658  S chneider & A ndersen : J ournal of AOAC I nternational V ol . 98, N o . 3, 2015


Determination of Triphenylmethane Dyes and Their Metabolites in Salmon, Catfish, and Shrimp by LC-MS/MS Using AOAC First Action Method 2012.25: Collaborative Study M arilyn J. S chneider U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Ln, Wyndmoor, PA 19038 W endy C. A ndersen 1 U.S. Food and Drug Administration, Office of Regulatory Affairs, Animal Drugs Research Center, Denver Federal Center Bldg 20, W. 6th Ave and Kipling St, Denver, CO 80225 Collaborators: H. An; C. Baker; R. Burger; Y.T. Cai; M. Conway; P. Couëdor; M. Crosswhite; M. Cumming; M. Filigenzi; A. Fong; W. Hammack; A. Harris; A. Hawkins; D. Hurtaud-Pessel; J. Kibbey; C.K. Lam; S.J. Lehotay; A.R. Lightfield; L. Lissemore; S. Lupton; R. Noonan; R. Potter; S. Prakash; S.R. Ruiz; J. Smith; C.D. Stephenson; E. Verdon

T riphenylmethane dyes have been used as a treatment against parasite and fungus infections in aquacultured fish, beginning with the use of malachite green (MG) in the 1930s (1, 2). Structurally related triphenylmethane dyes such as crystal violet (CV) and brilliant green (BG) have similar therapeutic properties, and the leuco metabolites of such dyes can persist in edible fish muscle for months (3). Concerns regarding the toxicity and mutagenicity of the dyes and leuco metabolites have resulted in triphenylmethane dyes being prohibited for the treatment of fish to be used for human consumption by many countries including the United States and the European Union (EU) member states (4–6). Although international regulations ban the use of triphenylmethane dyes in aquaculture, the dyes are inexpensive and readily available. Efficient monitoring of the food supply is needed to ensure these therapeutic dyes are not used in aquaculture, and numerous analytical methods have been published toward this goal. LC-MS/MS methods allow simultaneous determination of multiple triphenylmethane dye and leuco metabolite residues at and below a level of 1 µg/kg (7). One such LC-MS/MS method for determining three readily available triphenylmethane dyes (MG, CV, and BG) and the metabolites leucomalachite green (LMG) and leucocrystal violet (LCV) in seafood has recently been granted AOAC First Action Status (8). The method is based on a relatively simple acetonitrile extraction with hydroxylamine hydrochloride and magnesium sulfate. Residue quantification is normalized by the use of isotopically labeled internal standards and calibration based on extracted matrix standards. This paper describes the collaborative study of the AOAC First Action Method 2012.25 by 14 laboratories for the analysis of MG, CV, BG, LMG, and LCV residues in salmon, catfish, and shrimp.

Guest edited as a special report on “Methods of Analysis for Residues and Chemical Contaminants in Aquaculture” by Joe Boison and Sherri Turnipseed. 1 Corresponding author’s e-mail: DOI: 10.5740/jaoacint.14-263 Mention of brand or firm name does not constitute an endorsement by the U.S. Department of Agriculture or the U.S. Food and Drug Administration above others of a similar nature not mentioned. USDA is an equal opportunity provider and employer. qualitative identification of targeted analytes. Results from statistical analysis showed that this method provided excellent trueness (generally ≥90% recovery) and precision (RSDr generally ≤10%, HorRat <1). The Study Directors recommend Method 2012.25 for Final Action status. A collaborative study was conducted to evaluate the AOAC First Action 2012.25 LC-MS/MS analytical method for the determination of residues of three triphenylmethane dyes (malachite green, crystal violet, and brilliant green) and their metabolites (leucomalachite green and leucocrystal violet) in seafood. Fourteen laboratories from the United States, Canada, and the European Union member states participated in the study including national and state regulatory laboratories, university and national research laboratories, and private analytical testing laboratories. A variety of LC-MS/MS instruments were used for the analysis. Each participating laboratory received blinded test samples in duplicate of salmon, catfish, and shrimp consisting of negative control matrix; matrix fortified with residues at 0.42, 0.90, and 1.75 µg/kg; and samples of incurred matrix. The analytical results from each participating laboratory were evaluated for both quantitative residue determination and

Collaborative Study

Fourteen laboratories participated in this study. Laboratory participants were provided with intermediate standard solutions of the dyes, metabolites, and internal standards and homogenized salmon matrix for the purpose of method familiarization, as

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