Higher Confidence in Identification with QTRAP® LC/MS/MS
Systems when Screening and Quantifying Pesticides in Fruit
and Vegetable Samples
André Schreiber
AB SCIEX, Concord, Ontario, Canada
®
Figure 1. Ion path of a QTRAP LC/MS/MS System
Overview
®
QTRAP LC/MS/MS Systems provide in comparison to triple
quadrupole systems:
• MRM selectivity and sensitivity for quantitation with the lowest
limits of detection, excellent reproducibility and linear range
• Fast and high sensitivity Linear Ion Trap scan functions for
qualitative analysis and identification
• Rich MS/MS spectra because of fragment ion generation in
the LINAC® Collision Cell and the use of Collision Energy
Spread
• Combination of triple quadrupole and QTRAP ® System
functions without delay time in IDA experiments allowing
quantitation and identification with MS/MS spectra in a single
LC run
• Higher confidence of identification based on mass spectral
library search minimizing the risk of false positive and
negative results
This paper describes the use of QTRAP® Systems coupled to
liquid chromatography for the screening and identification of
pesticides in fruit and vegetable samples. The selectivity,
®
sensitivity, and accuracy of detection using QTRAP System and
conventional triple quadrupole mass spectrometers are
compared. The higher confidence of identification provided by
®
QTRAP linear ion trap MS/MS scans followed by library
searches which minimize the potential risk of false positive and
negative results is highlighted.
Introduction
Liquid Chromatography (LC) coupled to tandem Mass
Spectrometry (MS/MS) is a powerful analytical tool used in many
food, environmental and forensic testing laboratories to detect
targeted analytes, such as pesticides, drugs of abuse, steroids,
pharmaceuticals, personal care products, and many other
contaminants at trace levels.
A variety of ion sources including Electrospray Ionization (ESI),
Atmospheric Pressure Chemical Ionization (APCI), and
Atmospheric Pressure Photo Ionization (APPI) facilitates the
ionization of polar to non-polar compounds. Generated ions are
transferred following ionization through a vacuum interface into
the mass analyzer.
p1
1.14e5
1.10e5
174.2
5283
MRM chromatogram
5000
1.00e5
174.0
9.5e5
Product Ion Spectrum
(triple quadrupole MS/MS)
9.0e5
8.5e5
4500
Enhanced Product Ion Spectrum
(QTRAP® MS/MS)
8.0e5
9.00e4
7.5e5
4000
7.0e5
8.00e4
6.5e5
3500
96.2
7.00e4
6.0e5
104.2
104.0
5.5e5
3000
6.00e4
96.0
5.0e5
4.5e5
2500
5.00e4
4.0e5
79.0
4.00e4
3.5e5
2000
3.0e5
216.0
3.00e4
2.5e5
1500
79.0
132.0
5.0
146.4
1000
1.5e5
132.2
1.0e5
1.00e4
5.0e4
500
0.00
216.0
146.0
2.0e5
2.00e4
1
2
3
4
5
6
7
8
Time, min
9
10
11
12
13
14
15
70
80
90
100
110
120
130
140
150
160
170
m/z, amu
180
190
200
210
220
230
240
250
70
99.0
80
90
100
110.0
110
127.0
120
130
138.0
140
150
160
170
m/z, amu
180
190
200
210
220
230
240
250
®
Figure 2. Screening and identification of pesticides using triple quadrupole and QTRAP LC/MS/MS Systems. Left: detection 150 Multiple Reaction
Monitoring transitions to screen for pesticides. Middle: Product Ion spectrum of a triple quadrupole MS/MS. Right: Enhanced Product Ion spectrum of a
®
®
QTRAP MS/MS to identify the presence of a detected pesticide. (Note that the sensitivity of the QTRAP MS /MS spectrum is more than 100 times
higher than that of the triple quadrupole spectrum.)
Triple quadrupole (QqQ) mass analyzers operating in Multiple
Reaction Monitoring (MRM) are mostly used for the quantitation
of targeted analytes. In MRM mode, the first quadrupole filters a
specific precursor ion; the collision cell (second quadrupole)
generates fragments (product ions) which are filtered in the third
quadrupole (Figure 2).
In 2002, AB SCIEX introduced a new generation of LC/MS/MS
systems: hybrid triple quadrupole linear ion trap mass
spectrometers with axial ion ejection (QTRAP® System).
®
QTRAP Systems enable the unique capabilities provided by this
technology by coupling targeted triple quadrupole experiments,
such as MRM, precursor ion, and neutral loss scans, with high
sensitivity ion trap scan functions, such as enhanced MS,
enhanced product ion, enhanced resolution, enhanced multiple
2-4
charge and MS/MS/MS scan.
