MaterialsOTA, ±10 µg/mL in acetonitrile (ACN), aflatoxin mix containing AFB1, AFB2, AFG1, AFG2, ±20 µg/mL in ACN, DON, ±100 µg/mL in ACN, zearalenone (ZEN), ±100 µg/mL in ACN, fumonisin mix containing FB1, FB2, ±50 µg/mL in 50/50 ACN/H2O, NIV, ±100 µg/mL in ACN, neosolaniol (NEO), ±100 µg/ml in ACN, DOM, ±50 µg/mL in ACN, T2, ±100 µg/mL in ACN, HT2, ±100 µg/mL in ACN, 3-ADON, ±100 µg/mL in ACN, 15-ADON, ±100 µg/mL in ACN, diacetoxyscirpenol (DAS), ±100 µg/mL in ACN, fusarenon-X (FX), ±100 µg/mL in ACN, STC, ±50 µg/mL in ACN, all the above components were acquired from Biopure, Food Risk Management B.V., RomerLabs agent (Oostvoorne Nederland). Roquefortine C (ROQ-C), 0,5 mg, was purchased at Enzo Life Sciences, (Axxora Platform, Brussels, Belgium), ZAN, 10 mg, at Sigma (Darmstadt, Germany), AME, 5 mg, and alternariol (AOH), 5 mg, at Sigma (Darmstadt, Germany), FB3, 1 mg, at Medical Research Council (Swindow, UK), ENN B, ±1 mg/mL in methanol was acquired at Fermentek (Jerusalem, Israel). Acetic acid was provided by Merck (Overijse, Belgium), Methanol absolute LC-MS (for mobile phase) by Biosolve Chemicals (Valkenswaard, The Netherlands), ammonium acetate by Merck (Overijse, Belgium), n-hexane by BDH HiperSolv CHROMANORM (VWR International, Leuven, Belgium), ACN HPLC by Biosolve Chemicals (Valkenswaard, The Netherlands). All used solvents were HPLC or MS-grade.SamplesA total of 41 legume samples of 1 kg each from a legume processing company were included to examine the fluctuations in mycotoxin levels during processing. The samples originated from two production batches with different production dates and different raw material suppliers, and were followed from the raw material to the final products. Due to a non-disclosure agreement, the nature of the legume from the Fabaceae family and the company cannot be disclosed.For quantification, matrix matched calibration curves (MMCC) were constructed on blank legume flour. The blank sample must be similar to the products being analyzed and the mycotoxin concentration should be lower than the limit of detection (LOD)37.For the construction of the MMCC, five blank samples were spiked with a multi-mycotoxin mix and were stored in the dark for 15 min so the mycotoxins could be absorbed in the samples. Sample preparation was performed using a validated method as described by Chilaka et al.20. Briefly, each sample was thoroughly homogenized, and milled, from which a representative portion of 5 g was spiked with internal standards zearalanone (ZAN) and deepoxy-deoxynivalenol (DOM) at a concentration of 250 and 150 μg/kg, respectively. DOM was used as internal standard for DON, DON-3G, 3-ADON, and 15-ADON while ZAN was used for the other mycotoxins. The sample was extracted with 20 mL of extraction solvent [ACN/H2O/acetic acid (79/20/1, v/v/v)], vortexed, agitated and centrifuged at 4000 × g for 15 min. The supernatant was passed through a pre-conditioned C18 SPE column (Phenomenex, Utrecht, The Netherlands). The extraction was repeated by adding 5 mL of the extraction solvent ACN/H2O/acetic acid (79/20/1, v/v/ v) and the combined extract was made up to 25 mL with the extraction solvent (ACN/H2O/acetic acid (79/20/1, v/v/v)). The extract was transferred into the extraction tube and defatted using 10 mL of hexane. The defatted extract was divided into two parts, 10 mL of the first part was filtered through a glass filter, and 12.5 mL of the second part was mixed with 27.5 mL of ACN/acetic acid (99/1, v/v). Thirty milliliters of the mixture were further purified by passing through a MultiSep® 226 AflaZon+ multifunctional column (Romer Labs, Gernsheim Germany). The total Multisep 226 eluate was added to 2 mL of the glass filtered portion, and the mixture was evaporated to dryness under a gentle nitrogen flow in a thermostatic water bath heated at 40 °C. The residue was then redissolved in 150 μL of the injection solvent containing mobile phase A (H2O/MeOH/acetic acid (94/5/1, v/v/ v) +5 mM ammonium acetate) and mobile phase B (H2O/MeOH/ acetic acid (2/97/1, v/v/v) +5 mM ammonium acetate) mixed in the ratio of 3/2, v/v. The redissolved extract was centrifuged for 10 min at 14000 g, and further filtered using Ultrafree® PVDF centrifuge filters (Millipore Bedford, MA, USA) and analyzed using LC-MS/MS.LC-MS/MS analysisAn LC-MS/MS Waters Acquity HPLC coupled with a Micromass Quattro Premier XE triple quadrupole mass spectrometer was used. The column used was a Symmetry C18 (150 mm × 2.1 mm i.d. 5 µm) column with a guard column (10 mm × 2.1 mm i.d.) of the same material (Waters, Zellik, Belgium), and was kept at room temperature. During the run, a gradient elution program was employed. This gradient program involved varying amounts of mobile phase A and mobile phase B. These were used at a flow rate of 0.3 mL/min. The total analytical run time was 28 min. The gradient started at 95% mobile phase A with a linear decrease to 35% in 7 min. Mobile phase A decreased to 25% at 4 min, and an isocratic period of 100%, mobile phase B started at 11 min for 2 min. Initial column conditions were reached at 23 min using a linear decrease of mobile phase B, and the column was reconditioned for 5 min before the following injection. An injection volume of 10 μL was used. The capillary voltage was 3.2 kV and nitrogen was used as the desolvation gas. The mass spectrometer was operated with selected reaction monitoring (SRM) channels in the positive electrospray ionization (ESI+) mode. Source and desolvation temperatures were set at 150 and 350 °C, respectively. The instrumental control and data processing were performed with Masslynx™ version 4.1 and Quanlynx® version 4.1 software (Micromass, Manchester, UK). Prior to the study, the cone voltage at which the precursor ion is most abundant for each component was determined, as well as the corresponding precursor ion. Then, the collision energy and fragment ions formed, and their corresponding ionization mode were determined. More information about the MS/MS transition of each mycotoxin and the validation parameters according to Commission Regulation (EC) No. 401/2006 (68) and Commission Decision (EC) No. 2002/657 are described in refs. 20,37,38,39 and in the supplementary material. The performance of the method, including the LOD, LOQ, accuracy and precision, expressed as apparent recovery (Rapp), repeatability (RSDr), and intermediate precision (RSDR) are included in the Supplementary Material 2 and 3. The Waters QuanLynx software was used to determine the mycotoxin concentration in the food samples. For each set of analyses, a MMCC was constructed. When the identification criteria for mycotoxins in food and feed stated in the Guidance document SANTE/12089/201640 were met, but the concentration was lower than the LOD or LOQ, the sample was considered positive, but no concentration is given.
