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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
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:
wendy.andersen@fda.hhs.govDOI: 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.
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.
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