EU LEGISLATION REQUIRES AN OPTIMIZED/VALIDATED METHOD FOR THE OFFICIAL CONTROL OF BENTONITES AS AFLATOXIN INACTIVATORS

In Europe, bentonites are allowed as feed additives for aflatoxin mitigation (1m558) provided they havespecific mineralogical characteristics and an aflatoxin-binding capacity (BCAfB1) >90%. BCAfB1 isdetermined 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. Inthis work, we addressed this challenge and performed a robustness study by analysing six bentonitesthat met the mineralogical requirements for claim code 1m558. Fixing experimental conditions of the EUofficial assay, main leading parameters of this method were analysed and varied one-by-one. Leadingfactors selected for robustness testing were: (i) preparation mode of bentonite suspension; (ii) residualamount of acetonitrile in the test trial; (iii) acetate buffer concentration; (iv) incubation time; and (v)centrifugation. In some cases, different levels of each parameter were evaluated. Thereof, bentoniteswere first tested according to the experimental condition described in the EU method. Then, they weretested again by making some changes, each one in independent, triplicate experiments. It wasstatistically evinced that although some variations in the protocol, i.e., the optimization of acetonitrileconcentration in the working solutions, centrifugation conditions, and buffer concentration, produced aneffect on the AFB1 adsorption by materials, only a combination of all variations determined a strong,significant effect on BCAfB1 values. Due to its weakness, the method excluded four out of six bentonitesfrom being marketed in the EU because their BCAfB1 values were >90%. A new protocol was developedby keeping the main experimental parameters of the official assay and was in-house validated accordingto international harmonized guidelines. This protocol yielded BCAfB1 values >90% for all test bentonitesand showed satisfactory precisions with a RSDI of 3.4% and HorRat<2. This value falls within theacceptability criteria stated in the AOAC guidelines for standard method performance requirements. Thevalidity of the optimized method was proven by the Langmuir isotherm approach, which allowed thecalculation of the maximum adsorption capacity (Bmax) and affinity (Kd) of test bentonites. Theseadsorption parameters were compared with BCAfB1 values measured with both the official and optimizedassays. The study helped us rank the best aflatoxin-adsorbing bentonites and confirm the suitability ofthe optimized protocol for BCAfB1 measurement. An interlaboratory study for the validation of this methodis recommended. Application of the protocol to bentonites other than montmorillonite was demonstrated.Acknowledgements. This work was supported by the FoodSafety4EU Project (European Union'sHorizon 2020 Research and Innovation Programme) under Grant Agreement No. 101000613.