HPLC Determination and MS Confirmation of Malachite Green, Gentian Violet, and Their Leuco Metabolites in Catfish Muscle

Authors: Chen, Guoying; MIAO, SHUI - Shanghai Institute For Food And Drug Control

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/8/2010
Publication Date: 5/19/2010
Citation: Chen, G., Miao, S. 2010. HPLC Determination and MS Confirmation of Malachite Green, Gentian Violet, and Their Leuco Metabolites in Catfish Muscle. Journal of Agricultural and Food Chemistry.(58):7109-7114.

Interpretive Summary: Malachite green (MG) and gentian violet (GV) have been used in aquaculture since 1936 as effective fungicides, parasiticides, and antiseptics. They are rapidly absorbed by fish and extensively converted into leuco forms. Due to high toxicity, these drugs are banned for aquacultural use, but are still used illegally. To protect public health, sensitive and robust detection methods are needed. A liquid chromatographic method was developed to detect MG, GV, and their leuco residues in catfish muscle using visible and fluorescence detectors, and to confirm their identities by mass spectrometry. This detection scheme obviated lead oxide that converts leuco forms to chromatic forms for effective detection, thus eliminated uncertainties in oxide depletion and data integrity. Sample preparation was simplified to only one extraction step and one cleanup column. By excluding harmful organic solvents and lead oxide, this method is friendly to both workers and the environment, saves cost and time, and meets both the FDA and European Union regulatory requirements.

Technical Abstract: Residues of malachite green (MG), gentian violet (GV), and their leuco metabolites in catfish muscle were individually determined by HPLC using visible and fluorescence detectors. This detection scheme obviated a PbO2 column that converts leuco forms to chromatic forms for visible detection, thus eliminated band broadening and uncertainties in oxidant depletion and data integrity. Extraction was performed once in pH 3 McIlvaine buffer and acetonitrile, followed by cleanup using cation exchange columns. Liquid-liquid extraction was excluded to provide a friendly and relatively rapid protocol. Spectrometric detection of MG at 620 nm and GV at 588 nm achieved 0.38 and 0.26 ng/g (S/N=3) limit of detection (LOD), and 44.5-49.2% and 92.2-101.4% recovery at 1-10 ng/g (n=6), respectively. Fluorometric detection of leuco metabolites at 350 nm under 265 nm excitation achieved 0.10 and 0.09 ng/g LOD, and 74.3-84.5% and 80.6-86.5% recovery at 1-10 ng/g (n=6), respectively. Analyte identities were further confirmed by mass spectrometry.