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Enniatins (ENNs) and beauvericin (BEA), known as emerging mycotoxins, are the toxic secondary metabolites produced by various Fusarium species. Most grain and grain-based products are contaminated with ENNs and BEA. Animals have been exposed to ENNs and BEA primarily due to consumption of cereal grains and cereal by-products. ENNs and BEA have been detected in animal-derived food and human breast milk, and they pose significant threats to public health. Therefore, more contamination data are urgently needed for the risk assessment of ENNs and BEA present in animal-derived food. To ensure the quality of animal-derived food, a method has been developed for the simultaneous detection of five emerging mycotoxins (viz. enniatin B, enniatin B1, enniatin A, enniatin A1, and beauvericin) in eggs by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) coupled with cold-induced liquid-liquid extraction (CI-LLE) and dispersive solid phase extraction (DSPE). The main factors governing the response, recovery, and sensitivity of the method, such as the type of extraction solvent, the temperature and duration of cold treatment in CI-LLE, the type and dosages of adsorbents, and apparatus conditions and the type of mobile phase used, were optimized during sample pretreatment and instrument analysis. The mycotoxin residues in eggs were extracted using 20 mL acetonitrile-water-acetic acid (79∶20∶1, v/v/v) mixture for 20 min by the vortex shock method. After mixing, the mixture was frozen for 30 min in a freezer at -40 ℃ and centrifuged for 10 min at 10000 r/min. A 2 mL aliquot of the upper acetonitrile layer was purified by using 70 mg of C18 adsorbents. After whirling, the mixtures were centrifuged at 10000 r/min for 5 min. The purified solution was then concentrated to nearly dry in nitrogen atmosphere at 40 ℃. The residues were dissolved in 1.0 mL 80%(v/v) acetonitrile aqueous solution. The target analytes were separated on an ACQUITY UPLC BEH C18 chromatographic column (100 mm×2.1 mm, 1.7 μm) at a column temperature of 40 ℃, with a flow rate of 0.3 mL/min. The injection volume was 5 μL, and gradient elution was conducted using acetonitrile and 5 mmol/L ammonium formate solution as the mobile phases. Multiple reactions monitoring (MRM) was conducted in the positive electrospray ionization (ESI + ) mode. The isotope internal standard method was used for quantification of BEA, and the matrix-matched external standard method was used for quantification of four ENNs. The results of the optimized method showed that the five analytes were completely separated by using the above-mentioned chromatographic column. Good linear relationships were obtained for the five mycotoxins in the concentration range of 0.1-50.0 μg/L; the correlation coefficient ( r  2 ) ranged from 0.9983 to 0.9997. The limits of detection (LODs) ranged from 0.05 to 0.15 μg/kg, while the limits of quantification (LOQs) ranged from 0.20 to 0.50 μg/kg. Accuracy and precision experiments were conducted by spiking egg samples with known amounts of analytes at three concentration levels (0.5, 5.0, and 25.0 μg/kg, in compliance with the current legislation) with six replicates. The average recoveries of the five analytes ranged from 81.1% to 106%, and the relative standard deviations (RSDs) were between 0.27% and 9.79%. The matrix effects of the analytes were between 2.70% and 45.1% in egg samples after pretreatment by CI-LLE coupled with DSPE. The developed method was applied to the determination of five mycotoxins in rural eggs and commercial eggs. BEA was detected in most rural egg samples, with detection rates of 30.4%. None of the four ENN residues were detected. Therefore, we can conclude that the method described herein has the advantages of sensitivity, stabilization, accuracy, good recovery, and easy operation, and is suitable for the simultaneous and rapid determination of BEA and ENN residues in eggs.

sepu/chinese journal of chromatography

Simultaneous determination of beauvericin and four enniatins in eggs by ultra-performance liquid chromatography-tandem mass spectrometry coupled with cold-induced liquid-liquid extraction and dispersive solid phase extraction