Agriculture Reference
In-Depth Information
considered. The presence of this compound had never been con
rmed in previous
studies [28]. We then screened the
eshtotrackthepresenceofother
metabolites derived from the BG dye. The experiment involved a batch of trout
treated with BG compared to a batch of BG-untreated trout. Samples of trout
sh
esh
were collected and subjected to a chemical extraction step prior to their analysis by
high-resolution LC
Orbitrap mass spectrometer, a hybrid MS
with linear trap and orbital trap. The source of ionization was an electrospray
ionization probe set in the positive ion mode. The instrument operated in full scan
mode from
m
/
z
100 to 1000 at a resolving power of 60,000 (full width at half
maximum (FWHM)). Prior to the LC
-
MSusinganLTQ
-
-
MS analysis, the mass spectrometer was
calibrated using the manufacturer
s calibration solution (consisting of caffeine, the
tetrapeptide MRFA
,andUltramark
) in order to ensure reaching mass accuracies
in the range of 1
'
3ppm.
There were two different ways to process the resulting high-resolution LC
-
MS raw
data: the targeted way and the nontargeted way. In the targeted approach, the
suspected analyte ions, with their exact masses, were extracted from the total ion
chromatogram(TIC) for their identi
-
cation. The nontargeted approach consisted of
using all the signals obtained from the samples of the same batch of trout treated with
BG and comparing these with the signals obtained from the control batch. This
comparison was conducted using Sieve
software.
In the
first targeted approach, the presence of BG and of its leucobase LBG was
identi
ed in the treated
sh
flesh sample under high-resolution mass spectral
conditions. This identi
cation was processed by requesting extracted ion chromato-
grams of the targeted compounds, as shown in Figure 7.2. As can be seen in the
gure,
all the metabolites were separated in
15min. The mass spectra of BG (theoretical
mass M
+
: 385.26382) and its leukobase LBG (theoretical mass MH
+
: 387.27947)
show
<
1.2 ppm in mass accuracy. In addition, the resulting isotopic patterns of the
targeted compounds adequately matched with the theoretical
<
isotopic patterns.
H]
+
ion was subjected to high-resolution MS/MS
analysis and the resulting product ion spectrum was compared with MS/MS data
obtained from its pure custom-made standard form derived by chemical synthesis,
con
Furthermore, the LBG [M
+
rming the presence of LBG unambiguously.
In the second approach (nontargeted analysis) high-resolution LC
MS raw data of
BG extracted from both treated and untreated trout samples were all processed
through Sieve software for their comparison. For this, a set of six BG-treated trout
-
sh
samples and a set of six BG-untreated trout
fish samples were used. The Sieve
software extracted signi
cant differences in signals between the groups of treated and
untreated
fishes, thus providing a list of ions (compounds) present in one group and
absent in the other group. With this approach (Figure 7.2), we were able to
rst screen
and subsequently con
rm from all the high-resolution mass chromatograms obtained
the presence of both BG and LBG. Second, we even extracted and characterized the
presence of several other metabolized compounds, one of which was formally
identi
ed as desethyl LBG and another one, probably in a lower concentration,
with a mass between 441.32759 and 441.33641, whose molecular structure has not
yet been identi
ed with enough certainty [27].
