Development and application of deep eutectic solvent-based dispersive liquid-liquid microextraction for mycotoxin analysis in livestock feed by LC-MS/MS

Deep eutectic solvent-based dispersive liquid-liquid microextraction (DES-DLLME) has been developed, optimised, validated, and applied for the analysis of mycotoxins in livestock cereal feed. A two-step experimental design approach was utilised, namely full factorial design for initial screening, followed by a central composite design (CCD) for optimisation of quantifiable factors. The mycotoxin extraction efficiencies were significantly affected by extraction time, solvent volume and donor phase volume. A composite desirability function of 0.867 was achieved, with p -values from the lack-of-fit (LOF) test ranging from 0.064 to 0.854. Method validation was in accordance with Commission Implementing Regulation (EU) 2023/2782 of 14 December 2023, Commission Regulation (EC) No. 401/2006 of 23 February 2006 and the Commission Implementing Regulation (EU) 2021/808 dated 22 March 2021. The coefficient of determination ( R 2 ) values varied from 0.9908 to 0.9982. The limit of detection (LOD) and limit of quantification (LOQ) ranged between 0.36 and 10.1 µg/kg and 1.20 and 33.9 µg/kg, respectively. The repeatability (RSDr) CVs ranged from 2.77 to 8.90%, and the reproducibility (RSDR) CVs ranged from 0.30 to 9.80% were within acceptable range, whereas recoveries ranged from 86.6% to 99.2%. The method was applied to 43 animal cereal feed samples, traces of aflatoxins were observed, whereas fumonisin B 1 and fumonisin B 2 detected but were compliant.