Screening and Identification of Unknown Contaminants in
Untreated Tap Water Using a Hybrid Triple Quadrupole
Linear Ion Trap LC-MS/MS System
M.T. Baynham1, St. Lock1, D. Evans2, and P. Cummings 2
1
AB SCIEX Warrington Cheshire (UK), 2 ALcontrol Laboratories Rotherdam Yorkshire (UK)
Introduction
Protection of our drinking water resources from contaminants is
a major responsibility for both government and water producing
bodies. The response taken to a potential drinking water
emergency will depend upon both the composition and the
nature of the identified contaminant(s). Furthermore it is
essential that there is a high degree of confidence in the correct
and rapid identification of the problem before remedial action is
taken. To date it has been a necessity to employ a combination
of multiple analytical techniques to meet this end.
Screening Using Accurate Mass
Measurements and MS/MS
In this example two structural related but different pesticides
(Prometryn and Terbutryn) produce the same molecular ion
because they have identical molecular formulae. In the
environment there are hundreds of compound with the same
mass (Figure 2). Thus, a complete identification of unknown
contaminants by accurate mass alone may not yield to a
complete answer as this does not provide any structural
information. In the example above separation of these two
pesticides by HPLC was not clear-cut as they eluted with very
similar retention times (Figure 3). However, Prometryn and
Terbutryn have different MS/MS fragmentation patterns (Figure
2). Therefore product ion spectra are essential for confident
identification of unknown contaminants.
2000
frequency of occurrence
One method of detecting contaminants is the use of accurate
mass as a way to predict the formula and identity of a
contaminant. In this approach the mass spectrometer has to be
accurately calibrated because the greater the error the more
potential contaminants would be a match for the detected peak,
as <2ppm mass error is ideal.
more than 1500
compounds have a similar
molecular weight of
~250amu
1500
1000
500
200
400
600
800
1000
molecular weight
Figure 1. Abundance of compounds over molecular weight range of 1001000 amu
p1
Table 1. Comparison of sensitivities between the General Unknown Screening (GUS) and Multi Target Screening (MTS) approaches
Multi Target Screening
General Unknown Screening
Compound Name
Compound
Class
Polarity
MRM
Intensity at
1 µg/mL
~LOD (µg/mL)
Q3 Mass
Intensity at
10 µg/mL
~LOD (µg/mL)
Brodifacoum
Rat poison
Negative
521.0/79.0
5.80E+04
0.05
521.0
7.70E+05
5.00
Chlorophacinone
Rat poison
Negative
373.0/201.1
1.23E+04
0.20
373.0
3.40E+05
15.0
Difenacoum
Rat poison
Negative
443.1/135.0
1.40E+04
0.25
443.1
1.80E+06
1.25
Difethialone
Rat poison
Negative
537.0/79.0
6.00E+04
0.07
537.0
1.40E+06
5.00
Flocoumafen
Rat poison
Negative
541.1/161.0
1.30E+04
0.12
541.1
1.40E+06
2.00
Warfarin
Rat poison
Negative
307.0/161.1
1.80E+04
0.20
307.0
1.80E+05
40.0
Endothal
Rat poison
Negative
185.0/141.0
6.00E+03
2.0
185.0
-
100
DNOC
Cresol
Negative
197.0/137.1
5.00E+04
0.10
197.0
2.00E+06
1.25
Azinphos-ethyl
Organophosphorus
Positive
346.0/160.1
5.13E+03
1.00
346.0
6.50E+04
200
Demeton-S-methyl
Organophosphorus
Positive
231.0/89.0
1.00E+04
0.50
231.0
2.30E+05
20.0
Dichlorvos
Organophosphorus
Positive
221.0/127.0
9.33E+02
10.0
221.0
4.00E+04
200
Disulfoton
Organophosphorus
Positive
275.1/89.0
2.00E+03
5.00
275.1
2.00E+04
2000
Propetamphos
Organophosphorus
Positive
282.1/156.0
2.20E+03
2.50
282.1
5.20E+04
200
Tebupirimfos
Organophosphorus
Positive
319.0/153.1
1.90E+04
0.50
319.0
2.90E+05
20.0
Parathion-ethyl
Organophosphorus
Positive
292.1/236.0
4.73E+03
2.00
292.1
1.00E+04
500
Parathion-methyl
Organophosphorus
Positive
281.1/264.3
5.00E+02
10.0
264.1
2.00E+04
400
General Unknown Screening and Multi
Target Screening
There are two possible approaches of screening methods. The
first would to screen for a complete unknown. This General
Unknown Screening (GUS) would use a single ‘universal’ survey
scan over a defined mass range and could either be a Time-ofFlight (TOF), quadrupole or ion trap scan. This survey scan can
be used to trigger automatically the acquisition of a product ion
spectrum if a signal of a detected compound is above a defined
threshold. Finally, this spectrum can be searched against a mass
spectral library for identification. Comparison of Total Ion
Chromatograms (TIC) of unknown samples to that of the control
reveal compounds that are either unique to the sample or those
that are present at significantly higher concentrations than in the
control.
