LC/MS/MS Research Method for 14
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Transcript LC/MS/MS Research Method for 14
Quick and Sensitive Analysis of Multiclass
Veterinary Drug Residues in Animal
Products Using a Novel Benchtop Orbitrap
Mass Spectrometry System
Olaf Scheibner,1 Maciej Bromirski,1 Markus Kellmann,1 Sebastian Westrup, 2 Charles Yang3
1
Thermo Fisher Scientific, Bremen, Germany; 2Thermo Fisher Scientific, Dreieich, Germany;
3
Thermo Fisher Scientific, San Jose, CA, USA
Overview
Results
Purpose: Fast and reliable analysis of veterinary drugs in various matrices.
Goal
Methods: A generic UHPLC method was used in combination with full scan/vDIA data
acquisition.
44 multi-class veterinary drug residue
Benzylpenicillin (G), Cefalexine, Cefa
Chlortetracyclin, Ciprofloxacin, Cloxa
Dimetridazole, Doramectine, Doxycyc
Erythromycine Flumequin,
Erythromycine,
Flumequin Ipronidaz
Metronidazole, Metronidazole-OH, M
Phenoxymethylpenicillin (V), Ronidaz
Sulfadimidine (Sulfamethazine), Sulfa
Sulfamethoxypyridazine,
ypy
, Sulfathiazol
Tylosin) from muscle, kidney, milk an
chromatographic and mass spectrom
with eight calibration points were pre
ng/mL (ppb). Spiked samples (muscl
and plasma for Nitroimidazoles) were
for quantitation, while up to five addit
qualification, achieving linear calibrat
Results: Excellent sensitivity (below 1 ppb) and linearity could be achieved for a broad
variety of compounds.
Introduction
A new scan mode termed variable Data Independent Acquisition (vDIA) is utilized to
acquire
i unknown
k
samples
l and
d is
i compared
d to
t a standard
t d d Data
D t Dependent
D
d tA
Acquisition
i iti
(DDA) mode. The vDIA mode utilizes larger isolation windows than are typically
employed for standard DIA in proteomics applications, ranging from a minimum of 50
Da to over 500Da. In the example described below, a smaller isolation window is used
at the low end of the mass spectra,
spectra and the isolation window is increased with m/z to
improve the duty cycle. For a normal acquisition, about 5 scan ranges are set to cover
the entire mass range.
Methods
Sample Preparation
Samples were prepared using liquid/liquid extraction for antibiotics (muscle and kidney
matrix) and a C18 solid phase extraction cartridge for nitroimidazoles and avermectins
(plasma and milk matrix).
Liquid Chromatography
A generic LC method was applied for all samples consisting of a 6 minute gradient from
aqueous conditions to 95 % organic, resulting in a 15 minute full chromatographic
cycle.
y
For separation,
p
, a Thermo Scientific™ Accucore™ C18 2.1x100 mm column was
employed, using water and acetonitrile, both containing 0.1% formic acid, at a flow rate
of 400 µL/min for elution.
Mass Spectrometry
A Th
Thermo Scientific™
S i tifi ™ Q Exactive™
E
ti ™ benchtop
b
ht Orbitrap™
O bit ™ mass spectrometry
t
t system
t
was used and operated at a resolution of R=70,000 for full scan measurements and
R=35000 for data dependent fragmentation scans, while R=17500 was used for vDIA
scans.
Data Analysis
Thermo Scientific™ TraceFinder™ 3.2 software was used for data analysis.
FIGURE 1. Isolation windows of a vDIA experiment.
