霉菌毒素是由真菌产生的有毒次级代谢产物,可引起急性,亚急性,以及对人类和动物的慢性毒性。因此,这些毒素对健康和安全构成重大威胁。由于农业产业缺乏有效的防霉措施,饲料原料,如玉米,花生,小麦,大麦,小米,坚果,油性饲料,牧草,它们的副产品容易被霉菌和霉菌毒素污染,会影响动物生产,产品质量,和安全。环吡嗪酸(CPA),主要由甲羟戊酸生物合成,色氨酸,和二乙酸酯单元,是由青霉和曲霉属真菌产生的肌毒性次级代谢产物。CPA作为与黄曲霉毒素的共同污染物广泛存在于各种作物中。与一些常见的霉菌毒素如黄曲霉毒素相比,伏马菌素,曲霉毒素,玉米赤霉烯酮,以及它们的代谢物,注册会计师没有得到很好的调查。在美国,一项调查显示,51%的玉米和90%的花生样品含有CPA,最高水平为2.9mg/kg。在欧洲,在受青霉污染的奶酪中发现CPA高达4.0mg/kg。一些研究表明,CPA可以对小鼠的肝脏和脾脏等器官造成不可逆的损害。因此,建立快速、高效的注册会计师分析方法对饲料注册会计师的风险评估具有重要意义,标准限值的制定,保障饲料产品质量安全。QuEChERS方法,一种快速的样品预处理方法,简单,便宜,有效,和安全,广泛用于食品中农药残留的分析。在这项研究中,改进的QuEChERS方法结合超高效液相色谱-串联质谱(UPLC-MS/MS)用于测定饲料中的CPA水平。CPA的色谱分离和MS检测以及影响CPA提取效率的关键因素,包括提取溶剂的类型,无机盐的类型,以及吸附剂的类型和用量,进行了详细优化。在色谱分离步骤的优化过程中,流动相的酸和盐浓度影响CPA的分离和检测。在QuEChERS方法的优化过程中,加入一定量的乙酸,提高了CPA的萃取效率,GCB和PSA显著吸附饲料提取物中的CPA。在最优条件下,用2mL水和4mL含有0.5%乙酸的乙腈(ACN)萃取进料样品(1.0g)中的CPA。用0.4gNaCl和1.6gMgSO4盐析后,通过使用150mgMgSO4和50mgC18的分散固相萃取纯化1mLACN上清液,并通过UPLC-MS/MS分析。样品在WatersHSST3色谱柱(100mm×2.1mm,1.8μm)使用含0.5%甲酸和ACN的2mmol/L乙酸铵水溶液作为流动相,然后在多反应监测模式下通过正电喷雾电离进行分析。CPA在2-200ng/mL范围内表现出良好的线性,具有较高的相关系数(r=0.9995)。CPA的检测限和定量限,计算为信噪比的3倍和10倍,分别,分别为0.6和2.0μg/kg,分别。添加10、100和500μg/kgCPA的饲料样品的平均回收率为70.1%至78.5%,日内精度低于5.8%,日内精度低于7.2%,表明该方法具有良好的准确度和精密度。最后,采用改良的QuEChERS-UPLC-MS/MS方法对10份武汉市售饲料样品中的CPA进行分析。分析结果表明,该方法对饲料样品中的CPA分析具有良好的适用性。总之,改进的QuEChERS方法首次应用于饲料中CPA的提取和纯化,评估,和饲料样品中CPA的标准限值设置。
Mycotoxins are toxic secondary metabolites produced by fungal species that can cause acute, subacute, and chronic toxicity in humans and animals. Thus, these toxins pose a significant threat to health and safety. Owing to the lack of effective antimold measures in the agricultural industry, feed ingredients such as corn, peanuts, wheat, barley, millet, nuts, oily feed, forage, and their byproducts are prone to mold and mycotoxin contamination, which can affect animal production, product quality, and safety. Cyclopiazonic acid (CPA), which is mainly biosynthesized from mevalonate, tryptophan, and diacetate units, is a myotoxic secondary metabolite produced by Penicillium and Aspergillus fungi. CPA is widely present as a copollutant with aflatoxins in various crops. Compared with some common mycotoxins such as aflatoxins, fumonisins, ochratoxins, zearalenones, and their metabolites, CPA has not been well investigated. In the United States, a survey showed that 51% of corn and 90% of peanut samples contained CPA, with a maximum level of 2.9 mg/kg. In Europe, CPA was found in Penicillium-contaminated cheeses as high as 4.0 mg/kg. Some studies have shown that CPA can cause irreversible damage to organs such as the liver and spleen in mice. Therefore, the establishment of a rapid and efficient analytical method for CPA is of great significance for the risk assessment of CPA in
feeds, the development of standard limits, and the protection of feed product quality and safety. The QuEChERS method, a sample pretreatment method that is fast, simple, cheap, effective, and safe, is widely used in the analysis of pesticide residues in food. In this study, a modified QuEChERS method combined with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to determine CPA levels in
feeds. The chromatographic separation and MS detection of CPA as well as the key factors affecting the extraction efficiency of CPA, including the type of extraction solvent, type of inorganic salt, and type and dosage of adsorbent, were optimized in detail. During the optimization of the chromatographic-separation step, the acid and salt concentrations of the mobile phase affected the separation and detection of CPA. During the optimization of the QuEChERS method, the addition of a certain amount of acetic acid improved the extraction efficiency of CPA because of its acidic nature; in addition, GCB and PSA significantly adsorbed CPA from the feed extract. Under optimal conditions, the CPA in the feed sample (1.0 g) was extracted with 2 mL of water and 4 mL of acetonitrile (ACN) containing 0.5% acetic acid. After salting out with 0.4 g of NaCl and 1.6 g of MgSO4, 1 mL of the ACN supernatant was purified by dispersive solid-phase extraction using 150 mg of MgSO4 and 50 mg of C18 and analyzed by UPLC-MS/MS. The sample was separated on a Waters HSS T3 column (100 mm×2.1 mm, 1.8 μm) using 2 mmol/L ammonium acetate aqueous solution with 0.5% formic acid and ACN as the mobile phases and then analyzed by positive electrospray ionization in multiple reaction monitoring mode. CPA exhibited good linearity in the range of 2-200 ng/mL, with a high correlation coefficient (r=0.9995). The limits of detection and quantification of CPA, which were calculated as 3 and 10 times the signal-to-noise ratio, respectively, were 0.6 and 2.0 μg/kg, respectively. The average recoveries in feed samples spiked with 10, 100, and 500 μg/kg CPA ranged from 70.1% to 78.5%, with an intra-day precision of less than 5.8% and an inter-day precision of less than 7.2%, indicating the good accuracy and precision of the proposed method. Finally, the modified QuEChERS-UPLC-MS/MS method was applied to the analysis of CPA in 10 feed samples obtained from Wuhan market. The analysis results indicated that the developed method has good applicability for CPA analysis in feed samples. In summary, an improved QuEChERS method was applied to the extraction and purification of CPA from
feeds for the first time; this method provides a suitable analytical method for the risk monitoring, assessment, and standard-limit setting of CPA in feed samples.