A novel method for real-time monitoring of the capacity of aflatoxin B1 immunoaffinity column in IAC-HPLC assay

Immunoaffinity column (IAC) cleanup, coupled with high performance liquid chromatography (HPLC) analysis, is one of the most reliable methods for aflatoxin B1 (AFB1) analysis. However, real-time monitoring of the IAC capacity remains unachievable in IAC-HPLC analysis, compromising the reliability of AFB₁ quantification. Two artificial structural analogues of aflatoxin (AFB1-OH and AFB2-OH) were synthesized as potential markers for IAC capacity monitoring. Competitive ELISA and recovery experiments demonstrated that AFB2-OH was more suitable as a marker. When AFB2-OH was used for real-time monitoring of the capacity of the IAC in AFB1 determination by HPLC, the recovery of AFB1 exceeded 95 %. The limit of detection (LOD) and the limit of quantification (LOQ) for AFB1 were determined to be 0.14 ng/mL and 0.46 ng/mL, respectively. The method also exhibited good precision and accuracy. In spiked recovery experiments of four grains, the recovery of AFB1 ranged from 85.16 % to 107.05 % with relative standard deviations of 0.89 %-4.98 %. More importantly, due to the addition of AFB2-OH, the capacity of the IAC was monitored in real-time, ensuring the reliability of AFB1 determination results.