The other approach is often called Multi Target Screening (MTS).
In this approach a predefined list of compounds is looked for in a
Single Ion Monitoring (SIM) or Multiple Reaction Monitoring
(MRM) experiment. MRM mode is generally preferred because
of higher selectivity and sensitivity. Once a compound is
detected above a defined threshold a product ion scan is
collected and compared against a library. Dynamic exclusion of
compounds where MS/MS spectra are already acquired allows
the data collection of co-eluting compounds (Figure 4).
p2
242.1433
2.3e4
2.2e4
2.1e4
2.0e4
1.9e4
1.6e4
Terbutryn
TOF-MS
Terbutryn
MS/MS
1.5e4
1.4e4
H3C
1.3e4
S
1.8e4
Experimental
186.0846
1.7e4
2.4e4
In order to maximize sensitivity two injections, one in positive
and the other in negative polarity, for both the GUS and MTS
approach, were done. Additionally, this allowed the mobile phase
to be optimum for either polarity.
1.2e4
1.7e4
1.1e4
1.6e4
N
1.5e4
N H3C
1.0e4
CH 3
1.4e4
9000.0
1.3e4
H3 C
1.2e4
NH
N
NH
8000.0
CH 3
1.1e4
1.0e4
7000.0
9000.0
6000.0
8000.0
5000.0
7000.0
6000.0
4000.0
5000.0
3000.0
4000.0
3000.0
158.0524
2000.0
HPLC
2000.0
1000.0
138.0800
1000.0
0.0
116.0294
240.5
241.0
241.5
242.0
242.5
243.0
m/z, Da
243.5
244.0
244.5
245.0
245.5
246.0
242.1435
2.4e4
2.2e4
110
120
130
242.1479
200.1001
144.0618
140
150
160
170
180
190
200
210
220
230
240
250
m/z, Da
158.0522
6699
2.8e4
2.6e4
0.0
100
6500
Prometryn
TOF-MS
Prometryn
MS/MS
6000
5500
H3C
S
A Shimadzu HPLC system with binary LC10ADvp binary
gradient pump and SIL-HT autosampler was used for all HPLC
separations. The mobile phase used in positive mode was:
200.1000
5000
2.0e4
4500
CH3
1.8e4
N
N
CH3
4000
1.6e4
NH
H3 C
1.4e4
N
NH
3500
CH 3
3000
1.2e4
A: H2O + 2 mM NH4CH 3COO
2500
1.0e4
242.1474
2000
8000.0
1500
6000.0
B: CH3OH + 0.1% HCOOH
1000
4000.0
116.0292
2000.0
500
110.0724
152.1093
240.5
241.0
241.5
242.0
242.5
243.0
m/z, Da
243.5
244.0
244.5
245.0
245.5
246.0
0
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250
m/z, Da
Figure 1. Accurate mass measurement of Prometryn (top) and Terbutryn
(bottom) using a Quadrupole quadrupole-Time-of-Flight system in MS
mode and MS/MS spectra of both pesticides
The mobile phase used in negative mode was:
A: H2O
B: CH3OH + 0.1% NH4OH
HPLC separation for Multi Target Screening was performed on a
C18 monolithic column (Merck). Samples were analyzed using a
rapid gradient over 1.5 minutes at a flow rate of 1200 μL/min
(without splitting of the flow prior to the mass spectrometer).
Injection volumes of 50 or 100 μL were used for analysis.
6.4
5.5e4
HPLC separation of
Prometryn (red) and
Terbutryn (blue)
5.0e4
4.5e4
4.0e4
3.5e4
An ACE C18 (50 mm 5 μm HICHROM) column was used for
HPLC separation for General Unknown Screening. The HPLC
flow was set at 1200 μL/min with a gradient used from 25% B to
100% B over 16 minutes. An injection volume of 50 μL was
used.
3.0e4
2.5e4
2.0e4
1.5e4
1.0e4
MRM Survey Scan
5000.0
0.0
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
5.6
5.8
6.0 6.2
Time, min
6.4
6.6
6.8
7.0
7.2
7.4
7.6
7.8
Figure 3. HPLC analysis of Prometryn and Terbutryn on a standard C18
reverse phase column, both compounds elute with a retention time
difference of less than 6s
The technology that lends itself to this application is the hybrid
®
triple quadrupole linear ion trap technology (QTRAP LC/MS/MS
systems). It allows the use of any triple quadrupole scan,
including MRM, to trigger the acquisition of Linear Ion Trap
MS/MS spectra by Enhanced Product Ion scanning. Enhanced
Product Ion scan spectra give maximum sensitivity for library
searching with a complete pattern characteristic for Collision
Induced Dissociation (CID).