Having a variety of new scan types and the possibility to
combine them in single LC runs enable completely new
workflows for many LC/MS/MS applications, including food,
environmental, and forensic testing, metabolite identification and
proteomics.5-15
QTRAP® System Technology
The mass analyzer region of a QTRAP® System is based on the
conventional ion path of a triple quadrupole mass spectrometer.
In comparison to QqQ systems, the third quadrupole (Q3) of a
QTRAP ® System can be operated as a quadrupole or as a
Linear Ion Trap (LIT). The dual functionality of Q3 provides the
QTRAP ® System complete functionality of QqQ mass
spectrometer, including true Multiple Reaction Monitoring for
selective and sensitive quantitation and additional LIT scan
functions. These additional LIT scan functions greatly enhance
the performance for screening, confirmation and identification
applications.
(1) Trapping
Exit lens
(2) Scanning
Entrance lens
Figure 3. Trapping and scanning of ions in a Linear Ion Trap (LIT)
The operation of Q3 as LIT can be divided into two basic steps:
(1) trapping and (2) scanning as shown in Figure 3. First
precursor ions and/or product ions, depending on the selected
scan mode, exit the collision cell and enter Q3 operating in LIT
mode. The ions are trapped in Q3 by the RF voltage in radial
direction and by a repulsive DC voltage applied to the ‘exit lens’
of Q3. After a fill time of typically a few milliseconds to
accumulate ions, a repulsive ‘entrance lens’ is used to stop ions
entering Q3. Trapped ions are then cooled for a short time and
subsequently ejected axially from the LIT using an auxiliary
voltage that is ramped in concert with the drive RF voltage.
p2
The most important LIT scan modes for screening, confirmation
and identification applications are Enhanced MS (EMS),
Enhanced Resolution (ER) and Enhanced Product Ion (EPI)
scans.
QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe)
16
procedure.
In EMS, the ion trap is filled with molecular ions generated in the
ion source to identify unknown compounds. The ER scan
measures the mass of a targeted ion more accurately and can
be used to determine charge states and isotopic patterns.
Finally, the EPI mode can be used to identify the presence of the
detected analyte by interpretation of fragment ions or mass
spectral library searching.
A Shimadzu Prominence LC system consisting of system
controller CBM-20A, 2 pumps LC-20AD, degasser DGU-20A3,
autosampler SIL-AC, and column oven CTO-20AC was used. A
gradient of eluent A (water with 5 mM ammonium formate) and
eluent B (methanol with 5 mM ammonium formate) from 80/20 to
10/90 (A/B) over 8 min was used on a Phenomenex Synergi 4u
Fusion-RP 80A 50x2 mm column. The column temperature was
set to 25°C. Sample volume of 20 μL was injected.
The Enhanced Product Ion scan of QTRAP® Systems has
several advantages over product ion scanning of QqQ mass
spectrometers, including:
MS/MS Detection
• Much higher sensitivity of Enhanced Product Ion spectra
because of ion accumulation in the LIT as shown in Figure 2
• More product ion information in a single MS/MS spectrum
because of Collision Energy Spread
• Lower cycle time due to faster scanning of linear ion trap
®
The Enhanced Product Ion scan of QTRAP Systems has also
several advantages over product ion scan of traditional ion trap
mass spectrometers, including:
• Fragment rich MS/MS spectra characteristic for Collision
Induced Dissociation (CID) in comparison to resonance
excitation spectra
• No inherent low mass cut-off due to fragment generation in
the collision cell rather than in the ion trap
• Lower cycle time than traditional ion traps because of
simultaneous precursor ion selection and fragmentation while
QTRAP® System is filled
HPLC
In QqQ mode 75 pesticides were detected in positive polarity
using 2 MRM transitions per analyte.
The detected quantifier and qualifier MRM with the respective
MRM ratio are listed in Table 1. All compound dependent
parameters were completely optimized for the detection of each
pesticide with the highest sensitivity. A dwell time of 5 ms was
used for all MRM transitions.
This typical triple quadrupole experiment was compared to a
unique QTRAP® Information Dependent Acquisition (IDA) where
only a single MRM transition was monitored with a dwell time of
10ms per compound. This MRM survey automatically triggered
an Enhanced Product Ion scan if the MRM signal was above a
threshold of 500 cps. EPI spectra were acquired from 50 to
550 amu with a scan speed of 4000 amu/s and a Collision
Energy (CE) of 35 V with Collision Energy Spread (CES) of 15 V.
