Agriculture Reference
In-Depth Information
7.4 APPLICATION NUMBER 1: IDENTIFICATION OF BRILLIANT GREEN
AND ITS METABOLITES IN FISH UNDER HIGH-RESOLUTION MASS
SPECTRAL CONDITIONS (TARGETED AND NONTARGETED APPROACHES)
To give a clear idea of the advances of analytical screening methods for detection of
chemical traces in biological matrices, the
first study we shall discuss, which was
conducted at the ANSES
Fougères Laboratory, concerns pharmacologically active
dyes that are not authorized in the EU due to their toxic properties. While this
—
rst
example is not yet optimized for high throughput, it illustrates the importance of
employing existing methods that are time consuming and require multiple steps, and
streamlining them so that more analytes can be screened in each run. It also
demonstrates the differences in using two different strategies: the targeted screening
and the nontargeted approach. The dyes considered here were widely used in
aquaculture and
ciency in the prevention
and/or treatment of bacterial or fungal infections in aquatic animals. Malachite green
(MG) is one of the most popular and widely used of these compounds; it belongs to
the triarylmethane family and is known to possess mutagenic, carcinogenic, terato-
genic, and genotoxic properties [26]. Because of the potentially severe toxic effects of
these dyes, most of the countries around the world restricted their use for the
production of
fisheries due to their low cost and ef
fish intended for human consumption. Because of these
restrictions, methods for their monitoring are regulated in the EU, and analytical
techniques such as low-resolution tandem LC
fish and shell
-
MS are already widely used to screen
farmed
fish for this dye, and importantly, its metabolized product, leuco-malachite
green (LMG). Recently, after suspicions were raised about the substitution of MG by
another dye in farmed
fish products imported from South America and Asia, European
of
cial control methods were extended to cover a second type of dye named gentian
violet (also known as crystal violet (CV) in English-speaking countries). The
Reference Laboratory at ANSES
Fougères thus developed a method for monitoring
both malachite green and gentian violet and their respective metabolized leucobases
LMG and LCV [27]. The analytes were isolated from the
—
sh
flesh matrix by liquid
-
liquid extraction with acetonitrile. Determination was performed using LC
MS/MS
with positive electrospray ionization, using SRM mode, and monitoring two transi-
tions for each compound. The chromatographic separation was performed on a
reversed-phase HPLC C18 column, length 100mm
-
m
silica particles. The mobile phase consisted of ammonium formate buffer (0.05M, pH
4.5) mixed with acetonitrile and was used in gradient mode. Very similar methods
have now been in use for more than three years in almost all of the EU
×
diameter 2.1 mm, with 3.5
μ
'
sof
cial
control laboratories. Very recently, the presence of brilliant green (BG) in certain
sh
farm products, a third dye belonging to the same family of triarylmethanes, has been
suggested to possibly be in use in aquaculture farming, but has not yet been evidenced
by the targeted methods currently used in of
cial controls. The screening for the BG
substance in aquaculture products was therefore studied. A new technique was
proposed for detecting the metabolites of this compound in farmed
sh
esh. At
first, because of its similarity to other triarylmethane compounds, the hypothesis that
the leucobase of brilliant green (LBG) would be found in trout treated with BG was
