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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.

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QTrap Screening and Identification of Unknown Contaminants in Untreated Tap Water  

more than 1500 compounds have a similar molecular weight of ~250amu Introduction One method of detecting contaminants is the use of accurate...

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