A novel method for high preconcentration of ultra trace amounts of B₁, B₂, G₁ and G₂ aflatoxins in edible oils by dispersive liquid-liquid microextraction after immunoaffinity column clean-up

Abstract

In the present study, a new approach which uses immunoaffinity column clean-up combined with dispersive liquid-liquid microextraction (DLLME) is proposed for the preconcentration of ultra trace amounts of aflatoxins (B₁, B₂, G₁ and G₂). The aflatoxins are then determined using a high-performance liquid chromatography coupled with fluorescent detector. Samples are extracted by immunoaffinity column (IAC) clean-up, and their eluents are used as dispersants of the subsequent DLLME, for further enrichment of aflatoxins. Various parameters (the type of elution solvent, the type and volume of extraction solvent and disperser solvent, extraction time, and centrifugation time) that affect the efficiency of the two steps are optimized. Under the optimum conditions (extraction solvent: 120 μL of chloroform, disperser solvent: 500 μL of acetonitrile, sample pH: 7.4, centrifugation time: 3 min), the calibration for B₁, B₂, G₁ and G₂ was found to be linear with coefficient of estimation (R²) of 0.9994, 0.9976, 0.9989, 0.9973 respectively and the limit of detection (LOD) was between 1.1 × 10⁻⁴ to 5.3 × 10⁻³ ng mL⁻¹ (3σ(b)/m, n=9). The recoveries at the two spiked levels ranged from 96.0 to 110.0% and the relative standard deviation (RSD) was less than 7.8% (n=9). The results show that dispersive liquid-liquid microextraction combined with HPLC is a selective, simple, sensitive, and effective analytical method for the preconcentration and determination of ultra trace amounts of aflatoxins. The proposed method was applied for preconcentration and determination of B₁, B₂, G₁ and G₂ aflatoxin in edible oils. Analysis of aflatoxins in FAPAS test material showed that the proposed method has good accuracy.