Determination of residues of malachite green in aquatic animals

Residues of malachite green (MG) were extracted from homogenized animal tissues with a mixture of McIlvaine buffer (pH 3.0)–acetonitrile, and purified over an aromatic sulfonic acid solid-phase extraction column followed by HPLC or LC–ESI-MS–MS analysis. Ascorbic acid and N,N,N′,N′-tetramethyl-1,4-phenylenediamine dihydrochloride were added to reduce de-methylation of the dye. Responses were recorded at 620 nm (HPLC) or by multiple-reaction-monitoring (LC–MS–MS) after post-column oxidation using PbO2. MG and its primary metabolite leuco-malachite green (LMG) were successfully determined at 2.5–2000 μg/kg in catfish, eel, rainbow trout, salmon, tropical prawns and turbot, with a limit of detection at 1 μg/kg (HPLC) and 0.2 μg/kg (LC–MS–MS) for both MG and LMG. Recoveries for LMG were between 86±15% (prawn) and 105±14% (eel). Freeze–thawing cycles, and storage at 4 °C and −20 °C affected the recovery of both MG and LMG. Analyses of eel, trout and (processed) salmon field samples collected at local retailers, fish-market and -shops demonstrated trace levels of MG-residues.