Hi h R
High
Resolution
l ti LC-MS
LC MS ffor Qualitative
Q lit ti and
dQ
Quantitative
tit ti Mycotoxin
M
t i
Analysis
Feed
Extracts
A ly i in
i Complex
C pl Food
F d and
dF
d Extracts.
E t t
C C
C.
Crone , M.
M Zeller,
Z ll M.
M B
Bromirski
i ki , M.
M K
Kellmann
ll
Thermo Fisher Scientific
Scientific, Bremen
Bremen, Germany
Overview
Results
A general screening set up and a quan/qual approach for
the
were
th analysis
l i off mycotoxins
t i iin complex
l matrices
ti
evaluated using
g a benchtop
p LC-MS/MS Orbitrap
pTM
instrument.
Extracted
E
d iion chromatograms
h
(EICs)
(EIC ) off the
h quasimolecular
i l
l
ions with a narrow mass tolerance were used to obtain
selectivity from the Full MS data
data. Using the fast polarity
switching,
switching analytes ionizing in positive mode as well as such
i i i in
i negative
ti mode
d could
ld be
b d
t t d while
hil maintaining
i t i i
ionizing
detected
mass accuracy
y ((see Figure
g
3).
)
Conventionally used triple quad multi-methods
multi methods need time
consuming procedures to optimize on each compound
expected
t d tto b
be presentt iin the
th sample.
l Also,
Al
d
due to
t this
thi fact,
f t
the compounds
p
have to be pre-selected
p
with no chance for
retrospective data analysis afterwards.
In this work,, the p
potential of a high
g resolution accurate mass
(HRAM) approach for mycotoxin analysis is presented.
presented
Polarity
Exact Mass
[[M+H]+
]
LOD Full MS
[ppb]]
[pp
LOD SIM
[ppb]]
[pp
Factor
Aflatoxin B1
C17H12O6
+
313.0707
0.13
0.07
2
Afloatoxin G1
C17H12O7
+
329.0656
0.07
0.016
4
Citreoviridin
C23H30O6
+
403.2115
5.33
2.67
2
Cit i i
Citrinin
C13H14O5
+
251 0914
251.0914
8
04
0.4
20
Deoxynivalenol (DON)
C15H20O6
+
297 1333
297.1333
26 7
26.7
1 33
1.33
20
DON -15-acetyl
C17H22O7
+
425.217
53.33
10.67
5
HT-2 Toxin
C22H32O8
+
325.0707
2.67
0.27
10
Elemental
Composition
Polarity
Exact Mass
[M+H]+ / [M-H]-
Alternariol
C14H10O5
-
257 046
257.046
Alternario l-methylether
C15H12O5
-
271.061
Zearalenol alpha-
C18H24O5
-
319.155
Z
Zearalenol
l
l betab t
C18H24O5
-
319 155
319.155
Sterigmatocystine
g
y
C18H12O6
+
325.071
Ochratoxin A
C20H18ClNO6
+
404.09
Sterigmatocystine
C18H12O6
+
467.2276
0.53
0.05
10
Citreoviridin
C23H30O6
+
403.212
T2 Toxin
C24H34O9
+
403.212
1.33
0.27
5
Compound Name
6.17
257.04553
257
04553
-
0
7.48
271.06119
0
-
7.00
319.15503
6.45
319 15512
319.15512
0
7 25
7.25
325.07043
0
TABLE 2.
2 Q
Quantitation
tit ti off mycotoxins
t i in
i wheat
h t extract:
t
t
p
p
Comparison
between Full MS and SIM method setup.
Both experiments performed at R = 70 k.
RT: 4.82 - 6.29
+
6.70
6
0
404.08932
+
0
4500000
+
6.0
6.2
6.4
6.6
6.8
7.0
Time (min)
0
100
3500000
0
5.8
134 ppt 50
5000000
4000000
7.20
403.21100
403 21100
7.2
7.4
7.6
7.8
8.0
3000000
2000000
Aflatoxin B1
1500000
FIGURE 3.
3 Screening approach including fast polarity
switching and ddMS2 scans for confirmation.
confirmation Exemplarily
Exemplarily,
f
d iionizing
i i iin positive
iti mode
d as well
ll as th
four
compounds
three
p
g in negative
g
compounds
ionizing
mode are shown.
