关键词: 固相萃取, 超高效液相色谱-串联质谱法, 链格孢霉毒素, 婴幼儿奶粉

Abstract:

Alternaria toxin is a general term for a class of toxic metabolites produced by Alternaria, which widely exists in soil, grain, vegetables, and fruits. This mycotoxin is extremely harmful to human health. It is well known that infant milk powder containing vegetable oil is easily contaminated by Alternaria alternata. Alternaria toxins have thus become an increasingly important focus in food. Rapid and accurate detection of Alternaria toxin residues in food is of great significance for food safety. This requires pretreatment to purify the target toxins and maximize the accuracy and precision of the analysis. In this study, a rapid method based on online solid phase extraction/purification and ultra-performance liquid chromatography-tandem mass spectrometry (online SPE UPLC MS/MS) was established to detect seven Alternaria toxins (alternariol monomethyl ether, altenuene, tenuazonic acid, alternariol, tentoxin, altenusin, and altertoxin Ⅰ) in infant milk powder. First, the mass spectrometry and chromatographic conditions were optimized. A BEH-C₁₈ column (50 mm×2.1 mm, 1.7 μm) was selected, with 0.1% formic acid aqueous solution-acetonitrile as the mobile phase. Then, the extraction conditions (extraction agent ratio and extraction method) and the solid phase extraction process (extraction column, type and volume of the eluent, and pH of the sample loading solution) were optimized. One gram of milk powder (accurate to 0.01 g) was weighed into a 50 mL tip and bottom plug centrifuge tube. Acetonitrile-water (84∶16, v/v) was set as the extraction agent for the first two cycles, and acetonitrile-methanol-water (45∶10∶45, v/v/v) was set as the third extraction agent. Horizontal shaking for 30 min was the best extraction method. The sample was centrifuged at 9500 r/min for 10 min, and the supernatant extracted many times was mixed and blown with nitrogen at 40 ℃. The sample was redissolved in first-order water (pH 5.5), purified on an HLB column, and successively activated with 6 mL methanol and 6 mL first-order water (pH 5.5). The solution was then loaded onto the column, and the SPE was adjusted to ensure that the water sample flowed through the column at the rate of 1 mL/min so that the column did not dry up during the analysis process before the end of sample loading. The column was rinsed with 12 mL of first-order water. After leaching, the negative pressure filtration was continued for approximately 5 min, followed by elution with 10 mL methanol, and the eluted solution was directly tested after passing through a 0.22 μm filter membrane, without concentration. The analytes were determined by electrospray ionization (ESI) with alternating positive and negative ions. Under the optimal analysis conditions, the linear relationships of the seven Alternaria toxins were good in the mass concentration range of 0.5-200 μg/L, with coefficients of determination (R²)>0.9903. The limits of detection and limits of quantification were 0.15-0.64 μg/kg and 0.54-2.24 μg/kg, respectively. The recoveries of the seven Alternaria toxins were 79.1%-114.3%, and the relative standard deviations were less than 8.87% at different concentrations. The method was applied to the determination and analysis of 60 samples of infant milk powder, and the results showed that no toxin was found in stage one or stage two of the milk powder. Only one sample of the stage three of milk powder was detected, which was tentoxin, and the content was 4.97 μg/kg. The developed method is accurate, rapid, simple, sensitive, repeatable, and stable. It can be used for the practical determination of seven Alternaria toxins in infant milk powder.

Key words: solid phase extraction (SPE), ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), Alternaria toxins (ATs), infant milk powder