Pet and food animal (hogs, chicken, and fish) feeds were recently found to be contaminated with melamine (MEL). A quantitative and confirmatory method is presented to determine MEL residues in edible tissues from fish fed this contaminant. Edible tissues were extracted with acidic acetonitrile, defatted with dichloromethane, and cleaned up using mixed-mode cation exchange solid-phase extraction cartridges. Extracts were analyzed by liquid chromatography with tandem mass spectrometry with hydrophilic interaction chromatography and electrospray ionization in positive ion mode. Fish and shrimp tissues were fortified with 10-500 microg/kg (ppb) of MEL with an average recovery of 63.8% (21.5% relative standard deviation, n = 121). Incurred fish tissues were generated by feeding fish up to 400 mg/kg of MEL or a combination of MEL and the related triazine cyanuric acid (CYA). MEL and CYA are known to form an insoluble complex in the kidneys, which may lead to renal failure. Fifty-five treated catfish, trout, tilapia, and salmon were analyzed after withdrawal times of 1-14 days. MEL residues were found in edible tissues from all of the fish with concentrations ranging from 0.011 to 210 mg/kg (ppm). Incurred shrimp and a survey of market seafood products were also analyzed as part of this study.

Although melamine and cyanuric acid appeared to have low toxicity when administered separately, they induced extensive renal crystal formation when administered together. The subsequent renal failure may be similar to acute uric acid nephropathy in humans, in which crystal spherulites obstruct renal tubules.

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.

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