*4.8. Detection of Spiked and Authentic Samples*

The recovery test of the spiked samples and the verification of the authentic samples by LC-MS/MS were used to evaluate the accuracy and precision of the developed ICA for ZEN detection. A total of 187 authentic cereal and its product samples, including 40 corn, 19 wheat, 39 wheat flour, 49 cereal product, and 40 feed samples, were collected from China in 2019 (Tables S3 and S4). The cereal product samples were mainly corn gluten meal, corn gluten, corn germ, soybean meal, peanut meal, and rice bran meal. All of the authentic samples were homogenized and stored at −20 ◦C before the detection procedure.

For the recovery test, the cereal and feed samples (5.0 g), which had been confirmed to be free of ZEN contamination, were completely crushed and spiked the ZEN standard at 0, 3, 20, and 50 ng/g, and were stored for 2 h at room temperature. The methanol/water (12.5 mL, 1:1, *v*/*v*) was used as an extracting solution. Vortex blending was performed on the mixture for 5 min, and was then centrifuged for 10 min at 4000 rpm/min. The supernatant solution was diluted four-fold with a working buffer and was adjusted to pH 6–8 and detected by the developed ICA method. After 5 min, the strip of

ICA flowed over the absorption pad, and the result was judged by the naked eye and the strip reader, respectively. The pretreatment and detection for the authentic cereal and its product samples were carried out in the same way as the above procedures. The correlation between the results of the ICA and LC-MS/MS was also evaluated.

To verify the reliability of the ICA method using the LC-MS/MS method [33,34], the completely crushed corn, wheat, or feed sample (5.0 g) was added in an extract solution of acetonitrile/water/formic acid (10 mL, 80: 19: 1, *v*/*v*/*v*). The mixture was mixed with an ultrasonic bath for 30 min, and then centrifuged at 4000 rpm/min for 5 min. The supernatant (10 mL) was transferred into another tube, followed by adding C<sup>18</sup> (100 mg) and MgSO<sup>4</sup> (200 mg). The mixture was vortexed for 3 min and centrifuged at 4000 rpm/min for 5 min. The supernatant (1 mL) was concentrated to dryness by nitrogen gas. The residue was redissolved in methanol/water (400 µL, 50:50, *v*/*v*) and filtered with a 0.22 µm nylon filter, then analyzed by the LC-MS/MS with an Agilent Zorbax SB C18 reverse-phase column (3.5 µm, 150 mm × 2.1 mm) with a column temperature of 40 ◦C. The mobile phase was the different volume ratio of the acetonitrile/water, and flowed on the gradient elution program at a flow rate of 0.2 mL/min. The analysis was performed by multi-reaction monitoring (MRM) technology, and the ion source was an electrospray ion (ESI). The capillary voltage was at 3.0 kV, and the argon collision pressure was 2.60 <sup>×</sup> <sup>10</sup>−<sup>4</sup> Pa. The mass-to-charge ratios of the ZEN parent ion, quantitative ion, and qualitative ion were 317.1, 174.9, and 273.9, respectively.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6651/12/8/521/s1, Table S1: The parameters of ELISA and ICA for ZEN, Table S2: The ZEN-positive levels in the authentic samples from China in 2019, Table S3: The information of the authentic samples from China in 2019, Table S4: The information of the authentic samples from China in 2019, Figure S1: The standard curve of ELISA for ZEN, Figure S2. The stability and sensitivity tests of the ICA strip for ZEN in the storage of 3 months, Figure S3. The correlation between the quantitative ICA and LC-MS/MS for ZEN in the authentic samples.

**Author Contributions:** X.H. executed and validated the experiments; Y.M. interpreted the results and analyzed the data; C.Y. collected the data; Z.L. drafted the manuscript; M.L. conceived and designed the experiments, and D.D. modified and validated the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the National Natural Science Foundation of China (31701687), the Natural Science Foundation of Jiangsu Province (BK20170537), the National Key Research and Development Program of China (2017YFC1200100), the Senior Talent Scientific Research Initial Funding Project of Jiangsu University (16JDG035), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD-2018-87) and the Jiangsu Collaborative Innovation Center of Technology and Material of Water Treatment.

**Conflicts of Interest:** The authors declare no conflict of interest.