Also dynamic exclusion was activated to minimize the risk of
missing co-eluting compounds.
XIC of +MRM (150 pairs): 278.2/1...
Max. 4.9e4 cps.
Standard 10ppb
2500
8.00e4
6.00e4
7.6
4.00e4
®
A 3200 QTRAP LC/MS/MS System with Turbo V™ Source and
an ESI probe was used in both triple quadrupole and QTRAP®
modes to investigate the use of these two mass spectrometers
for the screening and identification of pesticides.
5
6
10
Max. 4.9e4 cps.
1000
0
5
6
7
8
Time, min
XIC of +MRM (150 pairs): 278.2/...
1.0e4
9
10
Max. 2900.0 cps.
7.6
2500
2.0e4
0.0
1500
2900
Standard 10ppb
MRM of
Metazachlor
(ratio = 0.99)
I n t e n s it y , c p s
3.0e4
9
7.6
4.9e4
4.0e4
2000
500
7
8
Time, min
XIC of +MRM (150 pairs): 278.2/1...
I n te n s i ty , c p s
Fruit and vegetable samples (apple, apricot, banana, cucumber,
grape, grapefruit, kiwi, lemon, orange, pear, pepper, raisin,
strawberry, tea, and tomato) were extracted using a modified
0.00
Max. 2900.0 cps.
Extract of apricot
5.8μg/kg
(10x diluted)
1.00e5
2.00e4
Samples
XIC of +MRM (150 pairs): 278.2/...
2900
I n t e n s i ty , c p s
Experimental
1.20e5
I n t e n s i ty , c p s
• Remarkable immunity of LIT from space charge effects which
result in mass shift and incorrect isotopic pattern in the
2-3
spectrum
Extract of apricot
MRM of
Metazachlor
(ratio = 0.90)
2000
1500
1000
500
5
6
7
8
Time, min
9
10
0
5
6
7
8
Time, min
9
10
Figure 4. Quantitation and MRM ratio identification of 5.8 μg/kg
Metazachlor in an extract of apricot (278/134 and 278/210)
p3
Table 1. MRM transitions for the 75 studied pesticides, MRM ratios, Limits of Detection (S/N=6) and coefficients of variation at 10ng/mL (n=3)
LOD
%CV (n=3)Pesticide
(ng/mL)
Pesticide
MRM 1
MRM 2
Ratio
Acetamiprid
223/126
223/99
0.20
0.2
4.7
Aldicarb
208/89
208/116
0.60
1.0
Atrazine
216/174
216/104
0.51
Azoxystrobin
404/372
404/344
Benalaxyl
326/148
Bendiocarb
LOD
%CV (n=3)
(ng/mL)
MRM 1
MRM 2
Ratio
Irgarol
254/198
254/83
0.14
0.05
2.20
8.5
Isoproturon
207/72
207/46
0.25
0.20
3.90
0.1
5.5
Lenacil
235/153
235/136
0.24
0.20
5.00
0.45
0.1
1.5
Linuron
249/160
249/182
0.98
1.00
8.50
326/91
0.97
0.1
3.3
Lufenuron
511/141
511/158
0.50
5.00
11.90
224/109
224/167
0.95
0.5
7.9
Malathion
331/127
331/99
0.42
0.20
2.70
Bromuconazole
378/159
378/161
0.77
0.5
4.5
Mebendazole
296/164
296/105
0.26
0.20
1.10
Buprofezin
306/201
306/116
0.71
0.2
4.9
Metalaxyl
280/220
280/192
0.79
0.50
4.90
Carbaryl
202/145
202/127
0.40
0.2
5.4
Metamitron
203/175
203/104
0.72
0.20
4.30
Carbendazim
192/160
192/132
0.17
0.1
3.1
Metazachlor
278/134
278/210
0.98
0.10
2.40
Carbofuran
222/165
222/123
0.44
0.2
3.3
Methabenzthiazuron
222/165
222/150
0.35
0.10
3.50
Chlorfenvinphos
359/99
359/155
0.90
0.5
5.5
Methidathion
303/145
303/85
0.93
0.50
4.70
Chlorotoluron
213/72
213/46
0.21
0.2
3.2
Methiocarb
226/169
