Comprehensive and sensitive validation of the method and determination of measurement uncertainty for simultaneous specification of aflatoxin B1, B2, G1 and G2 in nuts

Abstract

In this study, a comprehensive and sensitive validation study of the legally used method was conducted to simultaneously determine the levels of aflatoxin B1, B2, G1, and G2 in nuts. The applied method has been extensively evaluated in terms of in-laboratory detection limit and quantification limit, linearity, selectivity, precision, recovery, accuracy, adequacy, and measurement uncertainty. Levels of aflatoxins were examined by a validated immunoaffinity column-high performance liquid chromatography-post column derivatization-fluorescence detection (IAC-HPLC-PCA-FLD) analytical method. Detection limits (LOD) for aflatoxin G2, G1, B2, and B1 were found to be 0.05 μg/kg, 0.13 μg/kg, 0.06 μg/kg, and 0.15 μg/kg, respectively. The regression coefficient (R²) was greater than 0.999 according to constructed calibration curves for all aflatoxin components. Thus, the curves exhibited satisfactory linearity within the analyzed range. The combined standard uncertainty values for precision (RSD%) studies were below 10%. It showed a robust and reliable sensitivity with <10% RSD with the test procedure. Average recovery values were detected as 67-84% for hazelnut and 60-89% pistachio. All aflatoxin components in hazelnut paste, which is the proficiency test material, were determined within an appropriate |Z|-score range (≤ 2). The relative expanded uncertainty of measurement was 0.80 μg/kg for AFB1 and AFG1, 0.83 μg/kg for AFB2 and AFG2, and 0.78 μg/kg for total AFs. The results showed that the approved method for the investigation and monitoring of aflatoxins in hazelnuts and pistachios is highly sensitive, robust, reliable, selective, and reproducible. Validation parameters can be successfully used in routine analysis to monitor aflatoxins in nuts for compliance with current regulations.