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Sains Malaysiana 54(12)(2025): 2897-2905
http://doi.org/10.17576/jsm-2025-5412-08
Determination of Ochratoxin A in
Brewed Coffee by Dispersive Liquid-Liquid Microextraction-High-Performance Liquid
Chromatography-Fluorescence Detection
(Penentuan Okratoksin A di dalam
Bancuhan Kopi dengan Pengekstrakan Mikro Cecair-Cecair Berserak-Kromatografi Cecair Berprestasi Tinggi-Pengesanan Pendarfluor)
NURUL QISTINA IZZATI MD ZAIHAN, WAN MOHD AFIQ WAN MOHD KHALIK, HAFIZA
MOHAMED ZUKI & SAW HONG LOH*
Faculty
of Science and Marine Environment, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu,
Malaysia
Received:
5 August 2025/Accepted: 16 December 2025
Abstract
Coffee, the most commercialised food
product and one of the top three most consumed beverages globally, faces
significant challenges due to the contamination of ochratoxin A (OTA), a
mycotoxin found in various foods. This presents a serious health concern
requiring a rapid and sensitive method for detecting OTA. The study examined
the analytical performance of dispersive liquid-liquid microextraction (DLLME)
for extracting OTA from brewed coffee before analysis with high-performance
liquid chromatography and fluorescence detection (HPLC-FLD). Various parameters
influencing the extraction efficiency of DLLME, including the volume of
extraction solvent and dispersive solvent, exposing time, centrifugation, and
the addition of acid, were investigated and optimised. Under the optimised
conditions (100 μL of 1-octanol as the
extraction solvent, 200 μL of acetonitrile as
the dispersive solvent, 1-minute exposing time, with centrifugation, and the
addition of 5 μL of glacial acetic acid), the
relative recoveries of OTA in spiked brewed coffee samples at 1 µg L-1 ranged from 82.0% to 110.4%. The method demonstrated excellent linearity (r=0.9966)
and acceptable limits of detection (0.06 µg L-1) and quantification
(0.19 µg L-1). OTA levels in locally sourced brewed coffee samples
ranged from 0.77-0.90 µg L-1, remaining below the European Commission
regulatory limit of 3 µg kg-1. The developed DLLME-HPLC-FLD method
achieved a high score of 8.63 using the sample preparation metric of sustainability
(SPMS), underscoring its environmental compatibility. The optimised
DLLME-HPLC-FLD method provides a reliable, rapid, and environmentally friendly
approach for OTA detection in brewed coffee, thereby enabling continuous
monitoring of OTA in brewed coffee to ensure consumer safety.
Keywords: Coffee; DLLME; HPLC-FLD; mycotoxin
Abstrak
Kopi, sebagai produk makanan yang paling banyak dikomersialkan dan salah satu daripada tiga minuman teratas yang paling banyak diambil di seluruh dunia, menghadapi cabaran besar sebagai akibat pencemaran oleh okratoksin A (OTA), sejenis mikotoksin yang terdapat di dalam pelbagai makanan. Ini merupakan satu isu kesihatan yang serius yang memerlukan satu kaedah yang pantas dan sensitif untuk mengesan OTA. Penyelidikan ini mengkaji prestasi analitikal pengekstrakan cecair-cecair berserak (DLLME) dalam mengekstrak OTA daripada kopi yang telah dibancuh. Pelbagai parameter yang mempengaruhi kecekapan pengekstrakan DLLME, termasuk isi padu pelarut pengekstrak dan pelarut penyerak, masa pendedahan, pengemparan dan penambahan asid telah dikaji dan dioptimumkan. Di bawah keadaan optimum (100 μL 1-oktanol sebagai pelarut pengekstrak, 200 μL asetonitril sebagai pelarut penyerak, masa pendedahan selama 1 minit dengan pengemparan serta penambahan 5 μL asid asetik glasial), perolehan semula relatif OTA daripada sampel kopi bancuhan yang terpaku pada 1 µg L-1adalah dalam julat 82.0% hingga 110.4%. Kaedah ini menunjukkan kelinearan yang sangat baik (r=0.9966) serta had pengesanan (0.06 µg L-1) dan had penguantitian (0.19 µg L-1) yang boleh diterima. Tahap OTA di dalam sampel kopi bancuhan yang diperoleh secara tempatan berada dalam julat 0.77-0.90 µg L-1 masih di bawah had peraturan Suruhanjaya Eropah 3 µg
kg-1. Kaedah DLLME-HPLC-FLD turut mencapai skor tinggi dalam metrik kelestarian penyediaan sampel (SPMS) iaitu 8.63 yang menunjukkan keserasian kaedah ini dengan prinsip kelestarian alam sekitar. Kaedah DLLME-HPLC-FLD
yang dioptimumkan ini menyediakan pendekatan yang boleh dipercayai, pantas dan mesra alam untuk pengesanan OTA di dalam kopi bancuhan, dengan itu membolehkan pemantauan berterusan terhadap OTA di dalam kopi bancuhan untuk memastikan keselamatan pengguna.