226/121
0.96
0.50
3.30
Chlorpyrifos-methyl 322/125
324/125
0.71
1.0
8.4
Methomyl
163/88
163/106
0.77
1.00
7.00
Cyanazine
241/214
241/104
0.24
0.5
6.9
Metolachlor
284/252
284/176
0.29
0.10
2.20
Cyproconazole
292/125
292/70
0.95
0.5
7.2
Metolcarb
166/109
166/94
0.30
0.50
3.80
Cyromazine
167/85
167/68
0.45
0.5
4.2
Metribuzin
215/187
215/84
0.70
1.00
8.20
Desethyl-atrazine
188/146
188/104
0.40
0.5
7.7
Molinate
188/126
188/55
0.52
2.00
7.90
Desisopropylatrazine
174/104
174/96
0.77
1.0
6.7
Monolinuron
215/126
215/99
0.68
0.50
6.00
Diazinon
305/169
305/97
0.94
0.1
3.1
Monuron
199/72
199/126
0.16
0.10
1.60
Dichlorvos
221/109
223/109
0.55
1.0
4.6
Nitenpyram
271/126
271/237
0.78
0.50
4.10
Difenoconazole
406/251
408/253
0.56
0.2
2.8
Oxadixyl
279/219
279/132
0.51
0.50
0.70
Difenoxuron
287/72
287/123
0.65
0.5
2.0
Pendimethalin
282/212
282/194
0.18
1.00
8.70
Diflubenzuron
311/141
311/158
0.70
1.0
6.6
Prochloraz
376/308
376/70
0.26
0.20
2.80
Dimethenamide
276/244
276/168
0.24
0.1
3.2
Profenofos
375/305
373/303
0.87
1.00
1.90
Dimethoate
230/125
230/199
0.76
0.2
2.8
Promecarb
208/109
208/151
0.94
1.00
6.40
Dimethomorph
388/301
388/165
0.52
0.5
4.3
Prometon
226/142
226/86
0.29
0.10
3.20
Diuron
233/72
235/72
0.70
0.5
5.1
Prometryn
242/158
242/200
0.43
0.20
0.20
Ethiofencarb
226/107
226/77
0.62
0.5
6.7
Propachlor
212/170
212/94
0.72
0.20
3.70
Fenamiphos
304/217
304/202
0.59
0.1
2.6
Propanil
218/127
218/162
0.96
1.00
3.60
Flufenacet
364/194
364/152
0.71
0.2
3.4
Propiconazole
342/159
342/69
0.25
0.50
1.00
Flufenoxuron
489/141
489/158
0.51
1.0
7.6
Prosulfocarb
252/91
252/128
0.21
0.20
7.30
Fluroxypyr
255/209
255/181
0.74
5.0
10.4
Simazine
202/132
202/124
0.90
0.50
3.10
Hexaflumuron
461/158
461/141
0.59
5.0
9.0
Spiroxamine
298/144
298/100
0.66
0.20
3.40
Imazalil
297/159
299/161
0.37
0.5
8.5
Terbutryn
242/186
242/68
0.18
0.02
2.00
p4
Table 1. continued
LOD
%CV (n=3)Pesticide
(ng/mL)
Pesticide
MRM 1
MRM 2
Ratio
Imazapyr
262/217
262/149
0.54
0.2
3.0
Imazaquin
312/199
312/267
0.99
0.5
4.5
Imidacloprid
256/175
256/209
0.73
1.0
4.6
MRM 2
Ratio
Thiabendazole
202/175
202/131
0.62
0.10
1.60
Thiacloprid
253/126
253/99
0.32
0.20
6.70
Max. 1.2e4 cps.
1.6
Cyromazine
1.00e4
5000.00
MRM Selectivity
0.00
®
4
6
8
Time, min
XIC of +MRM (150 pairs): 312.2/...
12
Imazaquin
5000
2
6
8
Time, min
XIC of +MRM (150 pairs): 203.2/1...
4
10
12
Metamitron
1.0e4
5000.0
0.0
2
4
6
8
Time, min
XIC of +MRM (150 pairs): 223.2/1...
10
12
Acetamiprid
1.0e4
MRM Sensitivity
6
8
Time, min
XIC of +MRM (150 pairs): 192.2/1...
5.5
10
12
Max. 9.2e4 cps.
Carbendazim
5.0e4
0.0
6
8
Time, min
XIC of +MRM (150 pairs): 216.1/1...
1.6e4
2
4
10
12
1.0e4
0.0
6
8
Time, min
XIC of +MRM (150 pairs): 388.2/3...
1.4e4
1.0e4
2
4
Dimethomorph
(E+Z isomer)
10
12
8.5
8.3
2
4
6
8
Time, min
XIC of +MRM (150 pairs): 254.2/1...
