In Europe, bentonites are allowed as feed additives for aflatoxin mitigation (1m558) provided they have specific mineralogical characteristics and an aflatoxin-binding capacity (BCAfB1) >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 analysing six bentonites that met the mineralogical requirements for claim code 1m558. Fixing experimental conditions of the EU official assay, main leading parameters of this method were analysed and varied one-by-one. Leading factors selected for robustness testing were: (i) preparation mode of bentonite suspension; (ii) residual amount 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, bentonites were first tested according to the experimental condition described in the EU method. Then, they were tested again by making some changes, each one in independent, triplicate experiments. It was statistically evinced that although some variations in the protocol, i.e., the optimization of acetonitrile concentration in the working solutions, centrifugation conditions, and buffer concentration, produced an effect 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 bentonites from being marketed in the EU because their BCAfB1 values were >90%. A new protocol was developed by keeping the main experimental parameters of the official assay and was in-house validated according to international harmonized guidelines. This protocol yielded BCAfB1 values >90% for all test bentonites and showed satisfactory precisions with a RSDI of 3.4% and HorRat<2. This value falls within the acceptability criteria stated in the AOAC guidelines for standard method performance requirements. The validity of the optimized method was proven by the Langmuir isotherm approach, which allowed the calculation of the maximum adsorption capacity (Bmax) and affinity (Kd) of test bentonites. These adsorption parameters were compared with BCAfB1 values measured with both the official and optimized assays. The study helped us rank the best aflatoxin-adsorbing bentonites and confirm the suitability of the optimized protocol for BCAfB1 measurement. An interlaboratory study for the validation of this method is recommended. Application of the protocol to bentonites other than montmorillonite was demonstrated. Acknowledgements. This work was supported by the FoodSafety4EU Project (European Union's Horizon 2020 Research and Innovation Programme) under Grant Agreement No. 101000613.
EU LEGISLATION REQUIRES AN OPTIMIZED/VALIDATED METHOD FOR THE OFFICIAL CONTROL OF BENTONITES AS AFLATOXIN INACTIVATORS
Vito D'Ascanio;Donato Greco;Mariagrazia Abbasciano;Antonio Moretti;Giuseppina Avantaggiato
2023
Abstract
In Europe, bentonites are allowed as feed additives for aflatoxin mitigation (1m558) provided they have specific mineralogical characteristics and an aflatoxin-binding capacity (BCAfB1) >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 analysing six bentonites that met the mineralogical requirements for claim code 1m558. Fixing experimental conditions of the EU official assay, main leading parameters of this method were analysed and varied one-by-one. Leading factors selected for robustness testing were: (i) preparation mode of bentonite suspension; (ii) residual amount 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, bentonites were first tested according to the experimental condition described in the EU method. Then, they were tested again by making some changes, each one in independent, triplicate experiments. It was statistically evinced that although some variations in the protocol, i.e., the optimization of acetonitrile concentration in the working solutions, centrifugation conditions, and buffer concentration, produced an effect 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 bentonites from being marketed in the EU because their BCAfB1 values were >90%. A new protocol was developed by keeping the main experimental parameters of the official assay and was in-house validated according to international harmonized guidelines. This protocol yielded BCAfB1 values >90% for all test bentonites and showed satisfactory precisions with a RSDI of 3.4% and HorRat<2. This value falls within the acceptability criteria stated in the AOAC guidelines for standard method performance requirements. The validity of the optimized method was proven by the Langmuir isotherm approach, which allowed the calculation of the maximum adsorption capacity (Bmax) and affinity (Kd) of test bentonites. These adsorption parameters were compared with BCAfB1 values measured with both the official and optimized assays. The study helped us rank the best aflatoxin-adsorbing bentonites and confirm the suitability of the optimized protocol for BCAfB1 measurement. An interlaboratory study for the validation of this method is recommended. Application of the protocol to bentonites other than montmorillonite was demonstrated. Acknowledgements. This work was supported by the FoodSafety4EU Project (European Union's Horizon 2020 Research and Innovation Programme) under Grant Agreement No. 101000613.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
