Simultaneous Quantification of Fumonisins and Their Hydrolyzed Metabolites in Donkey Matrices: A Tool for Exposure Assessment and Toxicokinetic Studies

A novel, sensitive, and robust LC-MS/MS method was developed and fully validated for the simultaneous determination of fumonisins (FB₁, FB₂, FB₃) and their hydrolyzed metabolites (HFB₁, HFB₂, HFB₃) in donkey plasma, urine, and feces-three critical matrices for toxicokinetic studies. Sample preparation was optimized for each matrix: salting-out assisted liquid-liquid extraction (SALLE) with perchloric acidification for urine and feces, and a dilute-evaporate-shoot (DES) approach for plasma. Chromatographic separation was achieved on a BEH C₁₈ column with water-ACN containing 0.5% formic acid. The method demonstrated excellent linearity (R² ≥ 0.99), acceptable accuracy (mean recoveries: 73.3-111.5%), and good precision (intra- and inter-day RSDs < 20%). The limits of quantification (LOQ) for FBs and HFBs were 0.1-0.15 μg/L in plasma, 1.0 μg/L in urine, and 60 μg/kg in feces. To our knowledge, this is the first reported method capable of quantifying this comprehensive panel of analytes across multiple biological matrices in donkeys, providing an essential tool for future exposure assessments and pharmacokinetic research in this species.