Mycotoxins have carcinogenic, mutagenic, hepatotoxic, nephrotoxic, immunotoxic, neurotoxic, and teratogenic properties. Thus, these substances have attracted significant attention because they pose a threat to human health. As research on mycotoxins deepens, new structural analogues of mycotoxins are constantly being discovered. In this study, a method based on high performance liquid chromatography-quadrupole/orbitrap mass spectrometry was established for the simultaneous determination of 22 mycotoxins in milk. A simple, effective, and rapid pretreatment method was optimized by focusing on the solvent type, extractant volume, and extracting salt based on the characteristics of the mycotoxins and sample matrix. The analytes were extracted using 0.5% formic acid acetonitrile solution and added with sodium chloride to separate fats from water. The samples were centrifuged at 8000 r/min (4 ℃) for 5 min using a centrifuge and then concentrated using nitrogen. The dry residue was dissolved with 50% methanol aqueous solution. Twenty-two mycotoxins were separated on a ZORBAX Eclipse Plus C18 chromatographic column (100 mm×2.1 mm, 1.7 μm), and quantitative analysis was performed using the isotope internal standard method. The analytes were determined by liquid chromatography-quadrupole/orbitrap mass spectrometry in positive electrospray ionization mode. Qualitative analyses of the compounds were performed in full mass spectrometry/data-dependent tandem mass spectrometry (MS/dd-MS2) mode. Good linearities in the range of 0.5-100.0 μg/L were observed for the 22 mycotoxins, and the correlation coefficients (R2) were greater than 0.999. The limits of detection (S/N=3) and quantification (S/N=10) ranged from 0.3 to 0.5 μg/kg and from 1.0 to 1.5 μg/kg, respectively. The average recoveries of the 22 mycotoxins at three spiked levels of 1.5, 5.0, and 15 μg/kg were between 84.7% and 100.8%, with relative standard deviations (RSDs) of 1.2%-9.9%. These findings indicate that the method has high sensitivity and accuracy as well as good precision. Finally, the method was applied to the detection and analysis of mycotoxins in 25 actual commercial milk samples. The results revealed that the selected samples were not contaminated with any of the mycotoxins analyzed. Thus, the proposed method is useful as a quick preprocessing and confirmatory method for the simultaneous determination of mycotoxins in milk.
真菌毒素因具有致癌性、致突变性、肝毒性、肾毒性、免疫毒性、神经毒性及致畸性等危害而广受关注。本研究建立了高效液相色谱-四极杆/静电场轨道阱质谱法同时测定牛奶中22种真菌毒素的分析方法。针对真菌毒素的特性和牛奶样品基质的特点选择简单、有效、快速的前处理方法,重点优化了提取溶剂种类、溶剂用量和提取盐用量。样品用0.5%甲酸乙腈提取,氯化钠分层,低温高速离心净化,上清液经氮吹浓缩后复溶,ZORBAX Eclipse Plus C18色谱柱(100 mm×2.1 mm, 1.7 μm)分离,同位素内标法定量。化合物在正离子模式下电离,在数据依赖型质谱扫描模式下对目标物进行定性。结果表明,22种目标物在0.5~100.0 μg/L线性范围内呈良好的线性关系,相关系数(R2)均大于0.999,该方法的检出限(S/N=3)为0.3~0.5 μg/kg,定量限(S/N=10)为1.0~1.5 μg/kg。以牛奶为基质,在1.5、5.0、15 μg/kg加标水平下,22种真菌毒素的回收率为84.7%~100.8%,相对标准偏差为1.2%~9.9%。应用本方法对25份实际牛奶样品进行分析检测,结果显示22种真菌毒素均未检出。该方法灵敏度好,准确度高,可作为牛奶中真菌毒素的高通量筛查和确证检测方法。
Keywords: liquid chromatography-quadrupole/orbitrap mass spectrometry; milk; mycotoxins; rapid pretreatment.