Development and Validation of a Method for Direct Analysis of Aflatoxins in Animal Feeds by Ultra-High-Performance Liquid Chromatography with Fluorescence Detection.

Abstract

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.