Rapid determination of illegal dyes in aquaculture products using LC-Q-Orbitrap HRMS: Method development, validation, and application

Abstract

This study presents a sensitive, reliable, and rapid analytical method for the determination of four illegal dyes, malachite green (MG), crystal violet (CV), leucomalachite green (LMG), and leucocrystal violet (LCV) in Nile tilapia (Oreochromis niloticus), salmon (Salmo salar), crawfish (Procambarus clarkii), and shrimp (Penaeus vannamei) matrices using liquid chromatography coupled with quadrupole-Orbitrap high-resolution mass spectrometry (LC-Q-Orbitrap HRMS). The method employs a simplified sample preparation procedure, enabling high-throughput analysis. Validation was performed in accordance with the Commission Implementing Regulation (CIR) EU 2021/808, using three concentration levels ranging from 0.25 to 0.75 μg/kg. The method demonstrated excellent performance, with recoveries ranging from 71 % to 98 % and both repeatability and reproducibility consistently below 9 %. Calibration curves exhibited good linearity, with coefficients of determination (R²) exceeding 0.9955. The limits of detection (LOD) and quantification (LOQ) ranged from 0.019 to 0.057 μg/kg and 0.061–0.173 μg/kg, respectively. The decision limit (CCα) and detection capability (CCβ) values ranged from 0.26 to 0.31 μg/kg and 0.28–0.35 μg/kg, respectively. The method was applied to analyze 200 samples, consisting of 50 samples each from the four aforementioned species, were collected from local markets in Egypt, revealing that 59.5 % of the samples contained illegal dye residues. The reliability of the method was further confirmed through successful participation in two proficiency testing (PT) rounds. This study represents one of the most comprehensive assessments of illegal dye residues in fish and crustacean products in the Egyptian market, highlighting the need for enhanced food safety monitoring in the region.