# Occurrence of Zeralenone and Enniatin B in Swiss Wheat Grains and Wheat Flours
## 2 Materials and Methods
### HPLC-MS/MS Analysis  
LC-MS/MS analyses were conducted on a system consisting of an Agilent 1290 Infinity II chromatographic system coupled to an Agilent 6530 Q-TOF mass spectrometer. Separation of analytes was performed using an Agilent Poroshell 120 EC-C18 (2.1 \(\times\) 100 mm, 2.7 \(\upmu\)m) column protected by a guard column (Agilent EC-18, 2.1 \(\times\) 5 mm, 2.7 \(\upmu\)m). For ZEA analysis, the flow rate was set to 0.28 mL/min, and the column temperature was set at 35 \({}^{\circ}\)C. The two elution mobile phases were made up of water + 0.1% acetic acid (mobile phase A) and methanol + 0.1% acetic acid (mobile phase B). Gradient elution was as follows: 0-0.5 min, 10% B; 6-15 min, 98% B; 15.10-17 min, 10% B. Re-equilibration time was 6 min. Injection volume was 10 \(\upmu\)L.  
For ENB analysis, the flow rate was set to 0.25 mL/min, and the column temperature was set at 35 \({}^{\circ}\)C. The two elution mobile phases were made up of water + 0.1% formic acid + 5 mmol ammonium formate (mobile phase A) and methanol + 0.1% formic acid + 5 mmol ammonium formate (mobile phase B). Gradient elution was as follows: 0-0.5 min, 10% B; 6-15 min, 98% B; 15.10-17 min, 10% B. Re-equilibration time was 5 min. Injection volume was 10 \(\upmu\)L.  
Both LC methods were used the same MS parameters. The MS analyses were performed using an Agilent 6530 Q-TOF instrument in negative ionization mode (ESI-) in the spectral range of 100-1000 Da. Nitrogen served as the nebulizer and collision gas. The MS parameters were as follows: gas temperature, 350 \({}^{\circ}\)C; drying gas, 10 L/min; nebulizer, 40 psi; sheath gas temperature, 350 \({}^{\circ}\)C; sheath gas flow, 11 L/min; capillary voltage, 3500 V (for ZEA)/3000 V (for ENB); fragmentor voltage, 100 V. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Samples** & **Form** & **Land of Origin** & **Type** & **Year of Harvest** & **Label** \\ \hline G1 & Grains & Switzerland & Whole grain & 2021 & Conventional (Quality: Class 2) \\ G2 & Grains & Switzerland & Whole grain & 2021 & Conventional (Quality: Class 2) \\ G3 & Grains & Switzerland & Whole grain & 2021 & Conventional (Quality: Class 1) \\ G4 & Grains & Switzerland & Whole grain & 2021 & IP-Suisse (Quality: Class 2) \\ G5 & Grains & Switzerland & Whole grain & 2021 & Conventional (Quality: Class Top) \\ G6 & Grains & Switzerland & Whole grain & 2020 & IP-Suisse (Quality: Class 2) \\ G7 & Grains & Switzerland & Whole grain & 2020 & Conventional (Quality: Class 1) \\ F1 & Flour & Germany & Whole grain & NC * & Organic (label Bioland) \\ F2 & Flour & Germany, Austria, & 405 & NC * & Organic (Demeter label) \\ F3 & Flour & Hungary, Austria & 1050 & NC * & Organic (Demeter label) \\ \hline \hline \end{tabular}
* NC: not communicated.  
\end{table} 
Table 1: List of samples analyzed during the study. The mycotoxins were identified in wheat extracts using Mass Hunter Qualitative Analysis Software (v 10.0, Agilent) by comparing their retention time and _m/z_ to those of the reference standards. For the quantification, a matrix-matched calibration was prepared by spiking 5 g of cleaned wheat grains (Zwicky, Mullheim-Wigoltingen, Switzerland) with different concentrations of ZEA and ENB standards. The grains spiked with the mycotoxins were mixed using a vortex and allowed to stand overnight under the fume hood. On the next day, the grains were ground and extracted using the same extraction protocol as used for the samples, in triplicate. The following linear regressions were used for quantification:  
ZEA: linear range: 200 \(\mathrm{\SIUnitSymbolMicro g}\)/kg-15 \(\mathrm{\SIUnitSymbolMicro g}\)/kg. Equation: \(y=1x10^{8}\)\(x-1473.3\); R\({}^{2}=0.9999\). LOD = 7.5 \(\mathrm{\SIUnitSymbolMicro g}\)/kg; LOQ = 15 \(\mathrm{\SIUnitSymbolMicro g}\)/kg. ENB: linear range: 2000 \(\mathrm{\SIUnitSymbolMicro g}\)/kg-3 \(\mathrm{\SIUnitSymbolMicro g}\)/kg. Equation: \(y=3x10^{8}\)\(x+518\),566; R\({}^{2}=1\). LOD = 1.5 \(\mathrm{\SIUnitSymbolMicro g}\)/kg; LOQ = 3 \(\mathrm{\SIUnitSymbolMicro g}\)/kg.