National Reference Laboratory, ICAR-National Research Centre for Grapes, P.O. Manjri Farm, Pune 412 307, India.
J AOAC Int. 2020 Jul 1;103(4):940-945. doi: 10.1093/jaoacint/qsz037.
Aflatoxin (AF) contamination is one of the major regulatory concerns for animal feed. As feed is a complex analytical matrix, validated methods on AFs in feed are scanty. The available methods involve a derivatization step before AF analysis by high-performance liquid chromatography (HPLC) with fluorescence detection (FLD). The aim of this study was thus to develop and validate a simple and rapid method for direct analysis of AFs (AFB1, AFB2, AFG1, AFG2) in a range of animal feed matrices.
Feed samples were extracted with 80% methanol, followed by dilution with water and immmunoaffinity column cleanup. AFs were estimated using an ultra-high performance liquid chromatography (UHPLC) instrument. Use of a large volume flow cell in FLD allowed direct analysis of all AFs with high sensitivity. The method was thoroughly validated in a range of feed matrices.
This sample preparation workflow minimized co-extractives, along with matrix interferences. In pigeon pea husk feed, the method provided a limit of quantification (LOQ) of 0.5 ng/g for each AF with recoveries of AF- B1, B2, G1, and G2 as 71.5, 75.6, 82.4, and 78.2%, respectively. The precision (relative standard deviation, RSD) was below 5%. A similar method performance was also recorded in other matrices, including wheat bran feed and poultry feed.
The optimized method is suitable for regulatory testing because it is simple, robust, cost-effective, and high throughput in nature, with high sensitivity and selectivity.
Our workflow has provided a straightforward method for the analysis of AFs in a wide range of animal feed matrices with high sensitivity, selectivity, throughput, and cost-effectiveness. The method allowed a direct analysis of AFs by UHPLC-FLD without a step of derivatization.
黄曲霉毒素（AF）污染是动物饲料的主要监管问题之一。由于饲料是一种复杂的分析基质，因此针对饲料中 AF 的验证方法很少。现有的方法涉及在高效液相色谱（HPLC）与荧光检测（FLD）分析 AF 之前进行衍生化步骤。因此，本研究旨在开发和验证一种简单快速的方法，用于直接分析各种动物饲料基质中的 AF（AFB1、AFB2、AFG1、AFG2）。
采用 80%甲醇提取饲料样品，然后用水稀释，再经免疫亲和柱净化。使用超高效液相色谱（UHPLC）仪器估计 AFs 的含量。FLD 中使用大体积流通池可直接分析所有 AFs，具有高灵敏度。该方法在一系列饲料基质中进行了彻底验证。
这种样品制备工作流程最大限度地减少了共提取物和基质干扰。在木豆壳饲料中，该方法对每种 AF 的定量限（LOQ）为 0.5ng/g，AF-B1、B2、G1 和 G2 的回收率分别为 71.5%、75.6%、82.4%和 78.2%。精密度（相对标准偏差，RSD）低于 5%。在其他基质中，包括麦麸饲料和禽饲料，也记录到了类似的方法性能。
该优化方法适用于监管测试，因为它简单、稳健、具有成本效益且高通量，同时具有高灵敏度和选择性。
我们的工作流程为分析各种动物饲料基质中的 AF 提供了一种简单、灵敏、选择性高、高通量且经济有效的方法。该方法允许通过 UHPLC-FLD 直接分析 AF，无需衍生化步骤。