full scan
m/z 100 - 1000
vDIA
m/z 100 - 200
vDIA
m/z 200 - 300
vDIA
m/z 300 - 400
vDIA
m/z 400 - 500
vDIA
m/z 500 – 1000
Measurements were carried out in tw
scan – data dependent MS2 (FS-ddM
sensitivity of full scan HRAM detectio
narrow isolation window, triggered fro
occurrence in the full scan. This appr
t be
to
b analyzed,
l
d b
butt needs
d predefinitio
d fi iti
scan mode chosen is the variable DIA
combination of a full scan with severa
the MS2 windows vary between 100 D
the gap between FS-ddMS
FS ddMS2 experime
scan as all ion fragmentation, for exa
TABLE 1. Analyzed compounds wi
classical data dependent MS2 appr
Compound
FS-vDIA FS-ddMS2
[ppb]
[ppb]
Abamectin*
5.0
50.0
Amoxicillin
10
1.0
10
1.0
Ampicillin
0.5
0.5
Cefalexin
0.5
0.5
Cefalonium
0.5
0.5
Cefaperazone
1.0
1.0
Cefapirim
0.1
0.5
Cefquinome
5.0
5.0
Chlorotetracycline
1.0
5.0
Ciprofloxacin
0.5
0.5
Cloxacillin
0.1
0.5
Danofloxacin
5.0
1.0
Dapsone
05
0.5
01
0.1
Difloxacin
0.5
0.5
Dimetridazol
5.0
5.0
Doramectin*
10.0
500.0
Doxycyclin
0.5
0.5
Enrofloxacin
1.0
0.5
Eprinomectin
5.0
10.0
Erythromycine
1.0
0.5
Flumequine
1.0
0.5
Ipronidazol-OH
0.5
0.1
Such full range fragmentation exper
of the sample including all possible f
dynamic range and selectivity. The v
comparable to narrow isolation MS2
sample.
2 Quick and Sensitive Analysis of Multiclass Veterinary Drug Residues in Animal Products Using a Novel Benchtop Orbitrap Mass Spectrometry System
Results
gs in various matrices.
bination with full scan/vDIA data
rity could be achieved for a broad
Acquisition (vDIA) is utilized to
dard
d d Data
D t Dependent
D
d tA
Acquisition
i iti
windows than are typically
s, ranging from a minimum of 50
smaller isolation window is used
window is increased with m/z to
ut 5 scan ranges are set to cover
or antibiotics (muscle and kidney
nitroimidazoles and avermectins
sisting of a 6 minute gradient from
minute full chromatographic
e™ C18 2.1x100 mm column was
g 0.1% formic acid, at a flow rate
Goal
44 multi-class veterinary drug residues (Abamectin, Amoxicillin, Ampicillin,
Benzylpenicillin (G), Cefalexine, Cefalonium, Cefapririm, Cefoperazon, Cefquinom,
Chlortetracyclin, Ciprofloxacin, Cloxacillin, Danofloxacin, Dapson, Difloxacine,
Dimetridazole, Doramectine, Doxycycline, Enrofloxacine, Eprinomectine,
Erythromycine Flumequin,
Erythromycine,
Flumequin Ipronidazol-OH,
Ipronidazol OH Ivermectin,
Ivermectin Marbofloxacine,
Marbofloxacine
Metronidazole, Metronidazole-OH, Moxidectin, Nafcilline, Oxacilline,
Phenoxymethylpenicillin (V), Ronidazol, Sarafloxacine, Sulfadiazine, Sulfadimethoxine,
Sulfadimidine (Sulfamethazine), Sulfadoxine, Sulfamethoxazole,
Sulfamethoxypyridazine,
ypy
, Sulfathiazole,, Tetracycline,
y
, Thiamphenicol,
p
, Trimethoprime,
p
,
Tylosin) from muscle, kidney, milk and plasma were analyzed in one standardized
chromatographic and mass spectrometric method. For quantification, standard curves
with eight calibration points were prepared covering the range 100 pg/mL (ppt) to 500
ng/mL (ppb). Spiked samples (muscle and kidney for antibiotics, milk for avermectins
and plasma for Nitroimidazoles) were analyzed. Quasi molecular ions were monitored
for quantitation, while up to five additional fragment ions were monitored for
qualification, achieving linear calibration curves over the ranges described above.
Measurements were carried out in two scan modes. First, the classical approach of full
scan – data dependent MS2 (FS-ddMS2) was chosen. This scan mode combines the
sensitivity of full scan HRAM detection with the selectivity of a MS2 survey scan with
narrow isolation window, triggered from a predefined list of compounds upon
occurrence in the full scan. This approach is not limited to the number of components
t be
to
b analyzed,
l
d b
butt needs
d predefinition
d fi iti off components
t for
f confirmation.
fi
ti
The
Th second
d
scan mode chosen is the variable DIA (vDIA) scan mode. As Figure 2 shows, this is a
combination of a full scan with several wide range MS2 scans. The isolation widths of
the MS2 windows vary between 100 Da and 500 Da. This way this scan mode bridges
the gap between FS-ddMS
FS ddMS2 experiments on the one hand and full range fragmentation
scan as all ion fragmentation, for example.