Kata kunci: DLLME; HPLC-FLD; kopi; mikotoksin
REFERENCES
Antep, H.M. & Bozkurt, S.S. 2015. Enrichment of
ochratoxin A in cereals using ionic liquid based dispersive liquid-liquid
microextraction. Hacettepe Journal of Biology and Chemistry 43(2): 99-104.
Ballis, A.S. 2019. Coffee a worldwide trend with health
benefits. Acta Scientific Nutritional Health 3(3): 70-73.
Banahene, J.C.M., Ofosu, I.W., Odai, B.T., Lutterodt,
H.E., Agyemang, P.A. & Ellis, W.O. 2024. Ochratoxin A in food commodities:
A review of occurrence, toxicity, and management strategies. Heliyon 10(20): e39313.
Bedaso, Z.A. & Jilo, K.R. 2022. Coffee consumption
benefits and risks on human health. Journal of Medicine and Biology 4(1):
41-52.
Cina, M., del Valle Ponce, M., Fernandez, L. &
Cerutti, S. 2022. A green approach for ochratoxin A determination in coffee
infusions. Journal of Food Composition and Analysis 114: 104777.
Commission Regulation (EU) 2022/1370 2022. Official
Journal of the European Union 206, 8.8.2022. pp. 11–14. EUR-Lex.
Drunday, V. & Pacin, A. 2013. Occurrence of
ochratoxin A in coffee beans, ground roasted coffee and soluble coffee and
method validation. Food Control 30(2): 675-678.
Elik, A., Ablak, Ö., Haq, H.U., Boczkaj, G. &
Altunay, N. 2023. Combination of homogeneous liquid–liquid extraction and
vortex assisted dispersive liquid–liquid microextraction for the extraction and
analysis of ochratoxin A in dried fruit samples: Central composite design
optimization. Journal of Food Composition and Analysis 124: 105656.
Fakhri, Y., Ranaei, V., Pilevar, Z., Belaia, O.F.,
Kolaeva, N.V., Sarafraz, M. & Mousavi Khaneghah, A. 2024. Prevalence
and concentration of ochratoxin A in beer: A global systematic review,
meta-analysis, and health risk assessment. Food Science and Nutrition 12(11): 8503-8514.
Faraji, H. 2024. Advancements in overcoming challenges in
dispersive liquid-liquid microextraction: An overview of advanced strategies. Trends
in Analytical Chemistry 170: 117429.
Ferrone, V., Genovese, S., Carlucci, M., Tiecco, M.,
Germani, R., Preziuso, F., Epifano, F., Carlucci, G. & Taddeo, V.A. 2018. A
green deep eutectic solvent dispersive liquid-liquid micro-extraction
(DES-DLLME) for the UHPLC-PDA determination of oxyprenylated phenylpropanoids
in olive, soy, peanuts, corn, and sunflower oil. Food Chemistry 245: 578-585.
Freire, L., Braga, P.A.C., Furtado, M.M., Delafiori, J.,
Dias-Audibert, F.L., Pereira, G.E., Reyes, F.G., Catharino, R.R. &
Sant’Ana, A.S. 2020. From grape to wine: Fate of ochratoxin A during red, rose,
and white winemaking process and the presence of ochratoxin derivatives in the
final products. Food Control 113: 107167.
Gomes, A.L., Sherudillo, A., Shagurin, A., Antina, L.,
Moskaieva, O., Dubois, J., Kalugin, O.N., Sauvage, F., Miannay, F.A. &
Idrissi, A. 2025. Photophysical modulation of D205 dye in acetonitrile-butanol
mixtures: interplay of solvent polarity, hydrogen bonding, and conformational
dynamics. Journal of Molecular Liquids 435: 128109.
Gonzalez, A.L., Lozano, V.A., Escandar, G.M. & Bravo,
M.A. 2020. Determination of ochratoxin A in coffee and tea samples by coupling
second-order multivariate calibration and fluorescence spectroscopy. Talanta 219: 121288.
González-Curbelo, M.A. & Kabak, B. 2023. Occurrence of mycotoxins in dried fruits
worldwide, with a focus on aflatoxins and ochratoxin A: A review. Toxins 15(9): 576.
González-Martín, R., Gutiérrez-Serpa, A., Pino, V. &
Sajid, M. 2023. A tool to assess analytical sample preparation procedures:
Sample preparation metric of sustainability. Journal of Chromatography A 1707: 464291.
Humaid, A.A.H., Alghalibi, S.M.S. & Al-Khalqi, E.A.A.
2019. Aflatoxins and ochratoxin a content of stored yemeni coffee beans and
effect of roasting on mycotoxin contamination. International Journal of
Molecular Microbiology 2(1): 11-21.
International Agency for Research on Cancer (IARC). 1993.