Threshold
Dynamic
Exclusion
Dependant Scans
Figure 4. Experimental setup of a Multi Target Screening (MTS)
approach
p3
MS/MS
0.7
0.7
1.8e8
1.7e8
1.6e8
®
A 4000 QTRAP LC/MS/MS system was used for both MTS and
GUS experiments which triggered dependant Enhanced Product
Ion scanning (mass range of 50 to 750 amu at 4000 amu/s) with
a Collision Energy (CE) of 35 V and Collision Energy Spread
(CES) of 20 V. The MTS survey scan used MRM transitions
which have been optimized for each targeted analyte while the
GUS screen used a Q3 scan with a mass range of 90 to 750
amu and a Declustering Potential (DP) of 60 V.
1.5e8
1.4e8
1.7e8
Mineral water
MRM 230/146
1.6e8
1.5e8
1.4e8
1.3e8
1.3e8
1.2e8
1.2e8
1.1e8
1.1e8
1.0e8
1.0e8
9.0e7
9.0e7
8.0e7
8.0e7
7.0e7
7.0e7
6.0e7
6.0e7
5.0e7
5.0e7
4.0e7
4.0e7
3.0e7
3.0e7
2.0e7
2.0e7
1.0e7
1.0e7
0.0
0.1
0.2
0.3
0.4
1.00e7
9.00e6
0.6
0.7
0.8
0.0
0.9
230.0
174.0
1.16e7
1.10e7
0.5
Time, min
MS/MS of
Terbuthylazine
1.10e7
1.00e7
9.00e6
7.00e6
6.00e6
6.00e6
5.00e6
25 psi
Gas 1
50 psi
Gas 2
60 psi
80
0.8
0.9
174.0
95.9
103.8
146.0
100
120
140
78.9
160
180
200
m/z, amu
220
240
260
2 80
300
132.0
95.9
1.00e6
138.1
80
109.9
100
138.1
120
140
1 60
180
200
m/z, amu
220
240
260
280
300
Figure 5. 100 ng/mL Terbuthylazine spiked into mineral and tap water
analyzed in positive polarity MRM and EPI
0.6
3.2e7
Mineral water
MRM 213/141
3.2e7
3.0e7
2.8e7
0.6
3.4e7
3.8e7
3.4e7
10
0.7
MS/MS of
Terbuthylazine
2.00e6
78.9
3.6e7
CAD
0.6
230.0
132.0
109.8
Curtain gas
0.5
Time, min
3.00e6
146.0
2.00e6
Value
0 .4
4.00e6
103.8
3.00e6
Parameter
0.3
5.00e6
4.00e6
Table 2. Ion source and gas parameters
0.2
8.00e6
7.00e6
1.00e6
0.1
1.19e7
8.00e6
The source and gas settings for both MTS and GUS experiments
were the same (Table 2)
Tap water
MRM 230/146
Tap water
MRM 213/141
3.0e7
2.8e7
2.6e7
2.4e7
2.6e7
Temperature
IonSpray™ source voltage
650°C
-4500 V
2.2e7
2.4e7
2.0e7
2.2e7
2.0e7
1.8e7
1.8e7
1.6e7
1.6e7
1.4e7
1.4e7
1.2e7
1.2e7
+5500 V
1.0e7
1.0e7
8.0e6
8.0e6
6.0e6
6.0e6
4.0e6
4.0e6
2.0e6
2.0e6
0.0
0.1
0.2
0.3
0.4
0.5
Time, min
0.6
0.7
0.8
0.0
0.9
140.8
Results and Discussion
0.2
0.3
0.4
0 .5
Time, min
0.6
0.7
0.8
0.9
140.9
1.00e7
MS/MS of
MCPP
1.00e7
9.00e6
Figure 5 and 6 present data obtained for an injection of
100 ng/mL Terbuthylazine and MCPP in both mineral and tap
water, using the MRM to EPI MTS approach. The LINAC®
®
collision cell of the 4000 QTRAP system allows the
simultaneous monitoring of up to hundreds of MRM transitions
(contaminants) in a single sample injection. These MRM
transitions triggered Enhanced Product Ion scan spectra in a
cycle time of approximately 2.5 s without loss in sensitivity and
full spectral quality.
