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 0.14ng/g for MG, CV, LMG and LCV, respectively. The average recoveries in samples fortified with MG, CV, LMG and LCV over the range 0.5-10ng/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.