500000
0
200
400
ppt
600
50
13 ppt
0
800
0
5.0
RT: 5.26 - 6.77
Citrinin
Y = 124354+4.18085e+006*X R^2 = 0.9979 W: 1/X
100
5.80
NL: 9
NL
9.23E5
23E5
m/z= 251.09014-251.09266 F:
FTMS + p ESI SIM ms
[249.09-253.09] MS
110722_wheat_e_tsim_70k_01
8 ppb
400000000
5.82
NL: 2.11E5
m/z= 251.09014-251.09266 F:
FTMS + p ESI SIM ms
[249.09-253.09]
[249.09
253.09] MS
110722_wheat_f_tsim_70k_01
5.79
NL: 1.20E5
m/z= 251
251.09014-251.09266
09014-251 09266 F:
FTMS + p ESI SIM ms
[249.09-253.09] MS
110722 wheat g tsim 70k 01
110722_wheat_g_tsim_70k_01
50
350000000
0
100
Area
250000000
1 6 ppb
1.6
200000000
Citrinin
150000000
100000000
50
0
100
50000000
0
0
20
40
60
ppb
80
0.8
0 8 ppb
100
LOQ
50
0
5.5
6.0
Time (min)
6.5
FIGURE 6. Calibration curves for Aflatoxin B1 and
Citrinin in wheat extract
extract. SIM scan data.
data Concentration
levels
the llower end
as extracted
l
l att th
d are shown
h
t
t d ion
i
chromatograms
g
(5
( pp
ppm mass tolerance).
)
O
O
O
O
O
Elemental
Composition
p
Compound Name
Chromatography was performed using an Accela Open AS
and an Accela 1250.
1250 The mobile phase A consisted of 2 mM
ammonium formate and 20 uL/L formic acid.
acid The mobile
phase
h
B was composed
d off 2 mM
M ammonium
i
f
formate
and
d
20 uL/L formic acid in methanol. The stationaryy p
phase used
was Thermo Scientific Hypersil Gold 1.9um,
1 9um column
dimensions were 50 x 2
2.1
1 mm
mm. Separation was performed at
a flow
rate
fl
t off 300 uL/min
L/ i and
d a lilinear gradient
di t as follows:
f ll
2-45% B in 2 min,, 45-98% B in 6 min,, 98%B hold 11 min.
uL
Injection volume was 5 uL.
Exact Mass
[[M+H]+
]
Polarity
C17H12O6
+
C17H14O6
Stock
[[ng/mL]
g
]
313 0707
313.0707
+
LOD
(R=140k)
[ g/kg]
[µg/kg]
25
315.0863
LOD
(R=70k)
[ g/kg]
[µg/kg]
0 13
0.13
25
LOD
(R=35k)
[ g/kg]
[µg/kg]
0 13
0.13
0.13
0.13
0.67
C17H12O7
+
329.0656
25
0.07
0.07
0.07
Aflatoxine G2
C17H14O7
+
331.0812
25
0.67
0.67
0.67
Agroclavin
C16H18N2
+
239.1543
25
0.67
0.67
1.33
Beauvericin
C45H57N3O9
+
784 4168
784.4168
100
2 67
2.67
5 33
5.33
nd
n.d.
Citreoviridin
C23H30O6
+
403.2115
2000
5.33
5.33
5.33
C13H14O5
+
251.0914
3000
8
8
8
Deoxynivalenol (DON)
C15H20O6
+
297.1333
5000
26.67
26.67
26.67
DON -15-acetyl
C17H22O7
+
339.1438
2000
53.33
53.33
53.33
F
Fumagillin
illi
C26H34O7
+
459 2377
459.2377
2000
53 33
53.33
53 33
53.33
53 33
53.33
HT-2 Toxin
C22H32O8
+
425 217
425.217
500
2 67
2.67
2 67
2.67
2 67
2.67
Mycophenolic
y p
acid
C17H20O6
+
321.1333
2000
1.07
1.07
5.33
Neosolaniol
C19H26O8
+
383.17
500
1.33
1.33
2.67
Nivalenol
C15H20O7
+
313.1281
1000
26.67
26.67
53.33
Ochratoxin A
C20H18ClNO6
+
404.0895
250
0.67
1.33
1.33
R
Roquefortine
f ti C
C22H23N5O2
+
390 1925
390.1925
100
0 27
0.27
0 53
0.53
2 67
2.67
Sterigmatocystine
C18H12O6
+
325 0707
325.0707
100
0 53
0.53
0 53
0.53
0 53
0.53
T2 Toxin
C24H34O9
+
467.2276
500
1.33
1.33
1.33
TABLE 1. Results of quantitation
q
of mycotoxins
y
in
wheat extract done with Full MS.
