Methods to be Reviewed by OMB in 2016

658

chneider

ndersen

ournal of

AOAC I

nternational

. 98, N

. 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

arilyn

J. S

chneider

U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Ln,

Wyndmoor, PA 19038

endy

C. A

ndersen

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

Guest edited as a special report on “Methods of Analysis for

Residues and Chemical Contaminants in Aquaculture” by Joe Boison

and Sherri Turnipseed.

Corresponding author’s e-mail:

wendy.andersen@fda.hhs.gov

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.

SPECIAL GUEST EDITOR SECTION

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

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.

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.

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

Candidates for 2016 Method of the Year

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