0.1
1.04e7
1.10e7
MS/MS of
MCPP
9.50e6
9.00e6
8.50e6
8.00e6
7.50e6
8.00e6
7.00e6
7.00e6
6.50e6
6.00e6
6.00e6
5.50e6
5.00e6
5.00e6
4.50e6
4.00e6
4.00e6
3.50e6
3.00e6
3.00e6
2.50e6
213.0
2.00e6
213.0
2.00e6
1.50e6
1.00e6
1.00e6
104.7
80
100
5.00e5
120.9
120
140
160
180
2 00
220 240
m/z, amu
260
280
300
320
3 40
360
380
400
104.7
80
100
120
1 40
160
18 0
200
220
240
m/z, amu
2 60
280
300
320
340
360
380
400
Figure 6. 100 ng/mL MCPP spiked into mineral and tap water analyzed
in negative polarity MRM and EPI
Mineral water typically contains high levels of sodium, which may
affect sensitivity due to adduct formation. However, Figure 5 and
6 indicate that there is nearly no effect on S/N to detect
Terbuthylazine and MCPP in these water samples.
p4
Summary
The GUS approach shows the comparison of a blank control
sample to a sample that has been spiked with 0.1 μg/L of a
compound to be identified (Figure 7). The presence of the
compound with m/z=350 amu is detected in the sample by
comparing the two Q3 scan chromatograms. Acquisition of an
Enhanced Product Ion scan spectrum followed by library
searching allows to identification of Chlorpyrifos.
TIC: from Sample 1 (SAMPLE B) of Q3 5.wiff (Turbo Spray), Smoothed
®
Max. 8.7e7 cps.
I
n
8.0e7
t
6.0e7
0.3
Water sample
10.2
e
9.4
.
0.9
.
0.0
13.7 14.0
14.9
13.3
11.5
12.0
8.5
4.0e7
2.0e7
10.7
.
1
2
3
4
5
6
7
8
Time, min
9
9.8
10
11
12
13
14
+Q3: Exp 1, 12.046 min from Sample 1 (SAMPLE B) of Q3 5.wiff (Turbo Spray)
15
16
Max. 8.7e5 cps.
I
n
350.0
8.0e5
Survey Q3 spectrum at 12min
322.0
t
153.0
6.0e5
e
198.0
4.0e5
354.0
.
2.0e5
.
.
12 0
140
160
18 0
200
220
2 40
260
280
3 00
320
340
3 60
380
400
m/z, amu
+EPI (350.01) Charge (+0) CE (35) CES (20) FT (10): Exp 2, 12.058 min from Sample 1 (SAMPLE B) of Q3 5.wiff (Turbo Spra y)
4 20
440
460
4 80
500
520
540
Max. 2.7e6 cps.
I
n
197.8
EPI spectrum at 12min
2.5e6
t
2.0e6
e
1.5e6
1.0e6
.
114.9
5.0e5
.
.
80
100
133.8 150.7
120
140
179.8
160
213.8
180
200
220
260
280
349.7
321.7
293.7
275.7
240
m/z, amu
300
320
340
360
TIC: from Sample 1 (BLK) of Q3 2.wiff (Turbo Spray), Smoothed
380
The 4000 QTRAP LC/MS/MS system allows Multi Target
Screening (MTS) and General Unknown Screening (GUS) of
water samples to identify emerging contaminants. The MTS
approach is the most rapid and sensitive method to screen for
and detect the presence of targeted organic contaminants in
water. More than 2000 targeted compounds can be screened in
less than 20 minutes at low and sub μg/L level using the
described procedure and multiple sample injections. The GUS
method is an alternative to identify unknown compounds as it
does not rely on any knowledge of the analytes. Here, a sample
control comparison will detect unknown contaminants. In both
approaches automatically generated Enhanced Product Ion
spectra can be searched against a comprehensive mass spectral
library and the fragmentation information can be used for
identification and identification. However, the GUS approach is
lower in sensitive and requires significantly longer run times.
400
Max. 8.7e7 cps.
I
n
8.0e7
t
6.0e7
0.3
10.7
Control sample
13.3
11.4
e
8.4 8.5
4.0e7
13.7 14.0
14.6
12.6
9.8 10.2
.
2.0e7
.
0.0
.
1
2
3
4
5
6
7
8
Time, min
9
10
11
12
13
14
15
16
Figure 7. Comparison of a water sample to a blank control water with
resulting Q3 scan and EPI spectrum of Chlorpyrifos detected and
identified by library searching
In order to compare the relative sensitivities of both approaches,
GUS and MTS, over 70 compounds were tested including
compounds such as organophosphorus pesticides and rat
poisons. Limits of Detection (LOD) were determined to be the
triggering threshold of both approaches. In the GUS method the
LOD was set at 500,000 cps of the parent ion in Q3 scan
(background noise was generally lower than 500,000 cps). For
the MTS approach LOD was 5000 cps in MRM which was
determined as 2-3 times the background level of the most
intense MRM trace. The chromatographic conditions of MTS
were applied for this comparison work. Examples of results for
16 different compounds are given in Table 1 highlighting the
higher sensitivity of the MTS approach. An average of 2 orders
of magnitude comparing LOD of both approaches was found.
For Research Use Only. Not for use in diagnostic procedures.
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