Residue accumulation-guided preparation of grass carp matrix candidate reference materials for four nitrofuran metabolites using HPLC-MS/MS

A residue accumulation-guided strategy was developed for preparing grass carp matrix candidate reference materials. • Parent nitrofuran drugs were introduced by medicated bath to generate four marker metabolites in fish muscle. • HPLC–MS/MS enabled reliable quantification of nitrofuran marker metabolites in grass carp powder. • A 10 h bath exposure at 25 °C achieved target residue levels of 5–10 μg/kg. • The prepared candidate materials showed acceptable homogeneity, stability, and batch-to-batch reproducibility. The preparation of incurred aquatic matrix candidate reference materials remains challenging because residue formation is influenced by biological exposure processes and matrix interactions. In this study, a residue accumulation-guided strategy was developed for the preparation of a grass carp matrix candidate reference material containing four nitrofuran marker metabolites, including semicarbazide (SEM), 3-amino-2-oxazolidinone (AOZ), 1-aminohydantoin (AHD), and 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ). The relationship between medicated-bath exposure conditions and muscle residue levels was evaluated and used to determine practical preparation conditions. Under the optimized exposure conditions, grass carp samples were collected, homogenized, freeze-dried, ground, sieved, and blended to obtain a homogeneous candidate material. The prepared candidate material showed acceptable homogeneity and stability under the investigated conditions and yielded batch-to-batch reproducibility with RSD values of 1.9–3.2% for 0.4 g freeze-dried test portions. The developed material exhibited suitable characteristics for potential applications in analytical quality assurance, method comparison, and related scientific studies. This study provides an alternative strategy for preparing grass carp matrix candidate reference materials containing incurred nitrofuran marker metabolites through controlled biological exposure and residue accumulation-guided preparation. Download: Download high-res image (172KB) Download: Download full-size image