10
12
5.00e4
0.00
4
6
8
Time, min
10
12
2.0e6 68.0
85.1
1.0e6
125.2
60.1
50
100
150
200
250
m/z, amu
+EPI (312.20) Charge (+0) CE (3...
350
400
Max. 1.8e6 cps.
181.2 199.2
1.0e6
86.2
5.0e5
153.2
224.3
252.4
171.2
50
100
150
200
250
m/z, amu
+EPI (203.20) Charge (+0) CE (3...
300
350
400
Max. 2.9e6 cps.
203.3
2.0e6
104.2
1.0e6
175.3
89.0
50
100
150
200
250
m/z, amu
+EPI (223.20) Charge (+0) CE (3...
300
350
400
Max. 9.3e5 cps.
126.1
5.0e5
90.1
99.1
223.3
56.0
181.3
200
250
m/z, amu
+EPI (192.20) Charge (+0) CE (3...
50
100
150
300
350
400
Max. 1.3e6 cps.
160.2
1.3e6
1.0e6
5.0e5
105.1
0.0
50
132.1
100
192.2
150
200
250
m/z, amu
+EPI (216.10) Charge (+0) CE (3...
300
350
400
Max. 2.2e6 cps.
216.2
2.0e6
1.0e6
68.0
174.2
104.0
79.1
50
146.2
100
150
200
250
m/z, amu
+EPI (388.20) Charge (+0) CE (3...
165.2
1.5e6
114.1
400
258.2 273.2
242.2
208.3
300
350
400
Max. 6.2e6 cps.
198.2
6.0e6
254.3
4.0e6
2.0e6 91.1 108.1
50
350
Max. 1.5e6 cps.
388.3
139.1
0.0
50
300
301.2
100
150
200
250
m/z, amu
+EPI (254.20) Charge (+0) CE (3...
14
300
312.2
1.5e6
14
c p s
2
Max. 3.0e6 cps.
167.3
1.0e6
Max. 1.1e5 cps.
9.0
Irgarol
14
Max. 1.4e4 cps.
0.0
1.00e5
14
Max. 1.6e4 cps.
7.5
Atrazine
14
Icn p t es n s i t y ,
Despite the high selectivity of MRM detection, there is always a
risk of false positive or negative findings due to interfering matrix
signals. Accordingly, screening results have to be confirmed.
Typically a second MRM is monitored per analyte and the ratio of
quantifier to qualifier transition is calculated for each unknown
sample and compared to the MRM ratio of standards. Various
guidelines such as the European Commission Decision
2002/657/EC define MRM ratio tolerance levels for identification
(Table 2).17 Figure 4 shows an example of positive identification
based on MRM ratio of 5.8 μg/kg Metazachlor in apricot extract.
4
9.2e4
5000.0
Identification using MRM ratios
2
+EPI (167.20) Charge (+0) CE (3...
s i t y ,
QTRAP ® and triple quadrupole Systems operated in MRM allow
quantitation of trace levels with superior sensitivity. The Limits of
Detection (LOD) of 75 pesticides are summarized in Table 1.
Figure 5 gives example chromatograms of selected pesticides
spiked into a tomato extract at a concentration of 10 ng/mL. All
MRM transitions in the presented screening method were
detected using a short dwell time of 5 ms. The Linear Accelerator
®
(LINAC ) Collision Cell enables the use of such low dwell times
without loss in sensitivity.
0.0
14
Max. 1.7e4 cps.
5.0
1.7e4
14
Max. 1.5e4 cps.
4.4
1.5e4
14
Max. 7337.5 cps.
3.2
7338
0
10
s i t y , I cn pt es n s i t y , I cn pt es n s i t y , I cn pt es n s i t y , I cn pt es n
Detection in Multiple Reaction Monitoring (MRM) using QTRAP
and triple quadrupole Systems offers superior selectivity
because of double mass filtering in the mass analyzer. This
selectivity greatly assists the screening for unknown and
targeted analytes in complex samples such as extracts of fruits
and vegetables. Figure 4 shows an example of MRM detection of
5.8 μg/kg Metazachlor in apricot extract. No matrix signals
interfere with the quantitation of the identified contaminant
because of the high selectivity of MRM detection.
2
I n t e n s i t y , I cn pt es n s i t y , I cn pt es n
XIC of +MRM (150 pairs): 167.2/8...