All data was acquired
q
on a Thermo Scientific Q Exactive
benchtop Orbitrap system (Figure 1),
1) equipped with a HESI II
ionization source.
source
S
Spectrum
t
NL: 3.96E4
110722 Sow B 140k 01
110722_Sow_B_140k_01
0.9 ppm
9 35
9.35
100
R=69077
50
R 140k
R=140k
m/z= 784.4126-784.4204 F:
FTMS + p ESI Full ms
[230.00-1000.00] MS
110722_sow_a_70k_01
50
50
0
100
NL: 5.85E4
5 85E4
110722_sow_b_70k_01
784.41942
R=46911
3 3 ppm
3.3
Rela
ative Ab
bundan
nce
Rela
ative A
Abunda
ance
0
100
R=70k
0
100
NL: 1.04E5
110722_sow_b_35k_01
110722 sow b 35k 01
784.42885
15ppm
R=21124
m/z= 784.4126-784.4204 F:
FTMS
S + p ESI
S Full
u ms
s
[230.00-1000.00] MS
110722_sow_b_70k_01
50
0
100
R=35k
50
9 31
9.31
m/z= 784.4126-784.4204 F:
FTMS + p ESI Full ms
[230.00-1000.00] MS
110722_sow_b_35k_01
0
0
FIGURE 1.
1 Schematics of the instrument.
instrument
9.34
9 34
50
784.4
784.5
m/z
9.2
784.6
9.4
Time (min)
(
)
Compound Name
Elemental
Composition
Polarity
Exact Mass
[M+H]+
Stock
[ng/mL]
LOD (R=140k)
[µg/kg]
LOD (R=35k)
[µg/kg]
Beauvericin
C45H57N3O9
+
784.4168
100
2.67
n.d.
FIGURE
G
4. Beauvericin in sowfeed
f
matrix (concentration
(
e e 5
3 µg/
g) measured
easu ed with
t d
e e t resolution
eso ut o
level
5.3
µg/kg)
different
settings.
settings Full MS data.
data Mass tolerance of 5 ppm for EIC
could be maintained at R = 70 k
k.
EIC (5ppm mass tolerance)
S
Spectrum
t
313.07108
313
07108 313.07773
C 17 H 13 O 6
100
50
60
R=140k
Relattive Abundancce
50
100
R=70k
R
70k
60
5 64
5.64
40
5.04
NL: 0
m/z=
313.06913-313.07227
R 35k
R=35k
313.07
5.0
313.08
m/z
Aflatoxin B1
5.78
5.39
NL: 2
2.19E4
19E4
12 3 ppm
12.3
Compound Name
NL: 1.13E4
m/z=
313 06913 313 07227
313.06913-313.07227
0
50
O
90
5 04
5.04
0
100
O
Aflatoxin G1
5.08
329.06519
3
9 065 9
98.98470
80
70
311.05463
60
283.05975
50
40
30
215.06985
20
10
0
60
0
100
4.83
Aflatoxin G2
80
100
120
140
160
180
110722_Neat_A_70k_01 #1272 RT: 4.87 AV: 1 NL: 3.45E5
F: FTMS + p ESI d Full ms2 331.08@hcd45.00 [50.00-355.00]
4.87
240
O
4.9
5.0
5.1
5.2
5.3
5.4
Time (min)
5.5
5.6
5.7
280
300
O
O
O
O
90
O
OH
320
340
O
O
331.08096
O
O
70
O
O
60
O
O
O
O
O
O
50
313.06995
40
O
30
98.98484
221.04439
10
4.8
260
O
O
20
0
220
O
100
80
0
100
200
m/z
O
245.08075
257.08145
285.07556
240
280
303.08618
0
60
80
100
120
140
160
180
200
m/z
220
260
300
320
340
FIGURE 7. Confirmation of Aflatoxin species in wheat
extract by a HRAM ddMS2 scan. Annotation made with
Mass FrontierTM.