Some naturally occurring substances: Food items and constituents, heterocyclic
aromatic amines and mycotoxins.
Karami-Osboo, R., Miri, R., Javidnia, K., Kobarfard, F.,
AliAbadi, M.H.S. & Maham, M. 2015. A validated dispersive liquid-liquid
microextraction method for extraction of ochratoxin A from raisin samples. Journal
of Food Science and Technology 52(4): 2440-2445.
Lafferty, J. 2025. Most Consumed Beverages in the World:
What People Drink Most. https://mysticdrink.com/most-consumed-beverages-in-the-world-what-people-drink-most/
Accessed 11 December 2025.
Leitão, A.L. 2019. Occurrence of ochratoxin a in coffee: Threads
and solutions - A mini-review. Beverages 5(2): 36.
Loh, S.H., Chong, Y.T., Afindi, K.N.N. & Kamaruddin,
N.A. 2016. Determination of polycyclic aromatic hydrocarbons in beverage by low
density solvent based-dispersive liquid-liquid microextraction-high performance
liquid chromatography-fluorescence detection. Sains Malaysiana 45(10):
1453-1459.
Maham, M., Kiarostami, V., Waqif-Husain, S.,
Karami-Osboo, R. & Mirabolfathy, M. 2013. Analysis of ochratoxin A in malt
beverage samples using dispersive liquid–liquid microextraction coupled with
liquid chromatography-fluorescence detection. Czech Journal of Food Sciences 31(5): 520-525.
Mariño-Repizo, L., Goicoechea, H., Raba, J. &
Cerutti, S. 2018. A simple, rapid and novel method based on salting-out
assisted liquid–liquid extraction for ochratoxin A determination in beer
samples prior to ultra-high-performance liquid chromatography coupled to tandem
mass spectrometry. Food Additives and Contaminants - Part A 35(8): 1622-1632.
Mousavi, S.S., Shahsavani, A., Aladaghlo, Z. &
Fakhari, A.R. 2025. Solvent-assisted dispersive solid-phase extraction based on
salen as a new and efficient sorbent followed by GC-FID for determination of
polycyclic aromatic hydrocarbons in water, food, and soil samples. Journal
of the Iranian Chemical Society 22: 2005-2014.
Pakshir, K., Dehghani, A., Nouraei, H., Zareshahrabadi,
Z. & Zomorodian, K. 2021. Evaluation of fungal contamination and ochratoxin
A detection in different types of coffee by HPLC-based method. Journal of
Clinical Laboratory Analysis 35(11): e24001.
Pakshir, K., Mirshekari, Z., Nouraei, H., Zareshahrabadi,
Z., Zomorodian, K., Khodadadi, H., & Hadaegh, A. 2020. Mycotoxins detection
and fungal contamination in black and green tea by HPLC-based method. Journal
of Toxicology 2020(1): 2456210.
Sargazi, A., Aliabadi, A., Rahdari, A.,
Allahdini-Hesaroiyeh, S., Nejati-Yazdinejad, M. & Majd, M.H. 2017. A simple
and fast method for magnetic solid phase extraction of ochratoxin A. Journal
of the Brazilian Chemical Society 28(6): 950-959.
Schrenk, D., Bodin, L., Chipman, J.K., del Mazo, J.,
Grasl-Kraupp, B., Hogstrand, C., Hoogenboom, L., Leblanc, J.C., Nebbia, C.S.,
Nielsen, E., Ntzani, E., Petersen, A., Sand, S., Schwerdtle, T., Vleminckx, C.,
Wallace, H., Alexander, J., Dall’Asta, C., Mally, A., Metzler, M., Binaglia,
M., Horváth, Z., Steinkellner, H. & Bignami, M. 2020. Risk assessment of
ochratoxin A in food. EFSA Journal 18(5): e06113.
Sharifi, V., Abbasi, A. & Nosrati, A. 2016.
Application of hollow fiber liquid phase microextraction and dispersive
liquid-liquid microextraction techniques in analytical toxicology. Journal
of Food and Drug Analysis 24(2): 264-276.
Tan, Y.H., Chai, M.K. & Wong, L.S. 2018. A review on
extraction solvents in the dispersive liquid-liquid microextraction. Malaysian
Journal of Analytical Sciences 22(2): 166-174.
Wang, Q., Chen, R., Shatner, W., Cao, Y. & Bai, Y.
2019. State-of-the-art on the technique of dispersive liquid-liquid
microextraction. Ultrasonics Sonochemistry 51: 369-377.
Yazdanfar, N., Mahmudiono, T., Fakhri, Y., Mahvi, A.H.,
Sadighara, P., Mohammadi, A.A., & Yousefi, M. 2022. Concentration of
ochratoxin A in coffee products and probabilistic health risk assessment. Arabian
Journal of Chemistry 15(12): 104376.
*Corresponding author; email: lohsh@umt.edu.my
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