Hung-Yu Lin1, Dinesh Chandra Agrawal1, Wen-Goang Yang2, Wei-Jun Chien1*

1 Department of Applied Chemistry, Chaoyang University of Technology, Taichung, Taiwan, R.O.C.
2 Department of Leisure Services Management, Chaoyang University of Technology, Taichung, Taiwan, R.O.C.


Download Citation: |
Download PDF


Mycotoxins are naturally occurring toxins produced by certain molds and can be found in nuts, cereals, fruits, and oils. Furthermore, current food processing systems cannot wholly remove mycotoxins. The adverse health effects of chewing betel nut containing mycotoxins range from long-term consequences such as immune deficiency. In this study, a rapid solid-phase extraction (SPE) method to LC-MS/MS system was developed to determine nine mycotoxins in commonly used five types of betel nut products. The SPE eluate was injected into a Phenomenex UHPLC Kinetex Polar C18 Column (2.1 × 100 mm, 2.6 mm) and detected by an Agilent 6470 triple-quadrupole mass spectrometer. All mycotoxins were separated under 6.8 min with electrospray ionization in multiple reaction monitoring modes. The calibration curves ranged from 0.5 to 600 ng/mL (r2>0.99), LOQ values were between 0.25 to10 ng/mL, and LOD values were between 5 to 20 ng/mL. All the QCs have shown precision, and the accuracy range for intra-day and inter-day did not exceed 14.3% (RSD) and 91.2 to 117.6% (relative error). The matrix effect and extraction recovery for five types of betel nuts were in the range of 81.5 to 99.2% and 70.1 to 113.9%, respectively (except deoxynivalenol).

Keywords: LC-MS/MS, Mycotoxin, Multiple reaction monitoring (MRM).

Share this article with your colleagues



  1. Ałtyn, I., Twarużek, M. 2020. Mycotoxin contamination concerns of herbs and medicinal plants. Toxins, 12, 182–195.

  2. Azri, F.A., Sukor, R., Selamat, J., Abu Bakar, F., Yusof, N.A., Hajian, R. 2018. Electrochemical immunosensor for detection of aflatoxin B1 based on indirect competitive ELISA. Toxins, 10, 196–209.

  3. Bryła, M., Ksieniewicz-Woźniak, E., Waśkiewicz, A., Szymczyk, K., Jędrzejczak, R. 2018. Natural occurrence of nivalenol, deoxynivalenol, and deoxynivalenol-3-glucoside in polish winter wheat. Toxins, 10, 81–93.

  4. Chang, C.C., Kao, T.H., Zhang, D., Wang, Z., Inbaraj, B.S., Hsu, K.Y., Chen, B.H. 2018. Application of QuEChERS coupled with HPLC-DAD-ESI-MS/MS for determination of heterocyclic amines in commercial meat products. Food Analytical Methods, 11, 3243–3256.

  5. De Souza, G.D., Mithöfer, A., Daolio, C., Schneider, B., Rodrigues-Filho, E. 2013. Identification of alternaria alternata mycotoxins by LC-SPE-NMR and their cytotoxic effects to soybean (Glycine max) cell suspension culture. Molecules, 18, 2528–2538.

  6. Desmarchelier, A., Hamel, J., Delatour, T. 2020. Sources of overestimation in the analysis of acrylamide-in coffee by liquid chromatography mass spectrometry. Journal of Chromatography A, 1610, 460566.

  7. Escrivá, L., Font, G., Manyes, L., Berrada, H. 2017. Studies on the presence of mycotoxins in biological samples: An overview. Toxins, 9, 251–284.

  8. Fibigr, J., Šatínský, D., Solich, P. 2017. A UHPLC method for the rapid separation and quantification of anthocyanins in acai berry and dry blueberry extracts. Journal of Pharmaceutical and Biomedical Analysis, 143, 204–213.

  9. Gai, Q.Y., Jiao, J., Wang, X., Liu, J., Fu, Y.J., Lu, Y., Wang, Z.Y., Xu, X.J. 2020. Simultaneous determination of taxoids and flavonoids in twigs and leaves of three Taxus species by UHPLC-MS/MS. Journal of Pharmaceutical and Biomedical Analysis, 189, 113456.

