Determination of malachite green, crystal violet and their leuco-metabolites in fish by HPLC–VIS detection after immunoaffinity column clean-up

A high performance liquid chromatography method with visible detection (HPLC–VIS) for the determination of malachite green (MG), crystal violet (CV), leucomalachite green (LMG), and leucocrystal violet (LCV) in fish has been developed after clean-up through an immunoaffinity column (IAC). Residues were simultaneously extracted from fish muscle with acetonitrile and ammonium acetate buffer. The leuco-forms, LMG and LCV, were oxidized quantitatively to the chromic CV and MG by reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. Extracts were then purified on an IAC which prepared by immobilizing the anti-MG–CV antibodies by the sol–gel method. Finally, the eluents were analyzed by HPLC–VIS. The limits of detection were 0.15, 0.1, 0.18 and 0.14 ng/g for MG, CV, LMG and LCV, respectively. The average recoveries in samples fortified with MG, CV, LMG and LCV over the range 0.5–10 ng/g were from 71.6% to 96.8% with RSDs of 5.1–12.3% (n = 6). This novel method was confirmed by liquid chromatography–tandem mass spectrometry with electrospray interface in positive mode using multiple reaction monitoring.