High resolution accurate mass full MS2 spectra were used for
confirmation of the analytes by specific fragments. Due to the
data dependent settings MS2 in combination with an inclusion
list MS2 could be performed only once per analyte on top of the
list,
chromatographic
h
t
hi peakk in
i order
d to
t safe
f additional
dditi
l scan time
ti
((Figure
g
7).
)
Conclusion & Remarks
 Screening and quantitation including HRAM ddMS2 for
confirmation
fi
ti were performed
f
d without
ith t changing
h
i the
th instrument
i t
t
platform.
 SIM approaches
h were used
d to
t improve
i
sensitivity
iti it and
d quan
statistics.
/ 200 was necessary in
 For complex matrices, R = 70 k at m/z
order
o
de to a
avoid
o d false
a se negative
egat e results
esu ts and
a d to assure
assu e the
t e
required mass accuracy
accuracy, which could be distorted by
unresolved interferences in case of unsufficient resolution.
resolution
 Full MS data could be used for retrospective data analysis.
analysis
E
Even
th
though
h these
th
compounds
d were nott the
th focus
f
off this
thi
experiments,
p
it was p
possible to get
g the q
quantitative and
qualitative information for 140 additional spiked pesticides
from the same data set as well.
well
R f
References
[1] htt
http://www.fao.org/ag/agn/agns/chemicals_mycotoxins_en.asp
//
f
/ /
/
/ h i l
t i
5 78
5.64 5.78
5.45
5 45
20
313.07452
C 17 H 13 O 6
0
313.06
5 14 5.32
5.14
5 32
20
80
313.07852
0 9 ppm
0.9
0
40
0
100
NL: 5.81E3
313 07037
313.07037
C 17 H 13 O 6
NL: 9.72E3
m/z=
313.06913-313.07227
80
0
100
5.43
100
NL: 1.15E4
1.3 ppm
100
100
O
O
243.06511
EIC (5
(5ppm mass ttolerance)
l
)
784.41751
100
O
110722_ Neat_A _70k_01 #1333 RT: 5.06 AV: 1 NL: 2.83E5
F: FTMS + p ESI d Full ms2 329.23@hcd45.00 [50.00-355.00]
RT 4.76
4 76 - 5.77
5 77
RT:
1 33
1.33
Aflatoxine G1
Citrinin
Mass Spectrometry
FIGURE 2.
2 Method
M h d setup ffor a)) screening
i approach
h
including
g fast p
polarity
y switching
g b)) q
quan set up
p using
g
Full MS and c) quan set up using a SIM method setup.
setup
6.0
O
Liquid Chromatography
c))
5.5
Time (min)
450000000
IIn order
d tto avoid
id ffalse
l positive
iti or false
f l negative
ti results,
lt a
sufficient resolution is needed while working
g with complex
p
matrices.
matrices As shown for Beauvericin in sowfeed extract
(Figure 4) and for Aflatoxin in wheat matrix (Figure 5)
5),
unresolved
l d matrix
t i interferences
i t f
are leading
l di to
t false
f l negative
ti
results for the shown examples
p
when using
g R < 70 k at m/z
200. All compounds screened were detectable down to low
concentration levels (see Table 1 for Full MS for wheat extract
sample).
l )
Aflatoxine B2
b)
NL: 6.44E3
m/z=
313 06913
313.06913313.07227 MS
110722_wheat_f_tsi
m 70k 01
m_70k_01
5.63
1000000
Aflatoxin B1
For screening,
g, two Full MS scan events were p
performed
using opposite polarities (Figure 2).
