The Detection of Acidic Herbicides and Phenyl Ureas by LC- MS/MS with Large Volume Injection and Automated Column Switching

Home , Monolinuron

James Thomas1, Susan Struthers1, and Stephen Lock2 1 SEPA, East Kilbride, UK, 2 AB SCIEX Warrington, UK

Introduction

Acidic herbicides like Dicamba are used to kill broadleaf weeds before and after sprout. They control annual and perennial weeds in grain crops and highlands, are used to control brush and bracken in pastures, and in combination they are also used in pastures, range land, and noncrop areas (fence rows, roadways, and wastage) to control weeds. Phenyl urea pesticides such as Linuron are used as selective herbicides for pre- and post-emergence weed control in vegetables including potatoes, peas, carrots and beans; also on wheat, celery, parsnip and parsley. Both classes of pesticides are toxic to wildlife and some are suspected hormone-disrupting substances.

The provision of clean, uncontaminated drinking water is of paramount importance to the water industry. In recent times the requested limits of detection for such pesticides have been Experimental decreasing as methodologies improve. Typically water companies need to be able to have limits of quantitation for Sample Preparation pesticides between 0.1 – 1 µg/L (100 – 1000 part-per-trillion, ppt) River and ground water samples (10 mL) were filtered through a which often means that methods should have limits of detection Chromfil PET 20/25, 0.2 µm 25 mm filter. The filter was washed for certain pesticides in the range of 10 – 50 µg/L. by acetonitrile (0.85 mL) with the filter wash added directly into These low levels have often meant that water samples have to the sample. This filtered sample was directly injected onto the be prepared either by liquid/liquid or solid phase extraction in LC-MS/MS system. order to concentrate these contaminants to such a level where Chromatography they can be detected using traditional techniques such as GC- MS or HPLC with UV detection. Where GC-MC is used an Samples (200 µL) were directly injected and separated by additional derivatization step is often required before sample reversed-phase HPLC using a Dionex Ultimate 3000 system. A analysis. This sample pre-treatment used for traditional Gemini 3 µm, 150 x 2.0 mm C18 and a LUNA 3 µm C18 (2), 150 techniques can often be time consuming and add additional cost x 3 mm column from Phenomenex were used to analyze acid to the analyses. Therefore in this work the direct injection of herbicides and phenyl ureas respectively. Both columns were filtered samples was used for sample analysis, to reduce both kept at 40ºC and gradients from water containing 0.1% acetic cost and speed up the sample throughput. acid to acetonitrile containing 0.1% acetic acid were used to separate analytes. Automated column switching, involving a 10 port Valco switching valve, was used to switch between the column for acidic herbicide and the one for phenyl urea analysis (the gradient profiles are shown in Table 1).

p 1

Table 1. Gradient profiles used for the separation of acidic herbicides Table 2. MRM transitions to detect acidic herbicides and phenyl ureas and phenyl ureas using the AB SCIEX API 4000™ system

Acidic herbicides Phenyl ureas Pesticide Q1 Q3 DP CE

Flow Flow Time (min) % B Time (min) % B MCPA 199.0 141.1 -55 -20 (mL/min) (mL/min) Clopyralid A 189.9 146.0 -20 -12 0.0 10 0.4 0.0 10 0.2 Clopyralid B 191.9 148.0 -20 -12 1.5 10 0.4 5.0 10 0.2 2,4-D 218.9 161.1 -20 -20 10.0 95 0.7 9.0 100 0.3 Dicamba 218.9 175.1 -20 -8 18.0 95 0.7 16 100 0.3 2,4-DB 246.9 161.0 -20 -18 18.5 10 0.4 17 10 0.2 Dichlorprop 232.9 161.1 -25 -18 18.6 10 0.4 Bromoxynil 275.8 81.0 -50 -45

Ioxynil 369.7 127.0 -55 -50

Mass Spectrometry Bentazone 239.0 132.0 -50 -36

Analysis was performed on an AB SCIEX API 4000™ LC/MS/MS MCPB 227.1 141.1 -35 -25 system with Turbo V™ source electrospray ionisation (ESI) MCPP 213.0 141.1 -30 -22 probe in negative polarity (acidic herbicides) and positive polarity Triclopyr 253.9 196.0 -20 -16 (phenyl ureas). The MRM transitions for acidic herbicides and phenyl ureas are shown in Table 2. Fluroxypyr 253.0 195.0 -35 -20 Benazolin 242.0 170.1 -25 -20 Results and Discussion Aminopyralid 204.8 160.8 -55 -14

Examples of calibrations for both acidic herbicides and phenyl 2,4-DPA (S) 203.1 159.1 -35 -12 ureas are shown in Figures 1, 2 and 3. For both classes of 4-CAA (IS) 169.0 125.0 -20 -12 pesticides linear responses were obtained over the range tested with ‘r’ values never less than 0.998 (Table 3). 2,B-4,C-phenol (IS) 195.9 78.9 -45 -32 Isoproturon A 207.1 134.2 45 35