Results
LOD
%CV (n=3)
(ng/mL)
MRM 1
100
156.2 176.3
150
200
250
m/z, amu
300
350
400
Figure 5. Example chromatograms of pesticides spiked at 10 ng/mL into
a tomato extract (left) with corresponding Enhanced Product Ion spectra
at the same concentration (right)
Identification using QTRAP® MS/MS Spectra and Library
Searching
As an alternative, identification can be performed using full scan
MS/MS experiments and library searching to compare the
unknown with a standard spectrum. As shown in Figure 2,
identification based on MS/MS spectra is more efficient using
QTRAP® Systems because of higher sensitivity and faster
p5
scanning in comparison to QqQ. In addition, QTRAP® Systems
provide more characteristic MS/MS spectra and lower cycle
times than traditional ion traps.
QqQ: 1.5 s) although a longer dwell time was used per transition.
This shorter cycle improved reproducibility since more data
points were acquired across the LC peaks.
Figure 5 shows the Enhanced Product Ion (EPI) spectra of
selected pesticides at a concentration of 10 ng/mL spiked into
tomato extract. These spectra were acquired automatically using
IDA with a survey scan containing 75 MRM transitions. The
spectra shown illustrate that EPI spectra are highly selective and
they contain the complete molecular fingerprint of the analyte for
identification. Also EPI spectra have superior sensitivity and can
be searched against existing mass spectral libraries.
A mix of pesticides was spiked into tomato extract and analyzed
10 times using the developed QqQ and QTRAP ® methods. Data
presented in Table 3 and example spectra in Figure 8 clearly
show the better reproducibility of identification based on mass
spectral library searching of EPI spectra in comparison to MRM
ratio calculation.
XIC of +MRM (202 pairs): 202.0/1...
3.0e4
Max. 4.0e4 cps.
6.3
Std Thiabendazole 10ppb
(ratio = 0.63)
2.0e4
1.0e4
Table 2. MRM ratio tolerances of 2002/657/EC
0.0
2
4
6
8
Time, min
XIC of +MRM (202 pairs): 202.0/1...
Maximum Tolerance
> 0.5
± 20%
0.2 – 0.5
± 25%
0.1 – 0.2
± 30%
± 50%
4.0e4
2.0e4
3.0e6
6.5
12
14
Max. 4.1e6 cps.
175.2
131.2
65.0
60
92.2
104.2
80
143.2
100 120 140 160 180 200 220 240
m/z, amu
XIC of +MRM (150 pairs): 280.2/2...
Max. 1.7e4 cps.
7.7
1.6e4
1.4e4
Std Metalaxyl 10ppb
(ratio = 0.74)
Std Imazalil 10ppb
(ratio = 0.37)
6000
4000
2000
0
2
4
6
8
Time, min
XIC of +MRM (202 pairs): 297.0/...
10
12
14
Max. 1000.0 cps.
9.3
1000
15.2μg/kg Imazalil
in banana (10x diluted)
(ratio = 0.50)
500
0
2
4
6
8
Time, min
+EPI (297.00) Charge (+0) CE (3...
10
6.0e5
12
14
Max. 8.0e5 cps.
297.2
159.1
EPI
FIT = 90.4%
8.0e5
4.0e5
69.0
173.2
2.0e5
109.2 141.2
81.1
50
100
150
201.0 255.2
200
m/z, amu
250
+EPI (280.00) CE (35) CES (15):...
300
350
1.2e4
1.0e4
Library spectrum
Metalaxyl
5.0e7
4.0e7
3.0e7
145.1 192.2
2.0e7
132.1 148.1
162.2
220.3
4000.0
2000.0
0.0
1.0e7
6.2
2
4
6
8
Time, min
XIC of +MRM (150 pairs): 280.2/...
10
12
14
50
Max. 2100.0 cps.
7.9
2000
248.3
100
200
250
m/z, amu
+EPI (280.20) Charge (+0) CE (3...
1000
Reproducibility of quantitation and identification
500
2
4
6
8
Time, min
10
12
14
280.3
300
350
400
Max. 3.3e5 cps.
280.3
2.5e5
2.0e5
220.3
1.5e5
192.4
1.0e5
148.3
5.0e4
0
150
Unknown spectrum
FIT for Metalaxyl = 23.6%
3.0e5
I n t e n s it y , c p s
1500
134.0
105.1
3.3e5
13.3μg/kg Metalaxyl
in strawberry (10x diluted)
(ratio = 0.72)
Max. 6.6e7 cps.
160.1
6.0e7
6000.0
Performing identification analysis using an automatically
triggered EPI scan instead of MRM ratio calculation allows a
reduction of the number of monitored MRM transitions. Only 75,
instead of 150, MRM transitions have to be monitored using the
QTRAP ® screening method for detection and identification of 75
pesticides. The reduction in MRM transitions resulted in a shorter
cycle time of the MRM experiment (QTRAP®: 1.1 s and
Max. 7300.0 cps.