2) For the quantitative
approach a Full MS method and a timed targeted SIM
approach,
method
th d were used
d either
ith in
i positive
iti or negative
ti ionization
i i ti
mode. For confirmation byy fragments,
g
, each Full MS / SIM
scan event was followed by two data dependent (dd) MS2
events using higher energy collision induced dissociation
(HCD) and
off 4 u. Normalized
d an isolation
i l ti window
i d
N
li d collision
lli i
gy was set to 45. For the SIM method,, an isolation width
energy
of 4 u was used.
used Different resolution settings were evaluated
(R = 17.5
17 5 kk, 35 k and 70 k at m/z 200).
200)
LOQ
0
100
Relative Ab
bundance
Standards of mycotoxins and pesticides were spiked in wheat
extract
t t (intermediate
(i t
di t complex)
l ) and
d sowfeed
f d extract
t t (highly
(hi hl
complex).
p ) As described in [2],
[ ] the dryy sample
p was soaked
with water for 15 min before 3 parts acetonitril containing 1%
formic acid were added.
added The sample was shaken for 1h
b f
before
th supernatant
the
t t was taken
t k after
ft centrifugation.
t if
ti
I
In
order to keep
p the matrix concentration constant, the
standards were spiked into the matrix at a ratio of 1:10.
NL: 1.60E4
m/z=
m/z
313.06939313.07252 MS
110722 wheat e ts
110722_wheat_e_ts
im_70k_01
5.45
50
67ppt
67 t
2500000
Relativve Abundancce
Sample Preparation
NL: 2.65E4
m/z=
313.06913313.07227 MS
110722_wheat_d_ts
im_70k_01
5.52
100
Aflatoxin_B1
Y = 6351.99+5868.01*X R^2 = 0.9968 W: 1/X
300000000
Methods
a))
Elemental
Composition
p
Compound Name
Are
ea
Mycotoxins
M
t i in
i agricultural
i lt l products
d t have
h
the
th potential
t ti l to
t
seriouslyy affect the health of human and cattle. It is reported
p
by the Food and Agriculture Organization (FAO) that 25% of
all foodstuffs are contaminated with these highly toxic
substances.
b t
Therefore,
Th f
strict
t i t official
ffi i l regulations
l ti
are existing
i ti
g
p
y
with regards
to the presence
of mycotoxins
in feed and food.
Maximum residue levels (MRL) for the individual compounds
are deviating through different countries and commodities in
the range of 0
0.5
5 µg/kg to several hundred µg/kg.
µg/kg Due to the
usually quite complex matrices and time saving demands, the
development of highly selective and sensitive multiresidue
results
methods is necessary to ensure reliable results.
R
Relati
ive Ab
bunda
ance
Introduction
Using a SIM approach could even increase the dynamic
range off the
th instrument
i t
t by
b using
i the
th quadrupole
d
l as a
powerful mass filter restricting
p
g the ion population
p p
used for
detection. An increase in sensitivity (2-20x
(2 20x compared to the
Full MS) could be reached as shown for wheat extract
samples (Table 2).
2)
[2] KELLMANN ET AL
AL. “Full
Full Scan MS in Comprehensive
Q lit ti and
dQ
tit ti R
id Analysis
A l i in
i Food
F d and
d Feed
F d
Qualitative
Quantitative
Residue
Matrices: How Much Resolving
g Power is Required?”J
q
Am Soc
Mass Spectrom 2009,
2009 20,
20 1464–1476
1464 1476
A k
Acknowledgements
l dg
t The authors would like to thank
5.5
Time (min)
Elemental
Composition
Polarity
Exact Mass
[M+H]+
Stock
[ng/mL]
LOD (R=140k)
[µg/kg]
LOD (R=35k)
[µg/kg]
C17H12O6
+
313.0707
25
0.13
1.33
FIGURE 5. Aflatoxin in wheat matrix (concentration level
130 ng/kg) measured with different resolution settings.
Full MS data.
data Mass tolerance of 5 ppm for EIC could be
maintained at R = 70 k.
k
Hans Mol and Paul Zomer (Rikilt,
(Rikilt Wageningen) for their
contribution by supplying the standards for the experiments.
experiments
Mass FrontierTM is a trademark of HighChem, Ltd. All other 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
subsidiaries.
intellectual property rights of others. For Research Use only, not for Use in Diagnostic Procedures. Presented at DGMS
03/2012.