlovic, R., Gianoncelli, C., Sala, S., Zeni, V., Benelli, G., & Giorgi, A. (2023). Formal analyses are fundamental for the definition of honey, a product representing specific territories and their changes: the case of North Tyrrhenian dunes (Italy). Scientific Reports, 13(1), 17542. https://doi.org/10.1038/s41598-023-44769-1
25. Mazimba, O. (2017). Umbelliferone: Sources, chemistry and bioactivities review. Bulletin of Faculty of Pharmacy, Cairo University, 55(2), 223–232. https://doi.org/10.1016/j.bfopcu.2017.05.001
26. Gv, R., Sastri, T., & Burada, S. (2019). Bioactive Umbelliferone and its derivatives: An update. 59–66.
27. Lin, Z., Cheng, X., & Zheng, H. (2023). Umbelliferon: a review of its pharmacology, toxicity and pharmacokinetics. Inflammopharmacology, 31(4), 1731–1750. https://doi.org/10.1007/s10787-023-01256-3
28. Makni, Y., Diallo, T., Guérin, T., & Parinet, J. (2024). A proof-of-concept study on the versatility of liquid chromatography coupled to high-resolution mass spectrometry to screen for various contaminants and highlight markers of floral and geographical origin for different honeys. Food Chemistry, 436, 137720. https://doi.org/10.1016/j.foodchem.2023.137720
29. Wlodarska, M., Luo, C., Kolde, R., d’Hennezel, E., Annand, J. W., Heim, C. E., Krastel, P., Schmitt, E. K., Omar, A. S., Creasey, E. A., Garner, A. L., Mohammadi, S., O’Connell, D. J., Abubucker, S., Arthur, T. D., Franzosa, E. A., Huttenhower, C., Murphy, L. O., Haiser, H. J., … Xavier, R. J. (2017). Indoleacrylic Acid Produced by Commensal Peptostreptococcus Species Suppresses Inflammation. Cell Host & Microbe, 22(1), 25-37.e6. https://doi.org/10.1016/j.chom.2017.06.007
30. Li, X., Zhang, B., Hu, Y., & Zhao, Y. (2021). New Insights Into Gut-Bacteria-Derived Indole and Its Derivatives in Intestinal and Liver Diseases. Frontiers in Pharmacology, 12. https://doi.org/10.3389/fphar.2021.769501
31. Su, X., Gao, Y., & Yang, R. (2022). Gut Microbiota-Derived Tryptophan Metabolites Maintain Gut and Systemic Homeostasis. Cells, 11(15), 2296. https://doi.org/10.3390/cells11152296
32. Zhang, B.-X., Li, P.-S., Wang, Y.-Y., Wang, J.-J., Liu, X.-L., Wang, X.-Y., & Hu, X.-M. (2021). Characterization and synthesis of indole-3-acetic acid in plant growth promoting Enterobacter sp. RSC Advances, 11(50), 31601–31607. https://doi.org/10.1039/D1RA05659J
33. Sinha, A. K., Laursen, M. F., Brinck, J. E., Rybtke, M. L., Hjørne, A. P., Procházková, N., Pedersen, M., Roager, H. M., & Licht, T. R. (2024). Dietary fibre directs microbial tryptophan metabolism via metabolic interactions in the gut microbiota. Nature Microbiology, 9(8), 1964–1978. https://doi.org/10.1038/s41564-024-01737-3
34. Koga, J., Adachi, T., & Hidaka, H. (1991). IAA Biosynthetic Pathway from Tryptophan via Indole-3-pyruvic Acid in Enterobacter cloacae. Agricultural and Biological Chemistry, 55(3), 701–706. https://doi.org/10.1080/00021369.1991.10870678
35. Kuś, P. M., & Rola, R. (2021). LC-QqQ-MS/MS methodology for determination of purine and pyrimidine derivatives in unifloral honeys and application of chemometrics for their classification. Food Chemistry, 348, 129076. https://doi.org/10.1016/j.foodchem.2021.129076
36. Kim, T.-K., Atigadda, V. R., Brzeminski, P., Fabisiak, A., Tang, E. K. Y., Tuckey, R. C., Reiter, R. J., & Slominski, A. T. (2021). Detection of Serotonin, Melatonin, and Their Metabolites in Honey. ACS Food Science & Technology, 1(7), 1228–1235. https://doi.org/10.1021/acsfoodscitech.1c00119
37. Marcolin, L. C., de Oliveira Arias, J. L., Kupski, L., Barbosa, S. C., & Primel, E. G. (2023). Polycyclic Aromatic Hydrocarbons (PAHs) in honey from stingless bees (Meliponinae) in southern Brazil. Food Chemistry, 405, 134944. https://doi.org/10.1016/j.foodchem.2022.134944
38. Roman, S., Voaides, C., & Babeanu, N. (2023). Exploring the Sustainable Exploitation of Bioactive Compounds in Pelargonium sp.: Beyond a Fragrant Plant. Plants, 12(24), 4123. https://doi.org/10.3390/plants12244123
39. Albers, C., Manam, S. S., Bhatta, S., & Crasto, C. (2025). IJBLS 2025; 4(2):154-160 International Journal of BioLife Sciences Two-dimensional Molecular Fingerprints in Molecules Exhibiting the Sweet Flavor. 154–160. https://doi.org/10.22034/ijbls.2025.223573
40. Zhu, H., Liu, L., Wang, R., Wang, Z., Wang, Y., Dong, J., Wang, Q., Qiao, J., & Zhang, H. (2025). Unveiling the phytochemical profiles of Ziziphus jujuba honey: An authenticity assurance approach. Food Chemistry: X, 29, 102622. https://doi.org/10.1016/j.fochx.2025.102622
41. Giesbertz, P., Ecker, J., Haag, A., Spanier, B., & Daniel, H. (2015). An LC-MS/MS method to quantify acylcarnitine species including isomeric and odd-numbered forms in plasma and tissues. Journal of Lipid Research, 56(10), 2029–2039. https://doi.org/10.1194/jlr.D061721
Downloads
Published
Issue
Section
License
Copyright (c) 2026 La Ode Sumarlin, Etyn Yunita, Agustina Senjayani, Vetty Megantari
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.