9.1
7300
6.6e7
8000.0
Figure 7 shows another example demonstrating the advantage
of MS/MS library searching for identification. The MRM ratio
clearly identifies the presence of Metalaxyl in strawberry extract.
The corresponding EPI spectrum and the library search result
clearly identify a false positive finding. Metalaxyl was not present
in this strawberry sample.
XIC of +MRM (202 pairs): 297.0/...
Figure 6. Quantitation of 135 μg/kg Thiabendazole and 15.2 μg/kg
Imazalil in banana extract with positive identification of Thiabendazole
(0.55 with tolerance of 0.50-0.76) and negative identification of Imazalil
(0.50 with tolerance of 0.28-0.46). This false negative can be avoided by
identification based on Enhanced Product Ion (EPI) scan and library
search
I n te n s it y , c p s
This higher degree of Identification can be used to minimize the
potential risk of false positive and false negative detection based
on MRM ratio calculation. An example is presented in Figure 6.
The pesticides Thiabendazole and Imazalil were identified and
quantified above the MRL in a banana extract. However, only
Thiabendazole was identified by its MRM ratio. Imazalil could not
be identified based on the MRM ratio of 0.50 and an MRM ratio
tolerance of 0.28-0.46. In comparison, the EPI spectrum rich in
fragment ions searched against a library clearly identifies the
presence of both contaminants in the banana sample and the
false negative detection for Imazalil was avoided.
10
202.2
EPI
FIT = 92.9%
2.0e6
1.0e6
14
135μg/kg Thiabendazole
in banana (10x diluted)
(ratio = 0.55)
0.0
2
4
6
8
Time, min
+EPI (202.00) Charge (+0) CE (3...
4.0e6
12
Max. 5.8e4 cps.
In te n s ity , c p s
< 0.1
5.8e4
10
In te n s ity , c p s
Relative MRM Intensity
17
In te n s ity , c p s
4.0e4
50
134.0
100
150
202.3
200
250
m/z, amu
300
350
400
Figure 7. Quantitation of 13.3 μg/kg Metalaxyl in strawberry extract. This
result was identified by MRM ratio calculation (0.72 with tolerance of
0.59-0.89), but the library search clearly indicates a false positive finding
p6
Table 3. Reproducibility of identification based on MRM ratio calculation and mass spectral library search based on repeat injections of pesticides spiked
into tomato extract (n=10)
Pesticide
MRM ratio
%CV
Library FIT
%CV
Azoxystrobin
0.22
8.9
88.8
1.8
Dimethomorph
0.76
8.8
95.7
0.5
Flufenacet
0.90
11.8
91.2
1.3
Imazalil
0.52
13.6
94.7
1.3
Imidacloprid
0.39
25.3
78.1
5.5
Methabenzthiazuron
0.47
11.7
95.9
1.0
Thiabendazole
0.66
4.9
91.2
2.4
+EPI (297.00)...
Max. 2.3e6 cps.
+EPI (297.00)...
297.1
2.3e6
Max. 2.1e6 cps.
297.2
159.1
2.0e6
+EPI (297.00)...
Max. 2.3e6 cps.
297.2
2.3e6
159.1
2.0e6
2.0e6
159.1
1.5e6
1. inj. 96.4%
1.0e6
69.0
5.0e5
81.1
100
+EPI (297.00)...
Intensity, cps
Intensity, cps
1.5e6
3. inj. 96.6%
1.0e6
1.5e6
5. inj. 93.4%
1.0e6
69.0
5.0e5
173.2 255.1
201.1
81.1
109.2
200
300
m/z, amu
Max. 2.2e6 cps.
100
+EPI (297.00)...
69.0
200
300
m/z, amu
Max. 2.2e6 cps.
297.2
2.2e6
2.0e6
5.0e5
173.2 255.1
201.1
297.2
0.0
200
300
m/z, amu
Max. 2.2e6 cps.
+EPI (297.00)...
