Development and validation of a high-performance liquid chromatography method with post-column derivatization for the detection of aflatoxins in cereals and grains

A novel, reliable and rapid high-performance liquid chromatography (HPLC) method with post-column derivatization was developed and validated. The HPLC method was used for the simultaneous determination of aflatoxin B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁) and G₂ (AFG₂) in various cereals and grains. Samples were extracted with 80:20 (v/v) methanol:water and purified using C18 (40–63 μm) solid-phase extraction cartridges. AFs were separated using a LiChroCART–RP-18 (5 μm, 250 × 4.0 mm²) column. The mobile phase consisted of methanol:acetonitrile:buffer (17.5:17.5:65 v/v) (pH 7.4) delivered at the flow rate of 1.0 mL min⁻¹. The fluorescence of each AF was detected at λ_ex = 365 nm and λ_em = 435 nm. All four AFs were properly resolved within the total run time of 20 min. The established method was extensively validated as a final verification of the method development by the evaluation of selectivity (AFB₁, AFB₂, AFG₁ and AFG₂), linearity (R² ≥ 0.9994), precision (average SD ≤ 2.79), accuracy (relative mean error ≤ −5.51), robustness (p < 0.0080), ruggedness (p < 0.0100) and average recoveries (89.2–97.8%). The limits of quantification of AFB₁, AFB₂, AFG₁ and AFG₂ were 0.080, 0.073, 0.062 and 0.066 ng g⁻¹, respectively. Finally, the developed method was applied for the analysis of AFs in 45 samples comprising rice (n = 20), wheat (n = 15) and maize (n = 10). The results showed that 65% of rice, 20% of wheat and 80% of maize samples were found contaminated with AFs. Thus, according to the achieved results, it is suggested that the newly developed HPLC method could be effectively applied for the routine analysis of the AFs in different cereals and grains.