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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 ... 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，N9，N9-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 mg / kg in catfish， eel， rainbow trout， salmon， tropical prawns and turbot， with a limit of detection at 1 mg / kg (HPLC) and 0.2 mg / 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.

A McIlvaine solution at pH 3.0 was prepared by mixing 18.9 ml 0.2 M sodium hydrogen phosphate and 81.1 ml 0.1 M citric acid， whereas these volumes were 62.5 and 37.5 ml， respectively， to obtain a McIlvaine solution at pH 6.0. The SPE-eluent was prepared just before use and consisted of a mixture of 2.5 ml 25% (m/v) ammonium hydroxide， 2.5 ml 1.0 mg/ml methanolic ascorbic acid and 45 ml methanol. The sample-solvent was composed， just before use， from 2.5 ml 1.0 mg/ml methanolic ascorbic acid， 20 ml 50 mM sodium perchlorate containing 25 mM sodium acetate and 25 mM 1-pentanesulfonic acid adjusted to pH 4.0 with acetic acid， and 27.5 ml acetonitrile. Chemicals and solutions containing the dyes were protected from light.
Fish were bought at the fish market and local stores. Blank trout were collected from aquaria at
Utrecht University (The Netherlands). Tropical prawns were collected by the Dutch Inspectorate for Health Protection and Veterinary Public Health.