Sample pretreatment is a vital step in the detection of mycotoxins, and traditional pretreatment methods are time-consuming, labor-intensive and generate much organic waste liquid. In this work, an automatic, high-throughput and environmentally friendly pretreatment method is proposed. Immunomagnetic beads technology and dispersive liquid–liquid microextraction technology are combined, and the zearalenone in corn oils is directly purified and concentrated under the solubilization effects of surfactant. The proposed pretreatment method allows for the batch pretreatment of samples without pre-extraction using organic reagents, and almost no organic waste liquid is produced. Coupled with UPLC-FLD, an effective and accurate quantitative detection method for zearalenone is established. The recovery of spiked zearalenone in corn oils at different concentrations ranges from 85.7 to 89.0%, and the relative standard deviation is below 2.9%. The proposed pretreatment method overcomes the shortcomings of traditional pretreatment methods and has broad application prospects.

An immunoaffinity magnetic beads (IMBs) based automatic pretreatment method was developed for the quantitative analysis of deoxynivalenol (DON) by ultra-performance liquid chromatography and ultraviolet detector (UPLC-UV). First, N-hydroxysuccinimide-terminated magnetic beads (NHS-MBs) with good magnetic responsivity and dispersibility were synthesized and characterized by optical microscopy, scanning electron microscopy (SEM), and laser diffraction-based particle size analyzer. Then, the amino groups of anti-DON monoclonal antibody (mAb) and the NHS groups of NHS-MBs were linked by covalent bonds to prepare IMB, without any activation reagent. The essential factors affecting the binding and elution of DON were meticulously tuned. Under optimal conditions, DON could be extracted from a real sample and eluted from IMB by water, enabling environmentally friendly and green analysis. Hence, there was no need for dilution or evaporation prior to UPLC-UV analysis. DON in 20 samples could be purified and concentrated within 30 min by the mycotoxin automated purification instrument (MAPI), allowing for automated, green, high-throughput and simple clean-up. Recoveries at four distinct spiking levels in corn and wheat ranged from 92.0% to 109.5% with good relative standard deviations (RSD, 2.1–7.0%). Comparing the test results of IAC and IMB in commercial samples demonstrated the reliability and superiority of IMB for quantitatively analyzing massive samples.

In this study, a multicolor visual method based on a magnetic immunoassay and enzyme-induced gold nanobipyramids (Au NBPs) etching was developed for deoxynivalenol (DON) detection. The magnetic beads modified with high affinity DON monoclonal antibodies were used as a carrier for target enrichment and signal transformation and the Au NBPs with excellent plasmonic optical properties were served as enzymatic etching substrates. The oxidation state TMB, which was generated through catalysis of horseradish peroxidase (HRP), induced the etching of plasmonic Au NBPs, resulting in the longitudinal peak blue-shift of local surface plasmon resonance (LSPR). Correspondingly, Au NBPs with various aspect ratios displayed a variety of individual colors which were visualized by the naked eye. The LSPR peak shift was linearly related to the DON concentration in the range of 0~2000 ng/mL and the detection limit was 57.93 ng/mL. The recovery for naturally contaminated wheat and maize at different concentrations ranged from 93.7% to 105.7% with a good relative standard deviation below 11.8%. Through observing the color change in Au NBPs, samples with overproof DON could be screened preliminarily by the naked eye. The proposed method has the potential to be applied in on-site rapid screening of mycotoxins in grain. In addition, the current multicolor visual method only used for the simultaneous detection of multiple mycotoxins is in urgent need of a breakthrough to overcome the limitation of single mycotoxin detection.

A rapid and high-throughput fluorescence detection method for zearalenone (ZEN) based on a CuO nanoparticle (NP)-assisted signal amplification immunosensor was developed using an automated sample pretreatment and signal conversion system. CuO NPs with high stability and biocompatibility were used as carriers to immobilize anti-ZEN antibodies. The obtained CuO NP-anti-ZEN can maintain the ability to recognize target toxins and act as both a signal source and carrier to achieve signal conversion using automated equipment. In this process, target toxin detection is indirectly transformed to Cu²⁺ detection because of the large number of Cu²⁺ ions released from CuO NPs under acidic conditions. Finally, a simple and high-throughput fluorescence assay based on a fluorescent tripeptide molecule was employed to detect Cu²⁺, using a multifunctional microporous plate detector. A good linear relationship was observed between the fluorescence signal and the logarithm of ZEN concentration in the range of 16.0–1600.0 μg/kg. Additionally, excellent accuracy with a high recovery yield of 99.2–104.9% was obtained, which was concordant with the results obtained from LC-MS/MS of naturally contaminated samples. The CuO NP-based assay is a powerful and efficient screening tool for ZEN detection and can easily be modified to detect other mycotoxins.

The frequent detection of trichothecenes in grains highlights critical health risks to humans and animals. Based on the hybrid sorbent strategies, this study developed an innovative multifunctional column (ASAG563) integrating extraction, purification, and filtration to address limitations of existing methods, including cumbersome process, protracted duration, harmful to the environment, and significant matrix interference. Coupled with high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS), the ASAG563 column demonstrated superior recoveries (80.8–117.8%) and quantification limits (2.02~48.41 µg/kg) across cereals and feeds, with low relative standard deviations (<6.8%). Compared to commercial MFCs, the ASAG563 column simplified the process, reduced material consumption, saved 50% of analysis time, and effectively eliminated matrix effects. Analysis of 512 maize for feedstuff samples from Northeast China revealed significant contaminations with deoxynivalenol (DON) and its derivatives, emphasizing the necessity for enhanced regulatory measures. This novel integrated multifunctional pretreatment column presents a convenient, cost-effective, and eco-friendly solution for accurate TCT detection, significantly advancing analytical capabilities.

Matrix certified reference materials (CRMs) play a critical role in analytical method validation and the assurance of reliable measurement results. A certified reference material (GBW(E)100813) for whole-wheat flour was developed to ensure an accurate and reliable measurement of the main Fusarium mycotoxins (deoxynivalenol (DON), nivalenol (NIV), deoxynivalenol-3-glucoside (DON-3G), and zearalenone (ZEN)). CRM candidates were prepared using sun-drying, grinding, sieving, homogenising, packaging, and gamma irradiation. The final produced CRM was packaged at 50 g per unit and stored at 20 °C. Certification was performed using isotope dilution-liquid chromatography–tandem mass spectrometry. CRM characterization was performed in eight laboratories in accordance with the requirements of ISO Guide 35. The certified values and expanded uncertainties (at a confidence of 95%, k = 2) for DON, NIV, DON-3G, and ZEN were determined to be 0.98 ± 0.12 mg/kg, 1.37 ± 0.20 mg/kg, 242 ± 35 μg/g, and 382 ± 50 μg/g. The CRM was sufficiently homogeneous between and within bottles, and remained stable for up to 12 months at 20 °C and 9 days below 40 °C for transportation. Thus, CRM can be used for quality control and method validation to ensure the accurate and reliable quantification of the main Fusarium mycotoxins in whole-wheat flour.