*2.8. Investigation of Authentic Samples by the ICA and LC-MS*/*MS*

Using the ICA method, the ZEN contamination in the authentic samples was visually judged following the digitized detection. The results of the semi-quantitative and quantitative detection by the two models of visualization and digitization for ICA were in good agreement. Furthermore, the digitized detection of ICA could obtain accurate ZEN levels in the authentic samples, and some negative samples might have also detected ZEN contamination, which could further expand the detection ability and sensitivity of the developed ICA method. A total of 187 cereals and its product samples were investigated for ZEN contamination from China in 2019. The results of the ZEN levels using the quantitative ICA are shown in Table 3. The ZEN-positive rate was 62.6% in 117 out of the 187 total samples, which detected positivity in 28 out of 40 corn samples, 7 out of 19 wheat samples, 17 out of 39 wheat flour samples, 39 out of 49 cereal product samples, and 26 out of 40 feed samples. From the entire sample, the ZEN-positive levels ranged from 2.7 to 867.0 ng/g, with an average ZEN-positive level being 85.0 ng/g.

**Table 3.** The investigation of the ZEN in the original samples using the quantitative ICA from China in 2019.


The highest ZEN-positive rate was found in the cereal product samples (79.6%), which, along with the corn samples (70.0%) and the feed samples (65.0%), had above the average ZEN-positive rate (62.6%; Figure 6A). The average ZEN-positive levels for the cereal product and feed samples reached 95.9 ng/g and 173.0 ng/g, which were higher than the average ZEN-positive level of all of the samples (85.0 ng/g; Figure 6B). The lowest average ZEN-positive level was detected in the wheat flour samples, thus demonstrating the lower ZEN-exposure risk for wheat flour. To make a further assessment of the ZEN-exposure levels from China in 2019, the distribution of the ZEN-positive sample and ZEN-positive rate at different levels is shown in Figure 6C. The ZEN-positive levels were classified into five levels, namely, <10 ng/g, 10–60 ng/g, 60–100 ng/g, 100–350 ng/g, and >350 ng/g, according to the MLs from European Commission and the China National Standard. The ZEN-positive levels of 68.4% with 80 out of 117 ZEN-positive samples were lower than the ML value of China (60 ng/g), while 94% of samples had ZEN-positive levels less than the European Commission regulation of 350 ng/g. However, it is noteworthy that 6.0% in 117 of the ZEN-positive samples showed ZEN levels more than 350 ng/g, exceeding the ML value of China and the European Commission. In addition, the highest ZEN-positive

level was detected as 867.0 ng/g in a feed sample, which had been 14-fold and 2.5-fold higher than the ML values for China and the European Commission, respectively. These results indicate that ZEN contamination was a commonly occurring problem for the authentic cereals and their product samples from China in 2019, and most of the contamination levels were within the bounds of the control, but some samples were seriously ZEN-positive and needed to be focused on in order to ensure food safety and human health. *Toxins* **2020**, *x*, x FOR PEER REVIEW 7 of 12 LC‐MS/MS method for detecting the ZEN in the authentic samples was R2 = 0.9794 (Y = 0.94X + 4.8442; Figure S3), which further verified the reliability and accuracy of the proposed ICA method.

**Figure 6.** The distribution of ZEN‐positive rate (**A**), average ZEN‐positive level (**B**) and ZEN‐positive sample at different levels (**C**) from China in 2019. **Figure 6.** The distribution of ZEN-positive rate (**A**), average ZEN-positive level (**B**) and ZEN-positive sample at different levels (**C**) from China in 2019.

**3. Conclusions** To further enhance the detection ability and ensure food safety, a semi‐quantitative and a quantitative detection method of ICA were successfully established and used for detecting ZEN in cereal and feed samples. The results of the ICA could be judged visually by the naked eye or with a The LC-MS/MS detection showed that ZEN contaminations in the positive samples ranged from 3.2 to 761.7 ng/g (Table S2). The large correlation of results from the quantitative ICA method and LC-MS/MS method for detecting the ZEN in the authentic samples was R<sup>2</sup> = 0.9794 (Y = 0.94X + 4.8442; Figure S3), which further verified the reliability and accuracy of the proposed ICA method.

### digitized strip reader within 5 min. The visual LOD for ZEN was 0.50 ng/mL using the semi‐ **3. Conclusions**

*4.1. Reagents and Materials*

quantitative ICA. The quantitative ICA had a lower LOD of 0.25 ng/mL, and a wider detection range, which could obtain accurate ZEN levels. The powerful detection capability of the developed ICA was demonstrated by the evaluation of its sensitivity, specificity, stability, accuracy, and precision. The ICA could dramatically shorten the analytical procedure and the overall detection time when compared with the micro‐well based ELISA or chromatographic‐based HPLC method. A total of 187 samples of authentic cereals and their products from China in 2019 were investigated for ZEN contamination by both the developed ICA and the referenced LC‐MS/MS, in order to demonstrate the reliability of the proposed ZEN detection method. The ZEN‐positive rate was 62.6%, and the ZEN‐positive levels ranged from 2.7 to 867.0 ng/g, with an average ZEN‐positive level being 85.0 ng/g. The highest ZEN‐positive level was detected as 867.0 ng/g in a feed sample. It is noteworthy that the ZEN contamination levels of 6.0% in 117 ZEN‐positive samples exceeded the ML value of the China and European Commission. The results of this investigation suggest that ZEN contamination in China occurred widely and had a high detection rate. The efficiency and accuracy of the ZEN detection could have further improvement, and the study could provide an alternative approach and valuable information about ZEN contamination in China. **4. Materials and Methods** To further enhance the detection ability and ensure food safety, a semi-quantitative and a quantitative detection method of ICA were successfully established and used for detecting ZEN in cereal and feed samples. The results of the ICA could be judged visually by the naked eye or with a digitized strip reader within 5 min. The visual LOD for ZEN was 0.50 ng/mL using the semi-quantitative ICA. The quantitative ICA had a lower LOD of 0.25 ng/mL, and a wider detection range, which could obtain accurate ZEN levels. The powerful detection capability of the developed ICA was demonstrated by the evaluation of its sensitivity, specificity, stability, accuracy, and precision. The ICA could dramatically shorten the analytical procedure and the overall detection time when compared with the micro-well based ELISA or chromatographic-based HPLC method. A total of 187 samples of authentic cereals and their products from China in 2019 were investigated for ZEN contamination by both the developed ICA and the referenced LC-MS/MS, in order to demonstrate the reliability of the proposed ZEN detection method. The ZEN-positive rate was 62.6%, and the ZEN-positive levels ranged from 2.7 to 867.0 ng/g, with an average ZEN-positive level being 85.0 ng/g. The highest ZEN-positive level was detected as 867.0 ng/g in a feed sample. It is noteworthy that the ZEN contamination levels of 6.0% in 117 ZEN-positive samples exceeded the ML value of the China and European Commission. The results of this investigation suggest that ZEN contamination in China occurred widely and had a high detection rate. The efficiency and accuracy of the ZEN detection

membranes were from Millipore (Bedford, MA, USA). The absorbent pad, sample pad, polyvinyl chloride (PVC) sheet, and glass‐fiber conjugate pad were provided by Jiening Bio. Tech. Co., Ltd. could have further improvement, and the study could provide an alternative approach and valuable information about ZEN contamination in China.