Isoproturon B 207.0 72.0 56 35

Diuron 233.0 72.0 71 35

Isoproturon 207.0 72.0 56 35

Monolinuron 215.0 126.0 56 25

Chlorotoluron A 215.0 182.9 51 11

Chlorotoluron B 213.0 72.0 51 15

Metoxuron 229.0 72.0 106 35 Figure 1. Calibration for MCPB from 12.5 – 600 ng/L Fenuron 165.2 72.0 86 29

Pencycuron 329.0 124.8 90 39

Linuron 249.0 159.9 51 27

Isoproturon 207.1 134.2 45 35

Figure 2. Calibration for Dicamba from 12.5 – 600 ng/L

p 2

It can also be seen that every compound with the exception of Clopyralid gave a good signal-to-noise (> 15:1) from the lowest standard 12.5 ng/L (Table 3). Clopyralid, the least sensitive of all the compounds, gave a signal to noise of 25:1 at its lowest standard level of 25 ng/L. There was no carryover observed for either method.

This method has been validated and used routinely for testing Figure 3. Calibration for Isoproturon from 12.5 – 600 ng/L water samples as part of surveillance exercises. Normally such tests produce negative results but in certain instances positive results can be observed which normally result from the illegal Table 3. Signal-to-noise (S/N)* of the lowest calibration standard and r’ disposure of pesticides. values taken from calibration lines 12.5 – 600 ng/L

Pesticide S/N at 12.5 ng/L ‘r’ value XIC of -MRM (18 pairs): 239.000/132.000 Da ID: Bentazone from Sample 3 (Std 2) of AH100929MH.wiff (Turbo Spray) Max. 1.4e4 cps.

MCPA 86.7 0.99967 1.6e5 TIC of Standard at 12.5 ng/L

1.4e5 # Clopyralid 25.1 0.99769 1.2e5

1.0e5

2,4-D 51.3 0.99963 8.0e4 I n t en s i y , c ps 6.0e4

Dicamba 25.5 0.99856 4.0e4

2.0e4 11.76

2,4-DB 25.8 0.99936 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 Time, min XIC of -MRM (18 pairs): 242.... Max. 1041.7 cps. XIC of -MRM (18 pairs): 218.... Max. 1592.7 cps. XIC of -MRM (18 pairs): 239.0... Max. 1.3e4 cps.

Dichlorprop 76.5 0.99934 8.13 9.79 11.74 1.27e4 1000 1000 Benazoline Dicamba 1.20e4 Bentazone 900 900 1.10e4 1.00e4 Bromoxynil 50.3 0.99956 800 800 9000.00 700 700 8000.00 600 600 Ioxynil 148.5 0.99932 7000.00 500 500 6000.00 8.13

I n t en s i y , c ps 400 I n t en s i y , c ps 400 I n t en s i y , c ps 5000.00 8.31 4000.00 Bentazone 368.1 0.99888 300 300 10.05 12.42 11.00 13.23 3000.00 200 200 7.97 9.42 2000.00 6.72 7.21 8.96 10.06 MCPB 15.3 0.99868 100 8.54 100 1000.00 0 0 0.00 7.0 8.0 9.0 10.0 8.0 9.0 10.0 11.0 12.0 13.0 10 12 14 16 Time, min Time, min Time, min MCPP 102.1 0.99968 Figure 4a. 12.5 ng/L standard in negative polarity Triclopyr 27.6 0.99871

Fluroxypyr 22.3 0.99846

Benazolin 26 0.99876 XIC of -MRM (18 pairs): 239.000/132.000 Da ID: Bentazone from Sample 26 (1670444 Manhole d/s Crop Chemicals) of AH100929MH.wiff (... Max. 7.7e5 cps. 11.96 7.7e5 TIC of Manhole sample Aminopyralid 100.7 0.99955 7.0e5 6.0e5 Diuron 41.2 0.99816 5.0e5 4.0e5

Isoproturon 39.5 0.99864 I n t en s i y , c ps 3.0e5 2.0e5

Monolinuron 32 0.99904 1.0e5 0.90 1.93 2.71 3.67 4.11 4.345.41 5.82 6.71 6.89 7.59 8.05 9.15 9.36 9.98 10.90 12.63 13.68 14.39 15.41 15.91 16.36 17.20 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 Time, min Metoxuron 54.9 0.99882 XIC of -MRM (18 pairs): 242.... Max. 4875.0 cps. XIC of -MRM (18 pairs): 218.9... Max. 1.5e4 cps. XIC of -MRM (18 pairs): 239.0... Max. 7.2e5 cps.

