Determination of 4 Alternaria Mycotoxins in Sunflower Seeds by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry

The finely ground sunflower seed sample (2.5 g) was taken, and 500.0 μL of a mixed internal standard solution containing 100.0 μg·L^-1 of alternariol monomethyl ether-d₃ (AME-d₃), 1 000.0 μg·L^-1 of tentoxin-d₃ (TEN-d₃) and alternariol-d₂ (AOH-d₂), and 5 000.0 μg·L^-1 of tenuazonic acid-d₁₃ (TeA-d₁₃) was added. After mixing well and settling for overnight, the mixture was extracted in 25.0 mL of a mixture of acetonitrile, methanol, and 0.05 mol·L^-1 sodium dihydrogen phosphate solution (pH 3.0) at a volume ratio of 45∶10∶45, and then centrifuged. The supernatant was taken, and made its volume up to 30.0 mL with water. An aliquot (6.0 mL) was taken, and 15.0 mL of 0.05 mol·L^-1 sodium dihydrogen phosphate solution (pH 3.0) was added. The mixture was passed through Waters Oasis HLB solid phase extraction column (pre-activated with 5.0 mL of methanol and 5.0 mL of water), and washed with 20% (volume fraction) methanol solution. After drying the solid phase extraction column, it was eluted with 10 mL of a mixture of methanol containing 1% (volume fraction) ammonia and acetonitrile at a volume ratio of 1∶1. The eluent was blown to near dryness at 40 ℃ by nitrogen, and then the residue was redissolved in 200.0 μL of methanol and made its volume up to 2.0 mL with water. The 4 Alternaria mycotoxins including alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), and tentoxin (TEN) in the obtained solution were determined by ultra-high performance liquid chromatography tandem mass spectrometry, and internal standard method was used for quantitative analysis. As shown by the results, Waters Oasis HLB solid phase extraction column had the best purification effect, and adding ammonia to the eluant was beneficial for improving peak shape and making the baseline smoother. Linear relationships between values of mass concentration and peak area ratio of targets to internal standards were found in definite ranges, with lower limits of determination (10S/N) in the range of 0.15-1.50 μg·kg^-1. Test for recovery was made by standard addition method, giving results in the range of 75.0%-112%. 7 parallel tests were performed on the actual sample, with RSDs of the determined values less than 13%. This method was applied to the analysis of actual samples, and it was shown that the highest detection rate and detection amount were for TeA, with an average value of 1 890.16 μg·kg^-1, followed by TEN with an average value of 20.26 μg·kg^-1.