Rapid Determination of Typical Alternaria Toxins in Wheat by Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry

A dispersion liquid-liquid microextraction (DLLM) combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of seven Alternaria toxins (alternariol, alternariol monomethyl ether, altenuene, tenuazonic acid, altertoxin I, tentoxin and altenusin) in wheat. The samples were extracted with 3 mL of first-grade water and 10 mL of 1.5% formic acid acetonitrile-methanol (4:1, V/V), dehydrated with 2 g of MgSO₄ and salted out with 1 g of NaCl, and then the mixture was shaken for 15 min followed by centrifugation for 10 min. One mililiter of the supernatant and 100 μL of trichloromethane were used for DLLM. The seven Alternaria toxins were separated on a BEH C₁₈ (50 mm × 2.1 mm, 1.7 μm) column, and detected using an electrospray ionization source in the multiple reaction monitoring mode with positive and negative ions. The chromatographic separation was completed within five minutes. The limits of detection and quantification of this method were in the range of 0.11–0.16 μg/kg and 0.42–0.49 μg/kg, respectively. Good linearity was observed in the concentration range of 0.5–100 μg/L with coefficients of determination (R²) of greater than 0.987277. In wheat matrix spiked at three levels, the average recoveries of the seven Alternaria toxins were 70.7%–101.3%, with relative standard deviations of 1.22%–4.11%. This method was used to analyze the contamination status of the seven Alternaria toxins in rye, buckwheat, naked oat, barley, oat, and wheat. The results showed that tentoxin and tenuazonic acid were detected in the six cereal species at levels of 0.6–10.7 μg/kg and none detected to 31 μg/kg, respectively. The detection rate of altenusin was lower than that of tenuazonic acid and tentoxin, but its highest content (37.3 μg/kg) was comparable to tenuazonic acid. This method proved to be stable, accurate, sensitive and fast, and could meet the requirements for the analysis of multiple Alternaria toxin residues in a variety of cereal.