8.17 9.88 11.96 4875 1.5e4 Benazoline Dicamba 7.0e5 Bentazone Fenuron 53.1 0.99913 4500 1.4e4 4000 6.0e5 1.2e4 3500 5.0e5 Pencycuron 167.9 0.99982 1.0e4 3000 4.0e5 2500 8000.0

2000 3.0e5 Linuron 26.2 0.9993 I n t en s i y , c ps I n t en s i y , c ps 6000.0 I n t en s i y , c ps

1500 4000.0 2.0e5 8.44 8.10 1000 10.19 Chlorotoluron 50.5 0.99921 6.06 9.38 6.92 2000.0 1.0e5 500 9.41 12.02 11.05 10.74 11.98 12.47 13.49 0 0.0 0.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 10 12 14 16 * S/N was calculated in MultiQuant™ software version 2.0.1 Time, min Time, min Time, min # S/N of Chlopyralid at 25 ng/L Figure 4b. Manhole sample in negative polarity

p 3

Figures 4 and 5 show examples of where this method has Conclusion detected both the presence of certain acidic herbicides and The results show that both acidic herbicides and phenyl ureas phenyl ureas in samples of water from manholes. In each can be detected at the required limits set by the water industry in example the amount of pesticide detected varies with analyte the UK. The sample preparation used involved a simple filtration and is in the parts per trillion range but exceeds the lowest step which removed the cost and time associated with solid calibration standard. phase extraction and/or liquid liquid extraction traditionally used for GC-MS analysis. Acidic herbicides and phenyl urea pesticides ionise under different polarities and require different XIC of +MRM (14 pairs): 192.000/160.000 Da ID: Carbendazim from Sample 3 (Std 2) of UH101001MH.wiff (Turbo Spray) Max. 475.0 cps.

5.5e4 TIC of Standard at 12.5 ng/L HPLC conditions to obtain their best sensitivity. Using 5.0e4 4.5e4 conventional LC and a timed switching valve samples can be run 4.0e4 3.5e4 under the optimised LC conditions for either class of compounds, 3.0e4 2.5e4 without supervision. The automated column switching enables I n t e s i y , c ps 2.0e4 1.5e4 researchers to optimise the pH of the mobile phase and column 1.0e4 5000.0 chemistry to produce the best sensitivity for both compound 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 Time, min XIC of +MRM (14 pairs): 233.... Max. 2100.0 cps. XIC of +MRM (14 pairs): 213.... Max. 1414.2 cps. XIC of +MRM (14 pairs): 207.0... Max. 5.0e4 cps. classes.

12.26 12.10 11.09 1269 1400 5.0e4 1200 Diuron Isoproturon 4.5e4 1100 1200 Chlorotoluron Such a method has been shown to be robust and sensitive 1000 4.0e4 9.95 900 1000 3.5e4 8.80 9.67 800 enough to be applied to surveillance work, in the UK, needed to 10.18 3.0e4 700 800 2.5e4 600 maintain a safe water supply. 10.56 600 I n t en s i y , c ps 500 I n t en s i y , c ps I n t en s i y , c ps 2.0e4 400 11.28 400 1.5e4 300 12.10 1.0e4 200 200 11.41 11.07 12.46 5000.0 100 10.58 13.02 11.72 0 0 0.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 10.0 11.0 12.0 13.0 7.0 8.0 9.0 10.0 11.0 12.0 Time, min Time, min Time, min Figure 5a. 12.5 ng/L standard in positive polarity

XIC of +MRM (14 pairs): 213.000/72.000 Da ID: Chlortoluron from Sample 26 (1670444 Manhole d/s Crop Chemicals) of UH101001MH.wiff ... Max. 4.2e4 cps.

12.09 4.2e4 TIC of Manhole sample 4.0e4

3.5e4

3.0e4

2.5e4

2.0e4 I n t e s i y , c ps 1.5e4

1.0e4

5000.0

0.10 1.68 1.94 2.45 2.61 3.66 4.09 4.24 4.76 5.31 6.01 6.37 7.06 7.70 8.46 9.16 9.60 10.83 11.05 11.39 12.84 13.45 13.64 14.65 15.72 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 Time, min XIC of +MRM (14 pairs): 233.... Max. 8635.9 cps. XIC of +MRM (14 pairs): 213.0... Max. 4.0e4 cps. XIC of +MRM (14 pairs): 207.0... Max. 2.6e4 cps.

12.25 12.09 11.07 8636 4.0e4 2.6e4 8000 Diuron 2.4e4 Isoproturon 3.5e4 2.2e4 7000 Chlorotoluron 3.0e4 2.0e4 6000 1.8e4 2.5e4 1.6e4 5000 1.4e4 2.0e4 4000 1.2e4

I n t en s i y , c ps I n t en s i y , c ps 1.5e4 I n t en s i y , c ps 1.0e4 3000 8000.0 2000 1.0e4 6000.0 4000.0 1000 5000.0 2000.0 10.92 11.63 0 0.0 0.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 10.511.011.5 12.012.5 13.013.5 7.0 8.0 9.0 10.0 11.0 Time, min Time, min Time, min Figure 5b. Manhole sample in positive polarity

For Research Use Only. Not for use in diagnostic procedures.

Publication number: 3370611-01

Headquarters International Sales 353 Hatch Drive Foster City CA 353 Hatch Drive Foster City CA 94404 USA For our office locations please call the division Phone 650-638-5800 headquarters or refer to our website at www.absciex.com www.absciex.com/offices