TABLE 1. Analyzed compounds with LODs comparing vDIA acquisition with the
classical data dependent MS2 approach
Compound
p™
™ mass spectrometry
t
t system
t
r full scan measurements and
ile R=17500 was used for vDIA
used for data analysis.
t.
full scan
m/z 100 - 1000
vDIA
m/z 100 - 200
vDIA
m/z 200 - 300
vDIA
m/z 300 - 400
vDIA
m/z 400 - 500
vDIA
m/z 500 – 1000
Results
FS-vDIA FS-ddMS2
[ppb]
[ppb]
Abamectin*
5.0
50.0
Amoxicillin
10
1.0
10
1.0
Ampicillin
0.5
0.5
Cefalexin
0.5
0.5
Cefalonium
0.5
0.5
Cefaperazone
1.0
1.0
Cefapirim
0.1
0.5
Cefquinome
5.0
5.0
Chlorotetracycline
1.0
5.0
Ciprofloxacin
0.5
0.5
Cloxacillin
0.1
0.5
Danofloxacin
5.0
1.0
Dapsone
05
0.5
01
0.1
Difloxacin
0.5
0.5
Dimetridazol
5.0
5.0
Doramectin*
10.0
500.0
Doxycyclin
0.5
0.5
Enrofloxacin
1.0
0.5
Eprinomectin
5.0
10.0
Erythromycine
1.0
0.5
Flumequine
1.0
0.5
Ipronidazol-OH
0.5
0.1
Compound
Marbofloxacine
Metronidazole
Metronidazole-OH
Moxidectin
Nafcillin
Oxacillin
Penicillin G
Penicillin V
Ronidazol
Sarafloxacine
Sulfadiazine
Sulfadimethoxin
Sulfadimidin / Sulfamethazine
Sulfadoxin
Sulfamerazin
Sulfamethoxazole
Sulfamethoxypyridazine
Sulfathiazole
Tetracycline
Thiamphenicol
Trimethoprim
Tylosine
FS-vDIA FS-ddMS2
[ppb]
[ppb]
5.0
0.5
05
0.5
01
0.1
0.5
0.5
0.5
0.5
0.5
0.1
0.1
0.5
0.5
0.1
0.5
0.5
0.5
0.1
0.5
0.5
0.1
0.1
0.5
0.1
01
0.1
01
0.1
0.5
0.1
0.1
0.1
0.5
0.1
0.1
0.1
0.5
0.1
0.5
0.1
0.5
0.5
0.1
0.1
1.0
1.0
Detection and quantitation of all 44 compon
method. Table 1 shows the achieved LODs
th llowestt concentration
the
t ti level
l
l on which
hi h a co
one fragment was recognized as LOD. Whi
for both modes was the same, the sensitivit
component still can be confirmed by means
FIGURE 2.
2 Linearity of selected compoun
Ampicillin
Cefalonium
In most cases, the sensitivity of the two met
exceptions. In some cases the fact that a dd
fragmentation energy for each component s
ddMS2. In other cases the sensitivity on vDI
because on lower levels no MS2 scans were
confirmation are missing. In the cases of Ab
occurred. Due to the limited stability of the q
the sensitivity in the full scan detection was
missing full scan signal no MS2 was triggere
levels. In contrast, in vDIA mode several fra
much lower levels, so here one of the fragm
ion, leaving additional fragment ions for con
detection limits could be achieved than in d
FIGURE 3. Isotope pattern match for the
Such full range fragmentation experiments have the advantage of giving a full picture
of the sample including all possible fragments, but they suffer from limitations in
dynamic range and selectivity. The vDIA mode provides sensitivity and selectivity
comparable to narrow isolation MS2 scans while maintaining a full record of the
sample.
