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

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Abstract
Residues of malachite green (MG), gentian violet (GV), and their leuco metabolites in channel catfish muscle were individually determined by HPLC using diode array and fluorescence detectors and confirmed by tandem mass spectrometry. This detection scheme obviates a PbO2 reactor that converts leuco forms to chromatic forms for absorbance detection, therefore eliminating uncertainties in oxidant depletion and data integrity. Extraction was performed once in pH 3 McIlvaine buffer and acetonitrile, followed by cleanup using a polymeric strong cation-exchange column. Liquid−liquid extraction was excluded to provide an environmentally responsible and relatively rapid protocol. Spectrometric limits of detection (LOD; S/N = 3) for MG (λ = 620 nm) and GV (λ = 588 nm) were 0.38 and 0.26 ng/g with 44.5−49.2% and 92.2−101.4% recoveries (1−10 ng/g, n = 6), respectively. Fluorometric LOD (S/N = 3) for LMG and LGV (λex = 266 nm, λem = 360 nm) were 0.10 and 0.09 ng/g with 74.3−84.5% and 80.6−86.5% recoveries (1−10 ng/g, n = 6), respectively. This simplified protocol saves costs and meets the sensitivity requirements set by the Food and Drug Administration and the European Union.

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