Hydrophobic deep eutectic solvents as a greener alternative for extracting mycotoxins from milk and plant-based beverages

Hydrophobic deep eutectic solvents as a greener alternative for extracting mycotoxins from milk and plant-based beverages Raquel Torrijos1,2, Titiwan Changsan3, Chiara Dall’Asta1, Chongdee Buranachai3 1Department of Food and Drug, University of Parma, Italy; 2Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Spain; 3Department of Chemistry, Division of Physical Science, Faculty of Science, Prince of Songkla University, Hat Yai, Thailand. [email protected] / [email protected] Mycotoxins are harmful secondary metabolites produced by fungal species belonging to the Aspergillus, Penicillium, Fusarium, and Alternaria genera that can contaminate a wide range of food commodities, including milk and the increasingly popular plant-based beverages [1,2]. These toxins are routinely determined in foodstuffs using analytical procedures that typically involve hazardous organic solvents as extractants. In this regard, natural deep eutectic solvents (NADES) are a promising alternative to replace conventional solvents since they are biodegradable, present low toxicity, are easy to prepare, and inexpensive. Generally, NADES are formed by combining one or more hydrogen bond acceptors (HBAs) and one or more hydrogen bond donors (HBDs). These solvents are formed through specific molar ratios of natural components such as sugars, alcohols, amino acids, organic acids, choline derivatives, and terpenes. When combined, these constituents engage in strong hydrogen bonding interactions, resulting in a significant depression of the melting point and the formation of a stable liquid phase at room temperature [3]. Figure 3. Optimization volume of the HDES2. Figure 4. Mycotoxin recoveries from oat-based beverages using 900 µL of HDES2 as extractant. Although their extensive use for the extraction of biologically active compounds, DES has seldom been applied to mycotoxin extraction in foodstuffs. Thus, this work aimed to develop an environmentally friendly analytical method for mycotoxin determination in plant-based beverages and milk, combining hydrophobic deep eutectic solvent (HDES) (Figure 1) with liquid chromatography coupled to mass spectrometry in tandem (LC-MS/MS). Figure 1. Natural hydrophobic deep eutectic solvent (HDES) proposed in the study. Combinations of HBA and HBD tested (molar ratios): 2.3 LC-MS/MS analysis Menthol:Thymol (1:2) (HDES1) Menthol:Thymol:Acetic acid (1:2:1) (HDES2) Menthol:Thymol:Lactic acid (1:2:1) (HDES3) Stirred at 300 rpm and heated 70 ºC for 20 min. 1. INTRODUCTION HDES 2.1 Natural Hydrophobic Deep Eutectic solvent (HDES) preparation Menthol (HBA) Thymol (HBD) 2.2 Mycotoxin spiking and extraction procedure The use of 900 µL of the HDES2 (Menthol:Thymol:Acetic acid, at molar ratio 1:2:1) yielded recoveries of 93.6% for AFB1,95.4% for AFB2,88.2% for AFG1, 96.1% for AFG2,108.9% for AFM1, and 94.1% for OTA, with overall RSD <3.8%. Mycotoxin Rt Precursor ion (m/z) Molecular ion Product ion 1a(m/z)CE Product ion 2b(m/z) CE AFB17.67 313.1 [M+H]+285.1 25 241.2 42 AFB27.46 315.2 [M+H]+287.0 25 259.0 30 AFG17.25 329.0 [M+H]+243.0 25 311.0 20 AFG27.02 331.3 [M+H]+285.0 30 245.3 25 AFM16.96 329.0 [M+H]+273.0 35 229.0 59 FB18.70 722.4 [M+H]+334.0 43 352.0 42 FB29.49 706.4 [M+H]+318.0 42 354.0 42 OTA 8.98 404.5 [M+H]+238.7 21 101.7 68 ZEN 9.23 317.2 [M+H]-131.0 32 175.0 32 DON 4.63 355.2 [M+Ac]-295.0 13 265.0 17 3.1 Natural Hydrophobic Deep Eutectic solvent (HDES) optimization 3.2 Recovery results of HDES2 The application of a HDES prepared with Menthol:Thymol:Acetic acid (molar ratio 1:2:1) on oat beverages provided satisfactory recovery results for 6 mycotoxins, including the five aflatoxins (AFB1, AFB2, AFG1, AFG2, AFM1), and ochratoxin A (OTA). Recoveries ranging from 14.0% to 62.0% were obtained for the other tested analytes, suggesting that further optimization should be evaluated to increase the extraction efficiency for these mycotoxins. These preliminary findings reveal the promising potential of HDES as a sustainable alterative to conventional solvents for the simultaneous extraction of AFs and OTA from plant-based beverages, with possible applications extending to other liquid matrices, including milk. Spiking: AFB1, AFB2, AFG1, AFG2, FB1, FB2, OTA, ZEN, DON (10 µg L-1); AFM1 (1 µg L-1) 1. 5 mL oat-beverage + 9001200 µL HDES and vortex 10 s. Figure 1. LC-MS/MS chromatogram (quantifier ion displayed) in oat-based beverage at 10 µg L-1 for AFB1, AFB2, AFG1, AFG2,FB1,FB2, OTA, ZEN, DON; and 1µg L-1 for AFM1. The addition of acetic acid enhanced the extraction of some mycotoxins such as FB1, FB2, and OTA. As a result, HDES2 Menthol:Thymol:Acetic acid (1:2:1) was selected for the simultaneous extraction of 10 mycotoxins. The best recoveries were obtained when adding 800 and 900 µL of HDES2. Nevertheless, using 800 µL of HDES was complicated to collect from the beverage. Moreover, the recoveries decreased when HDES volume were 1000–1200 µL. Consequently, 900 µL of HDES2 was selected as optimal for mycotoxin extraction. 3. RESULTS AND DISCUSSION Table 1. MS/MS parameters for the mycotoxins studied. Multiple reaction monitoring (MRM) was used as acquisition mode. 2. Mycotoxin extraction by shaken 10 min. 3. Centrifugation 4000 rpm for 20 min. 4. Separation of HDES (top layer) and dilution with EtOH. 5. Filtration and injection into the LC-MS/MS system. Chromatography: Column: XSelect® HSS T3 column (2.1x150 mm, 2.5 µm) Mobile Phases: H2O 0.2 % acetic acid (v/v) 5 mM ammonium acetate (Phase A); MeOH 0.2% acetic acid (v/v) (Phase B) Gradient: 0 min, 5%B; 8 min, 90%B; 12 min, 5%B. Flow rate: 0.4 mL/min Column Temperature: 40 °C Acknowledgments References 4. CONCLUSIONS 2. MATERIALS AND METHODS Figure 2. Comparative analysis of HDES1, HDES2, and HDES3. AFB1 AFB2 AFG1 AFG2 AFM1 OTA This work has carried out in the frame of the ALIFAR project, funded by the Italian Ministry of University through the program ‘Dipartimenti di Eccellenza 2023-2027’; the Horizon Europe MSCA-SE MYCOBEANS project (Grant Agreement N. 101131125) funded by the European Union. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them. R.T. would like to thank the Conselleria de Educación, Universidades y Empleo of Generalitat Valenciana for the APOSTD grant (CIAPOS/2022/015). [1] N. Benkerroum, International Dairy Journal, 2016,62,63. [2] R. Torrijos, O. Mihalache, C. Dall’Asta, Food Control, 2025,170,111051. [3] K. Wu, J. Ren, Q. Wang, M. Nuerjiang, X. Xia, C. Bian, Foods, 2022,11(21), 3528