A Chemical-Derivatization Assisted Single-Point Isotopologue Multi-point Calibration Strategy for High-Throughput Quantification of Nitrofuran Metabolites in Aquatic Products by HPLC-HRMS

Abstract Construction multi-point calibration curves for quantifying nitrofuran metabolites (NFMs) via HPLC-HRMS is crucial and challenging, being laborious, time-intensive, and prone to inaccuracies. This study developed a chemical-derivatization assisted isotopologue multi-point calibration (IMC) strategy. It used 2-chloro-6-fluorobenzaldehyde (ClFBA) instead of 2-nitrobenzaldehyde (prescribed in GB 31656.13-2021) to derivatize NFMs. Owing to Cl’s distinctive isotopic signature, ClFBA derivatization enhanced the M + 1 ∼ M + 4 isotopic abundances of NFMs, facilitating HRMS detection. Integrating single-point IMC with internal calibration, multi-point calibration curves were constructed based on the correlation between the isotopic abundance (reflecting concentration) of ClFBA-derivatized NFMs and HRMS signals, applied to quantify NFMs in aquatic products. All NFMs exhibited excellent linearity (R2 ≥ 0.9991), with LOQs of 0.5 μg/kg. Recoveries ranged from 87.99% to 109.87%, while intra-day RSDs were between 4.19% and 11.88% and inter-day RSDs fell within 2.86% to 8.78%. This methodology holds potential for precise HRMS-based quantification of other amino-containing small-molecule drugs.