Agriculture Reference
In-Depth Information
obtained from standard injections are used to create an Excel
file containing each
compound de
ned by its formula, the exact molecular weight, and the expected
retention time. This list is the strategic heart of the automatic process of identi
cation
called ToxID
.
Using this software, a compound is positively identi
ed from HRMS analysis
when the following criteria are met simultaneously: measured retention time in
accordance with the expected retention time, measured high-resolution accurate mass
in accordance with the expected theoretical accurate mass with a tolerance of
<
5 ppm
(criterion to be de
ned arbitrary), and peak intensity higher than an arbitrary threshold
to be prede
ned (Figure 7.4). This method has been tested successfully on beef, pork,
and poultry meat samples. However, the limitation of the method is in the ef
cient
extraction step of those 60 antibiotic compounds from the matrices suitable at the
MRL level. A single extraction procedure did not extract all compounds at concen-
tration levels that could be high enough to be detected by the instrument. Thus, two
different extractions had to be carried out in parallel for each sample to analytically
cover the set of 60 antimicrobial compounds. Our next step in this work is to consider
developing a comprehensive analytical method that may be able to detect not only
multiple antimicrobial compounds but also multiple classes of veterinary drugs,
including, if possible, antiparasitics, anti-in
ammatories, anticoccidials, and tran-
quillizers. This particular approach for screening veterinary drug residues can be
considered as a
as the analytes are searched only after their
mass has been acquired in the full scan mode.
Another approach we are working on is the nontargeted approach aimed at
identifying metabolites of antimicrobials under high-resolution mass spectral
conditions. One of the
“
posttarget screening
”
cation of
trace metabolites/degradation products of antibiotics in beef. In a preliminary test
with a method involving microbiological analysis using agar diffusion, our beef
sample showed positive microbial
first studies for this approach was the identi
ed
principal antibiotic components in the beef meat that displayed microbial inhibition
by using MRM and high-resolution full scan MS. Second, we focused on
identifying unknown metabolites of antibiotics using a nontargeted approach
with high-resolution mass spectral conditions. In this approach, we compared
high-resolution mass spectral data of a microbial inhibition-active beef muscle
sample with a set of blank beef muscle samples taken from various batches of
animals of the same species that had not been subjected to any intentional veterinary
medication. For this, a set of four consecutive LC
inhibition activity. We initially identi
MS analyses of the single
microbial inhibition-active beef muscle sample and a single LC
-
MS analysis for
each of the six different blank beef samples were carried out using high-resolution
mass spectral conditions. The instrument was operated in full scan FTMS mode
over a range of 10
-
1200 Da at a resolving power of 60,000 FWHM, in electrospray
positive ionmode. LC separationwas performed using a C18 column (125mm
-
×
3mm)
with 5
m particle size. The mobile phase consisted of 1mM HFBA in 0.5% formic
acid solution and 0.5% formic acid in methanol/acetonitrile solution (50:50, v/v).
The LC-MS raw data from the blank samples and the positive sample controlled
were compared (Figure 7.5) with the help of Sieve software. Based on the Sieve
μ
