Mycotoxins are secondary metabolites produced by toxigenic fungi under specific environmental conditions. Fruits, owing to their high moisture content, rich nutrition, and improper harvest or storage conditions, are highly susceptible to various mycotoxins, such as ochratoxin A (OTA), zearalenone (ZEN), patulin (PAT), Alternaria toxins, etc. These mycotoxins can cause acute and chronic toxic effects (teratogenicity, mutagenicity, and carcinogenicity, etc) in animals and humans. Given the high toxicity and wide prevalence of mycotoxins, establishing an efficient analytical method to detect multiple mycotoxins simultaneously in different types of fruits is of great importance. Conventional mycotoxin detection methods rely on high performance liquid chromatography (HPLC) coupled with mass spectrometry (MS). However, fruit sample matrices contain large amounts of pigments, cellulose, and minerals, all of which dramatically impede the detection of trace mycotoxins in fruits. Therefore, the efficient enrichment and purification of multiple mycotoxins in fruit samples is crucial before instrumental analysis. In this study, a reliable method based on a QuEChERs sample preparation approach coupled with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established to determine 36 mycotoxins in fruits. In the optimal extraction method, 2.0 g of a sample was extracted with 10 mL of acetic acid-acetonitrile-water (1∶79∶20, v/v/v) in a 50 mL centrifuge tube, vortexed for 30 s, and ultrasonicated for 40 min. The mixture was then salted out with 2.0 g of anhydrous MgSO4 and 0.5 g of NaCl and centrifuged for 5 min. Next, 6 mL of the supernatant was purified using 85 mg of octadecylsilane-bonded silica gel (C18) and 15 mg of N-propylethylenediamine (PSA). After vigorous shaking and centrifugation, the supernatant was collected and dried with nitrogen at 40 ℃. Finally, the residues were redissolved in 1 mL of 5 mmol/L ammonium acetate aqueous solution-acetonitrile (50∶50, v/v) and passed through a 0.22 μm nylon filter before analysis. The mycotoxins were separated on a Waters XBridge BEH C18 column using a binary gradient mixture of ammonium acetate aqueous solution and methanol. The injection volume was 3 μL. The mycotoxins were analyzed in multiple reaction monitoring (MRM) mode under both positive and negative electrospray ionization. Quantitative analysis was performed using an external standard method with matrix-matched calibration curves. Under optimal conditions, good linear relationships were obtained in the respective linear ranges, with correlation coefficients (R2) no less than 0.990. The limits of detection (LODs) and quantification (LOQs) were 0.02-5 and 0.1-10 μg/kg, respectively. The recoveries of the 36 mycotoxins in fruits ranged from 77.0% to 118.9% at low, medium, and high spiked levels, with intra- and inter-day precisions in the range of 1.3%-14.9% and 0.2%-17.3%, respectively. The validated approach was employed to investigate mycotoxin contamination in actual fruit samples, including strawberry, grape, pear, and peach (15 samples of each type). Eleven mycotoxins, namely, altenuene (ALT), altenusin (ALS), alternariol-methyl ether (AME), tenuazonic acid (TeA), tentoxin (Ten), OTA, beauvericin (BEA), PAT, zearalanone (ZAN), T-2 toxin (T2), and mycophenolic acid (MPA), were found in the samples; three samples were contaminated with multiple mycotoxins. The incidence rates of mycotoxins in strawberry, grape, pear, and peach were 27%, 40%, 40%, and 33%, respectively. In particular, Alternaria toxins were the most frequently found mycotoxins in these fruits, with an incidence of 15%. The proposed method is simple, rapid, accurate, sensitive, reproducible, and stable; thus, it is suitable for the simultaneous detection of the 36 mycotoxins in different fruits.
基于QuEChERS-超高效液相色谱-串联质谱法(UPLC-MS/MS),建立了水果(草莓、葡萄、梨和桃)中36种真菌毒素的准确定量分析方法,并应用该方法对不同水果中真菌毒素的污染情况进行了分析。2.0 g样品用10 mL乙酸-乙腈-水(1∶79∶20, v/v/v)提取,无水硫酸镁(2.0 g)和氯化钠(0.5 g)盐析,离心后采用85 mg十八烷基硅烷键合硅胶(C18)和15 mg N-丙基乙二胺(PSA)对上清液进行吸附净化,浓缩复溶后过滤膜;采用Waters XBridge BEH C18色谱柱分离,以5 mmol/L醋酸铵水溶液和甲醇为流动相进行梯度洗脱,采用电喷雾离子源(ESI)正、负离子切换多反应监测模式测定,以空白基质匹配外标曲线法准确定量。结果表明,36种真菌毒素在8.5 min内完成色谱分离,并在各自的线性范围内具有良好的线性关系,相关系数(R2)≥0.990,检出限(LOD)和定量限(LOQ)分别为0.02~5 μg/kg和0.1~10 μg/kg。在低、中、高3个添加水平下,4种水果基质中36种真菌毒素的平均加标回收率为77.0%~118.9%,日内精密度为1.3%~14.9%,日间精密度为0.2%~17.3%。利用所建立的方法对60份水果样品(15份草莓、15份葡萄、15份梨和15份桃)进行检测,共检出11种真菌毒素,平均含量为0.13~35.85 μg/kg,其中3份样品存在多种真菌毒素共同污染的情况。本方法操作简便,快速准确,灵敏度高,重复性与稳定性良好,适用于草莓、葡萄、梨和桃中36种真菌毒素的同时检测。
Keywords: QuEChERS; fruits; mycotoxins; ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).