Abstract
Routine internal calibration is necessary to evaluate the performance of ultrahigh-performance liquid chromatography (UHPLC); therefore, false negative and false positive results are avoided. Herein, we report a detailed internal calibration protocol and validation procedure for the determination of aflatoxins in groundnuts and red chili plants via UHPLC to comply with the requirements of ISO/IEC: 17025. The validated parameters included the flow rate accuracy, injector accuracy, temperature accuracy, system precision, photodiode detector, detector linearity, injector linearity, gradient proportion test, fluorescence detector linearity and wavelength accuracy results. The obtained percent deviation, relative standard deviation and r2 values for injector accuracy, system precision and calibration curves were -0.77, 0.78 and ≥ 0.999, respectively. During the development of the aflatoxin analysis scope, internally calibrated UHPLC was used for separating the aflatoxin mixture at a concentration of 5 µg/kg in the ground nut and red chili matrices. Witness test results revealed that the quantification of individual aflatoxins at 0.5 µg/kg in the red chili matrix was achievable. A stability assessment of the aflatoxin standards solubilized in the solvent was achieved by generating control plots, and uncertainties were also measured.