297.1
2.0e6
159.1
159.1
1.5e6
1.5e6
8. inj. 94.3%
1.0e6
69.0
69.0
5.0e5
173.2
81.0 141.2 201.1
0.0
100
Intensity, cps
Intensity, cps
6. inj. 94.1%
1.0e6
5.0e5
255.1
201.1
141.2
100
2.2e6
159.1
2.0e6
173.2
109.2
164.3
255.1
200
m/z, amu
173.2
81.1
300
0.0
100
201.1 255.1
200
m/z, amu
300
1.5e6
10. inj. 94.8%
1.0e6
69.1
5.0e5 81.1
173.2
255.1
141.2
201.2
100
200
m/z, amu
300
Figure 8. Repeated analysis of banana extract with detection of 15.2
μg/kg Imazalil – Library search FIT values of Enhanced Product Ion
spectra indicate the high reproducibility of MS/MS identification using
®
QTRAP LC/MS/MS Systems
Summary
Multiple Reaction Monitoring (MRM) using triple quadrupole and
®
QTRAP LC/MS/MS Systems is widely used for targeted analyte
screening and quantitation because of its unmatched selectivity
and sensitivity. Double mass filtering in the mass analyzer allows
for the best Signal-to-Noise (S/N) and thus the lowest Limits of
Detection (LOD) with excellent reproducibility and linear range.
In many applications, such as food, environmental and forensic
testing, additional mass spectrometric information is required by
guidelines for identification of detected analytes. This
identification is typically performed by detecting two MRM
transitions per compound and the calculation of an MRM ratio.
of even state-of-the art triple quadrupole mass spectrometers,
such as AB SCIEX systems using the patented Linear
Accelerator (LINAC®) Collision Cell. This results in a limitation of
the number of compounds that can be screened for in a single
injection and/or less reproducibility and accuracy due to an
insufficient number of data points across the LC peaks.
Here Hybrid Triple Quadrupole Linear Ion Trap (QTRAP®)
Systems provide a novel workflow for the screening and
identification of a multitude of targeted analytes by combining
selective MRM detection with a highly sensitive MS/MS scan
using Q3 as Linear Ion Trap. In Information Dependent
Acquisition (IDA) experiments, the detection of an MRM signal
above a specified threshold automatically triggers an Enhanced
Product Ion (EPI) scan. These EPI spectra are as sensitive and
selective as MRM signals and contain the complete molecular
fingerprint because of precursor ion selection in Q1, product ion
generation in the collision cell, and ion accumulation in the LIT.
The rich product ion spectra are the result of the generation of
fragment ions in the collision cell. These spectra can be
searched against existing mass spectra libraries.
The information saved into a full scan MS/MS spectrum allows
identification with a higher degree of confidence minimizing the
risk of potential false positive and negative detection. In addition,
the improved cycle time for all confirmatory MRM transitions can
be used to increase the dwell time of all other MRM transitions to
improve S/N, resulting in better reproducibility and accuracy.
Alternatively, additional compounds can be screened in the MRM
survey scan.
However, with an increasing demand to test for more and more
contaminants in a larger variety of samples, the required number
of MRM transitions exceeds the possibilities
p7
References
1 A. Schreiber ‘Advantages of Using Triple Quadrupole over
Single Quadrupole Mass Spectrometry to Quantify and
Confirm the Presence of Pesticides in Water and Soil
Samples’ Application Note AB SCIEX (2006)
2 J. W. Hager: Rapid Commun. Mass Spectrom. 16 (2002)
512-526
3 J. W. Hager and J. C. Y. Le Blanc: Rapid Commun. Mass
Spectrom. 17 (2003) 1056-1064
4 B. A. Collings et al.: J. Am. Soc. Mass Spectrom. 14 (2003)
622-634
5 P. Marquet et al.: J. Chromatogr. B 789 (2003) 9-18
6 C. A. Mueller et al.: Rapid Commun. Mass Spectrom. 19
(2005) 1332-1338
7 S.M.R Stanley et al.: J. Chromatogr. B 836 (2006) 1-14
8 H. D. Hernando et al.: Anal. Bioanal. Chem. 389/6 (2007)
1815-1831
9 M. J. M. Bueno et al.: Anal. Chem. 79/24 (2007) 9372-9384
10 M. Gros et al.: J. Chromatogr. A 1189 (2008) 374-384
11 Hongying Gao et al.: Rapid Commun. Mass Spectrom. 21
(2007) 3683-3693
12 R. D. Unwin et al.: Molecular and Cellular Proteomics 4/18
(2005) 1134-1144
13 E. Ciccimaro et al.: Rapid Comm. Mass Spectrom. 20 (2006)
3681-3692
14 L. Anderson et al.: Molecular and Cellular Proteomics 5/4
(2006) 573-588
15 J. R. Whiteaker et al.: J. Proteome Research 6/10 (2007)
3962-3975
16 M. Anastassiades et al.: J. AOAC Int. 86 (2003) 412-431
17 European Commission Decision 2002/657/EC
For Research Use Only. Not for use in diagnostic procedures.
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