Optimization and in-house validation of the analytical procedure for official control of bentonites as aflatoxin inactivators

In Europe, bentonites are allowed as feed additives for aflatoxin mitigation (1m558) provided they have specific mineralogical characteristics and an aflatoxin-binding capacity (BCAfB1) above 90%. BCAfB1 is determined by an official adsorption assay using an aflatoxin solution (4 mg/L) in acetate buffer (pH 5.0) and a bentonite at 0.02% (w/v). To date, the robustness of this method has not been investigated. In this work, we addressed this challenge and performed a robustness study by analyzing six bentonite samples that met the mineralogical requirements for claim code 1 m558. Leading factors selected for robustness testing were (1) preparation mode of bentonite suspension, (2) residual amount of acetonitrile in the test trial, (3) acetate buffer concentration, (4) incubation time, and (5) centrifugation. It was statistically evinced that factors 2 and 5 affected the results. Due to its weakness, the method excluded 4 out of six bentonites to be marketed in EU, being BCAfB1 < 90%. A new protocol was developed by keeping the main experimental parameters of the official assay and was in-house validated. This protocol yielded BCAfB1 > 90% for all test bentonites and showed satisfactory precisions with a RSDI of 3.4% and HorRat < 2. Its validity was proven by the isotherm approach, comparing Langmuir adsorption parameters with BCAfB1 values. Application of the protocol to bentonites other than montmorillonite was demonstrated.