色谱

色谱 ›› 2024, Vol. 42 ›› Issue (5): 445-451.DOI: 10.3724/SP.J.1123.2023.10030

• 研究论文 • 上一篇    下一篇

改进的QuEChERS结合超高效液相色谱-串联质谱法检测饲料中的环匹阿尼酸

彭茂民1, 喻小兵2, 陈琳3, 熊青松4, 刘丽1, 郑丹1, 夏虹1, 余琼卫4,*(), 彭西甜1,*()   

  1. 1.湖北省农业科学院农业质量标准与检测技术研究所, 农产品营养品质与安全湖北省重点实验室, 湖北 武汉 430064
    2.荆州市农业农村局, 湖北 荆州 434020
    3.湖北省食品质量安全监督检验研究院, 湖北 武汉 430075
    4.武汉大学化学与分子科学学院, 湖北 武汉 430072
  • 收稿日期:2023-10-26 出版日期:2024-05-08 发布日期:2024-05-13
  • 通讯作者: *Tel:(027)68755087,E-mail:qwyu@whu.edu.cn(余琼卫);Tel:(027)87381082,E-mail:pxitian@aliyun.com(彭西甜).
  • 基金资助:
    湖北省重点研发计划项目(2020BBB078);湖北省重点研发计划项目(2021BBA227);湖北省重点研发计划项目(2022BBA0069)

Modified QuEChERS method combined with ultra performance liquid chromatography-tandem mass spectrometry for detection of cyclopiazonic acid in feeds

PENG Maomin1, YU Xiaobing2, CHEN Lin3, XIONG Qingsong4, LIU Li1, ZHENG Dan1, XIA Hong1, YU Qiongwei4,*(), PENG Xitian1,*()   

  1. 1. Institute of Agricultural Quality Standards and Testing Technology Research, Hubei Academy of Agricultural Science, Hubei Key Laboratory of Nutritional Quality and Safety of Agro-products, Wuhan 430064, China
    2. Jingzhou Agriculture and Rural Bureau, Jingzhou 434020, China
    3. Hubei Provincial Institute for Food Supervision and Test, Wuhan 430075, China
    4. College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China
  • Received:2023-10-26 Online:2024-05-08 Published:2024-05-13
  • Supported by:
    Research and Development Program of Hubei Province, China(2020BBB078);Research and Development Program of Hubei Province, China(2021BBA227);Research and Development Program of Hubei Province, China(2022BBA0069)

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摘要：

采用改进的QuEChERS前处理方法结合超高效液相色谱-串联质谱(UPLC-MS/MS)分离检测,建立了饲料中真菌毒素环匹阿尼酸(CPA)的快速分析新方法。详细优化了UPLC-MS/MS分离检测条件和影响CPA回收率的QuEChERS条件,优化后的分析方法如下:1.0 g饲料加入2 mL水和4 mL 0.5%乙酸乙腈溶液进行提取,再加入提取盐包(0.4 g氯化钠和1.6 g无水硫酸镁),离心分层后,取1 mL乙腈提取液,加入150 mg无水硫酸镁和50 mg C18吸附净化,上清液进行UPLC-MS/MS分离检测;采用Waters HSS T3柱(100 mm×2.1 mm, 1.8 μm),以含0.5%甲酸的2 mmol/L乙酸铵水溶液和乙腈作为流动相,梯度洗脱,电喷雾正离子模式下以多反应监测(MRM)扫描模式定量。CPA在2~200 ng/mL的范围内具有良好的线性,相关系数良好(r=0.9995),方法的检出限和定量限分别为0.6和2 μg/kg,饲料中CPA在10、100、500 μg/kg 3个加标水平下的回收率为70.1%~78.5%,日内RSD为4.1%~5.8%,日间RSD为6.1%~7.2%。将本方法应用于实际饲料样品中CPA的分析,取得了很好的效果。本研究将改进的QuEChERS方法应用于饲料中CPA的提取净化,为饲料中CPA的风险监测、评估和限量标准制定提供了一种有效的检测技术。

关键词: QuEChERS, 超高效液相色谱-串联质谱, 环匹阿尼酸, 饲料

Abstract:

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.

Key words: QuEChERS, ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), cyclopiazonic acid (CPA), feeds

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