Thermo Scientific Poster Note • PN-64223-IMSC 0814S 3
Results
ectin, Amoxicillin, Ampicillin,
Cefapririm, Cefoperazon, Cefquinom,
nofloxacin, Dapson, Difloxacine,
ofloxacine, Eprinomectine,
ermectin Marbofloxacine,
ermectin,
Marbofloxacine
, Nafcilline, Oxacilline,
oxacine, Sulfadiazine, Sulfadimethoxine,
Sulfamethoxazole,
ycline,
y
, Thiamphenicol,
p
, Trimethoprime,
p
,
were analyzed in one standardized
hod. For quantification, standard curves
vering the range 100 pg/mL (ppt) to 500
ney for antibiotics, milk for avermectins
d. Quasi molecular ions were monitored
ment ions were monitored for
s over the ranges described above.
odes. First, the classical approach of full
chosen. This scan mode combines the
e selectivity of a MS2 survey scan with
efined list of compounds upon
ot limited to the number of components
ponents
t for
f confirmation.
fi
ti
The
Th second
d
scan mode. As Figure 2 shows, this is a
nge MS2 scans. The isolation widths of
00 Da. This way this scan mode bridges
e one hand and full range fragmentation
comparing vDIA acquisition with the
und
oxacine
dazole
dazole-OH
ctin
n
n
nG
nV
zol
xacine
azine
methoxin
midin / Sulfamethazine
xin
erazin
ethoxazole
ethoxypyridazine
azole
cline
henicol
oprim
e
FS-vDIA FS-ddMS2
[ppb]
[ppb]
5.0
0.5
05
0.5
01
0.1
0.5
0.5
0.5
0.5
0.5
0.1
0.1
0.5
0.5
0.1
0.5
0.5
0.5
0.1
0.5
0.5
0.1
0.1
0.5
0.1
01
0.1
01
0.1
0.5
0.1
0.1
0.1
0.5
0.1
0.1
0.1
0.5
0.1
0.5
0.1
0.5
0.5
0.1
0.1
1.0
1.0
ave the advantage of giving a full picture
s, but they suffer from limitations in
e provides sensitivity and selectivity
hile maintaining a full record of the
Detection and quantitation of all 44 components was possible with one generic
method. Table 1 shows the achieved LODs in a dilution series for quantitation. Here,
th llowestt concentration
the
t ti level
l
l on which
hi h a componentt could
ld b
be confirmed
fi
d with
ith att lleastt
one fragment was recognized as LOD. While the full scan detection of the components
for both modes was the same, the sensitivity of the methods was given by the fact if a
component still can be confirmed by means of a sufficient fragment signal.
FIGURE 2.
2 Linearity of selected compounds.
compounds
Ampicillin
Dimetridazol
In addition, all components were de
shows an example of linear dynamic
spiked in muscle and kidney sample
in milk at a level of 0.5 pp
ppb and Nitro
In the TraceFinder processing softw
with identification through the retent
available. The first is the use of isoto
the result for Ciprofloxacin as an ex
extract at a concentration level of 5
confirmation.
Next stage, using the fragment infor
Figure 5 shows a comparison of frag
mode. Both modes show close to id
even showing similar ion ratios, so i
modes.
Cefalonium
Metronidazol
F
. IGURE 4. Fragment match for th
shows the result for vDIA mode, t
mode.
In most cases, the sensitivity of the two methods was on the same level, with a few
exceptions. In some cases the fact that a ddMS2 method can define a specific
fragmentation energy for each component served for a slightly higher sensitivity of the
ddMS2. In other cases the sensitivity on vDIA seems to be higher than on ddMS2,
because on lower levels no MS2 scans were triggered and so the fragments for
confirmation are missing. In the cases of Abamectin and Doramectin a special problem
occurred. Due to the limited stability of the quasi molecular ions of these compounds,
the sensitivity in the full scan detection was highly limited. While in FS-ddMS2 with
missing full scan signal no MS2 was triggered, nothing could be detected at lower
levels. In contrast, in vDIA mode several fragment masses could be detected down to
much lower levels, so here one of the fragment signals could be taken as quantifier
ion, leaving additional fragment ions for confirmation. With this, significant lower
detection limits could be achieved than in ddMS2 mode.
FIGURE 3. Isotope pattern match for the example of Ciprofloxacin.
Third stage is the match of the obta
T
TraceFinder
Fi d software,
ft
severall built
b ilt i
components. Figure 5 shows an exa
4 Quick and Sensitive Analysis of Multiclass Veterinary Drug Residues in Animal Products Using a Novel Benchtop Orbitrap Mass Spectrometry System
s possible with one generic
tion series for quantitation. Here,
ntt could
ld b
be confirmed
fi
d with
ith att lleastt
l scan detection of the components
methods was given by the fact if a
ficient fragment signal.
