Development and validation of a DLLME‐HPLC‐FLD method for determination of aflatoxins in Chrysanthemum morifolium based on quality by design principles

Abstract Introduction Aflatoxins, potent carcinogens produced by Aspergillus species, present significant health risks and commonly contaminate herbal products such as Chrysanthemum morifolium . Detecting these toxins in C. morifolium proves challenging due to the complex nature of the herbal matrix and the fluctuating levels of toxins found in different samples. Objectives This study aimed to develop and optimize a novel method for the detection of aflatoxins in C. morifolium using dispersive liquid–liquid microextraction combined with high‐performance liquid chromatography‐fluorescence detection based on quality by design principles. Methodology The method involved determining critical method attributes and parameters through the Plackett–Burman design, followed by optimization using the Box–Behnken design. Monte Carlo simulation was employed to establish a design space, which was experimentally verified. Method validation was performed to confirm accuracy, precision, and stability. Results The developed method exhibited excellent linearity ( R 2 > 0.9991) for aflatoxins B 1 , B 2 , G 1 , and G 2 across a range of concentrations, with recovery rates between 85.52% and 102.01%. The validated method effectively quantified aflatoxins in C. morifolium under different storage conditions, highlighting the impact of temperature and storage time on aflatoxin production. Conclusion This study successfully established a reliable and effective method for the detection of aflatoxins in C. morifolium , highlighting the importance of strict storage conditions to reduce aflatoxin contamination. Using a quality by design framework, the method demonstrated robustness and high analytical performance, making it suitable for routine quality control of herbal products.