  10. Geng, Z., Yang, D., Zhou, M., Zhang, P., Wang, D., Liu, F., Zhu, Y., Zhang, M. 2014. Determination of deoxynivalenol-3-glucoside in cereals by hydrophilic interaction chromatography with ultraviolet detection. Food Analytical Methods, 7, 1139–1146.

  11. Laganà, A. 2017. Introduction to the toxins special issue on LC-MS/MS methods for mycotoxin analysis. Toxins, 9, 325–328.

  12. Lechner, M., Breeze, C.E., Vaz, F., Lund, V.J., Kotecha, B. 2019. Betel nut chewing in high-income countries-lack of awareness and regulation. The Lancet Oncology, 20, 181–183.

  13. Liao, C.D., Wong, J.W., Zhang, K., Hayward, D.G., Lee, N.S., Trucksess, M.W. 2013. Multi-mycotoxin analysis of finished grain and nut products using high-performance liquid chromatography–triple-quadrupole mass spectrometry. Journal of Agricultural and Food Chemistry, 61, 4771–4782.

  14. Lin, H.Y., Liang, X.T., Yang, W.G., Chien, W.J. 2020. High-performance liquid chromatography-tandem mass spectrometry with polar C18 for rapid quantification of anthocyanin and flavonoid in black soybean extracts. International Journal of Applied Science and Engineering, 17, 363–371.

  15. Liu, H., Luo, J., Kong, W., Liu, Q., Hu, Y., Yang, M. 2016. UFLC-ESI-MS/MS analysis of multiple mycotoxins in medicinal and edible Areca catechu. Chemosphere, 150, 176–183.

  16. Muñoz-Solano, B., González-Peñas, E. 2020. Mycotoxin determination in animal feed: An LC-FLD method for simultaneous quantification of aflatoxins, Ochratoxins and Zearelanone in This Matrix. Toxins, 12, 374–390.

  17. Pantano, L., La Scala, L., Olibrio, F., Galluzzo, F.G., Bongiorno, C., Buscemi, M.D., Macaluso, A., Vella, A. 2021. QuEChERS LC–MS/MS screening method for mycotoxin detection in cereal products and spices. International Journal of Environmental Research and Public Health, 18, 3774–3791.

  18. Rodríguez, M., Núñez, F. 2020. Novel approaches to minimizing mycotoxin contamination. Toxins, 12, 216–219.

  19. Santos Pereira, C.C., Cunha, S., Fernandes, J.O. 2019. Prevalent mycotoxins in animal feed: Occurrence and analytical methods. Toxins, 11, 290–352.

  20. Sun, J., Li, W., Zhang, Y., Hu, X., Wu, L., Wang, B. 2016. QuEChERS purification combined with ultrahigh-performance liquid chromatography tandem mass spectrometry for simultaneous quantification of 25 mycotoxins in cereals. Toxins, 8, 375–393.

  21. Tolosa, J., Barba, F.J., Font, G., Ferrer, E. 2019. Mycotoxin incidence in some fish products: QuEChERS methodology and liquid chromatography linear ion trap tandem mass spectrometry approach. Molecules, 24, 527–538.

  22. Zhang, L., Dou, X.W., Zhang, C., Logrieco, A.F., Yang, M.H. 2018. A review of current methods for analysis of mycotoxins in herbal medicines. Toxins, 10, 65–104.

  23. Zhang, S., Zhao, Y., Li, H., Zhou, S., Chen, D., Zhang, Y., Yao, Q., Sun, C. 2016. A simple and high-throughput analysis of amatoxins and phallotoxins in human plasma, Serum and urine using UPLC-MS/MS combined with PRiME HLB μElution platform. Toxins, 8, 128–141.


Received: 2021-05-21
Revised: 2021-07-07
Accepted: 2021-07-08
Publication Date: 2021-09-01

Cite this article:

H.-Y., Lin, Agrawal, D.C., Yang, W.-G., Chien, W.-J. 2021. A simple HPLC-MS/MS method for the analysis of multi-mycotoxins in betel nut, International Journal of Applied Science and Engineering. 18, 2021145.

  Copyright The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are cited.