Quantitative and Confirmatory Analyses of Malachite Green and Leucomalachite Green Residues in Fish and Shrimp

Abstract

Liquid chromatographic methods are presented for the quantitative and confirmatory determination of malachite green (MG) and leucomalachite green (LMG) in catfish, trout, tilapia, basa, salmon, and shrimp. Residues were extracted from tissues with ammonium acetate buffer and acetonitrile, and isolated by partitioning into dichloromethane. Leucomalachite green was quantitatively oxidized to the chromic malachite green by reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. Samples were cleaned-up by solid phase extraction with alumina and propylsulfonic acid phases. Extracts were analyzed for total MG by liquid chromatography with visible detection (LC-VIS) at 618 nm, using isocratic elution and a C18 column. The method was validated in each species fortified with LMG at 1, 2, 4, and 10 ng/g (ppb). Average recoveries were 85.9 % (± 8.5 % RSD) for channel catfish, 87.8 % (± 5.1 % RSD) for rainbow trout, 93.9 % (± 8.9 % RSD) for tilapia, 90.8 % (± 11.0 % RSD) for basa, and 89.9 % (± 8.4 % RSD) for tiger shrimp. A slight variation to this extraction procedure was previously validated for LMG and MG in salmon; 1,2 this report contains additional salmon data showing that the two extraction procedures produce equivalent results for salmon analysis. MG was confirmed in fish extracts by ion trap LC-mass spectrometry (LC-MS n ) with no discharge-atmospheric pressure chemical ionization (ND-APCI). In addition to providing confirmatory data, the LC-MS n method provides an alternative quantitative method for MG in fish. The recoveries of LMG, measured as MG by this LC-MS n method, at all fortification levels (1-10 ppb) were high (75-116 %) with relative standard deviations of 21 % or less. The results agreed very closely with those obtained from the same extracts using the LC-VIS analysis procedure, indicating that matrix suppression was not an issue with LC-MS n . This study also included the determination of MG and LMG residues in tissues from catfish, trout, tilapia, and salmon that had been treated with MG. Malachite green was quantitatively determined at the LC-VIS method detection limit of 1.0 ppb; detection limits of approximately 0.25 ppb are possible with LC-MS n .The Laboratory Information Bulletin is a communication from the Division of Field Science, Office of Regulatory Affairs, U.S. Food and Drug Administration for the rapid dissemination of laboratory methods (or scientific regulatory information) which appear to solve a problem or improve an existing problem. In many cases, however, the report may not represent completed analytical work. The reader must assure, by appropriate validation procedures, that the reported methods or techniques are reliable and accurate for use as a regulatory method.

Certain Dyes as Pharmacologically Active Substances in Fish Farming and Other Aquaculture Products

“…This method included the most common and effective therapeutic dyes used in aquaculture (MG, CV, BG, and MB) and expanded the list to include other dyes that might be substituted for these to avoid regulatory detection, including pararosaniline, ethyl violet, the trinaphthylmethyl Victoria blue dyes, azure B, and new MB. Similar to other procedures, 145 the dyes were extracted from salmon using ammonium acetate buffer at pH 4.5, acetonitrile, and alumina followed by liquid-liquid extraction with dichloromethane, oxidation with DDQ, and cation exchange SPE. Because leuco metabolites are only available for MG and CV, the inclusion of a DDQ oxidation process drives leuco metabolites of triarylmethane and phenothiazine dyes to their chromic parent dye for simplified analysis.…”

“…144 This permitted sensitive analysis of the sum of MG and LMG in a variety of seafood products with HPLC-VIS and quantification and confirmation of residue identity by LC-MS n with no-discharge atmospheric pressure ionization at and below concentrations of 1 μg/kg for complete regulatory monitoring. 145,146 The method was later extended to include CV, LCV, and BG residues 30 and adapted for other analytical procedures including LC-MS/MS analysis. 147 Though extract clean-up procedures for triphenylmethane compounds often include similar procedures based on acid/organic solvent extraction with partitioning into dichloromethane and cation exchange SPE cartridge clean-up, many variations exist.…”

Current trends in sample preparation for growth promoter and veterinary drug residue analysis

“…A number of groups have developed alternative methods for isolating TPM residues using McIlvaine buffer and ACN prior to purification on aromatic sulphonic acid [413], SAX [418] or alumina-MCX SPE sorbents [415,419]. Other groups have used an alternative ammonium acetate and ACN extraction solvent prior to PRS [409,420] or alumina-SCX [410] clean-ups.…”

Novel stilbene‐based Fischer base analog of leuco‐TAM – (2E,2′Z)‐{2‐(4‐(E)‐styrylphenyl)propane‐1,3‐diylidene}bis(1,3,3‐trimethylindoline) – derivatives: synthesis and structural consideration by 1D NMR and 2D NMR spectroscopy

“…It has long been used to control fungal and protozoan infections in fish. [17,18] Although the use of MG has now been prohibited for the control of fungal infections in commercial fisheries, it is still used worldwide because it is readily accessible and economical. [19] As a result, there is a strong demand for the substitutes for MG compounds.…”