Determination of nitrofuran metabolites and nifurpirinol in animal tissues and eggs by ultra-high performance liquid chromatography-tandem mass spectrometry validated according to Regulation (EU) 2021/808.

In this work, an analytical method for the determination of eight non-allowed nitrofurans, including nifurpirinol and the metabolites of furazolidone, furaltadone, nitrofurantoin, nitrofurazone, nifursol, nitrovin, and nifuroxazide in animal tissues, including muscle (poultry, bovine, ovine, porcine, rabbit, and fish), kidney (bovine, ovine, porcine), and eggs, has been developed and validated according to Regulation (EU) 2021/808. The method was based on derivatization with 2-nitrobenzaldehyde in acid medium, followed by vortex-assisted liquid-liquid extraction and solid phase extraction for sample purification prior to ultra-high performance liquid chromatography-tandem mass spectrometry. Under selected conditions, the method was validated showing satisfactory relative matrix effects (CV ≤ 20 %), linearity (R ≥ 0.98), trueness (≤20 %, expressed as bias), accuracy (83-120 %), repeatability (1.7-19.9 %), reproducibility (1.9-25.7 %), specificity (blank signal ≤30 % at the LCL), and ruggedness. The decision limit for confirmation (CCα) for the target analytes ranged from 0.27 to 0.35 μg kg, all below the current reference point for action (RPA) of 0.5 μg kg for the studied compounds. This validated method is currently accredited according to UNE-EN ISO/IEC 17025 by the Spanish National Accreditation Body (ENAC) to be implemented for official control analyses in the Public Health Laboratory of Valencia (Spain).