Dimetridazol
In addition, all components were detected in spiked samples at low levels. Figure 3
shows an example of linear dynamic range for selected compounds. Antibiotics were
spiked in muscle and kidney samples at levels of 5 ppb, while Abamectins were spiked
in milk at a level of 0.5 pp
ppb and Nitroimidazoles in p
plasma at a level of 0.1 pp
ppb.
FIGURE 5. Library match for the examp
shows the result for vDIA mode, the lo
mode .
In the TraceFinder processing software, besides component detection in the full scan
with identification through the retention time, three stages of confirmation are
available. The first is the use of isotopic information from the full scan. Figure 4 shows
the result for Ciprofloxacin as an example. Even in the complex matrix of muscle
extract at a concentration level of 5 ppb the match is clear and give an unambiguous
confirmation.
Next stage, using the fragment information, is the confirmation with fragment masses.
Figure 5 shows a comparison of fragment confirmation in vDIA mode and ddMS2
mode. Both modes show close to identical results with clear matches of the fragments,
even showing similar ion ratios, so ion ratios can be used for confirmation in both
modes.
Metronidazol
F
. IGURE 4. Fragment match for the example of Ciprofloxacin. The upper panel
shows the result for vDIA mode, the lower panel shows the result for ddMS2
mode.
as on the same level, with a few
ethod can define a specific
or a slightly higher sensitivity of the
s to be higher than on ddMS2,
ed and so the fragments for
and Doramectin a special problem
olecular ions of these compounds,
mited. While in FS-ddMS2 with
ng could be detected at lower
masses could be detected down to
als could be taken as quantifier
n. With this, significant lower
ode.
Conclusion
The Q Exactive MS with a combination o
UHPLC
C system was used to create a gen
class veterinary drug residues. The single
confirmation in RT, isotopic ratio and frag
requirements.
le of Ciprofloxacin.
processing
g was accomplished
p
wi
All data p
identification and three stages of confirma
A new scan type called variable DIA (vDIA
unknown screening.
Acknowledgements
We thank Dr
Dr. Thorsten Bernsmann from C
providing the samples measured in this s
Third stage is the match of the obtained spectra against a spectral library. For
T
TraceFinder
Fi d software,
ft
severall built
b ilt in
i lib
libraries
i are available,
il bl consisting
i ti off 6000
components. Figure 5 shows an example of library match.
All trademarks are the property of Thermo Fisher Scie
encourage use of these products in any manners that
Thermo Scientific Poster Note • PN-64223-IMSC 0814S 5
ked samples at low levels. Figure 3
selected compounds. Antibiotics were
of 5 ppb, while Abamectins were spiked
in p
plasma at a level of 0.1 pp
ppb.
FIGURE 5. Library match for the example of Ciprofloxacin. The upper panel
shows the result for vDIA mode, the lower panel shows the result for ddMS2
mode .
s component detection in the full scan
ee stages of confirmation are
tion from the full scan. Figure 4 shows
n in the complex matrix of muscle
tch is clear and give an unambiguous
he confirmation with fragment masses.
rmation in vDIA mode and ddMS2
lts with clear matches of the fragments,
n be used for confirmation in both
of Ciprofloxacin. The upper panel
anel shows the result for ddMS2
Conclusion
The Q Exactive MS with a combination of a Thermo Scientific™ UltiMate™ 3000
UHPLC
C system was used to create a generic method to detect and quantitate 44 multiclass veterinary drug residues. The single methods proved to have both sensitivity and
confirmation in RT, isotopic ratio and fragment ions to exceed the EU regulatory
requirements.
processing
g was accomplished
p
with easy
y to use TraceFinder software both for
All data p
identification and three stages of confirmation, including spectral library matching.
A new scan type called variable DIA (vDIA) for presented to be a useful approach for
unknown screening.
Acknowledgements
We thank Dr
Dr. Thorsten Bernsmann from CVUA-MEL
CVUA MEL in Muenster
Muenster, Germany
Germany, for
providing the samples measured in this study.
a against a spectral library. For
re available,
il bl consisting
i ti off 6000
ary match.
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to
encourage use of these products in any manners that might infringe the intellectual property rights of others.
PO64223-EN 0814S
6 Quick and Sensitive Analysis of Multiclass Veterinary Drug Residues in Animal Products Using a Novel Benchtop Orbitrap Mass Spectrometry System
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