high sensitivity detection of pesticides in water using
TRANSCRIPT
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High Sensitivity Detection of Pesticides inWater Using Online SPE Enrichment
Author
Sarah Gledhill
South East Water
Frimley Green
England
Application Note
Environmental
Abstract
An online solid phase extraction (SPE) method for analyzing 17 chlorinated phenoxy
acid herbicides and pentachlorophenol has been developed on an Agilent 6410
Triple Quadrupole LC/MS system. This method meets the performance requirements
set by the UK Drinking Water Inspectorate for standard deviation, bias, recovery and
total error, and it is accredited by the United Kingdom Accreditation Service (UKAS).
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Introduction
Chlorinated phenoxy acid herbicides and their derivatives arewidely used for control of broadleaf weeds in crops and brushalong roads. While runoff from treated areas can contaminatesurface and groundwater, some chlorinated phenoxy acid herbicides have been directly applied to waterways and reservoirs for the control of aquatic weeds and algae.
Traditionally, these pesticides were monitored in water byextracting 1 L of sample using liquid-liquid or solid phaseextraction followed by methylation using diazomethane. The derivatized sample was then analyzed by gas chromatog-raphy/mass spectrometry (GC/MS). However, liquid chromatography/mass spectrometry (LC/MS) methods elimi-nate the need for derivatization and offer the ability to signifi-cantly reduce the amount of sample required, depending onthe sensitivity of the mass spectrometer used. An LC/MSmethod using off-line solid phase extraction was previouslydeveloped at South East Water in the United Kingdom using50 mL of sample.
This application note describes the development and valida-tion of a new method for 17 chlorinated phenoxy acid herbi-cides and pentachlorophenol (PCP) using online solid phaseextraction, which enables sensitive detection with only 1.5 mLof water. The required amount of sample has been reduced byalmost three orders of magnitude over the original GC/MSmethod. The method uses a polymeric solid phase extractioncartridge that is attached online to an Agilent 1200Quaternary LC pump, which in turn is coupled to an Agilent6410A LC/MS Triple Quadrupole system upgraded with ahotbox. The method meets the performance requirements setby the UK Drinking Water Inspectorate for standard deviation,bias, recovery, and total error and is accredited by the UKAS.
Experimental
Reagents and StandardsReagents were obtained as follows: formic acid 98%, LC/MSgrade from Fluka; glacial acetic acid, HPLC grade, FisherScientific; acetonitrile, HPLC gradient grade, JT Baker; acetone and methanol, JT Baker. The PLRP-S SPE cartridgewas obtained from Agilent (p/n 5062-8547). All pesticidestandards and internal standards were obtained as solids with certified purity from QMX Laboratories.
Stock standards were prepared for each pesticide using 50 mgof solid standard weighed into a 50-mL volumetric flask andmade to volume with acetone, for a final concentration of1,000 mg/L. Mixed Intermediate standards were prepared byadding 10 µL of each individual stock standard to a 100-mLvolumetric flask and made to volume with methanol, for afinal concentration of 100 µg/L of each pesticide standard.
Stock internal standard was prepared using 10 mg ofdichlorophenyl acetic acid (DCPAA) weighed into a 100-mLvolumetric flask and made to volume with acetone, to a finalconcentration of 100 mg/L. The working internal standardwas prepared by adding 250 µL of stock internal standardadded to a 50-mL volumetric flask and brought to volume withmethanol for a final concentration of 500 µg/L.
Calibration standards were prepared by first acidifying 1 L ofultrapure water (from Milli-Q system) by adding 5 mL offormic acid. A 50-mL amount of acidified water was thenadded to each of five 60-mL amber bottles, and 50 µL of inter-nal standard were added to each bottle. The calibration stan-dards were then prepared in the five 60-mL bottles per the following matrix:
Bottle numberVolume of mixed intermediate standard added (µL)
Final concentration of standard (µg/L)
1 100 0.20
2 50 0.10
3 20 0.04
4 10 0.02
5 0 0.00
All bottles were shaken well, and 2 mL was removed fromeach bottle and placed in a 2-mL labeled vial for analysis. Thecalibration range was 0.0–0.20 µg/L, and spiking of AnalyticalQuality Control samples was done at 0.10 µg/L.
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InstrumentsThe system was built using Agilent 1200 Infinity Series LCmodules coupled to a 6410A Triple Quadrupole LC/MS with‘hotbox’ upgrade. The hotbox upgrade kit (G2573A) comprisedan additional MS turbo-pump with controller and replacemententrance and exit lenses for the collision cell. The onlineenrichment used the 1200 Quaternary LC pump, an AgilentAutosampler with 900 µL metering device and multidraw
capability, and a programmable 6-position selection valve. Asecond programmable 6-port/2-position valve is used toselect between loading sample on a re-useable solid phaseextraction cartridge or elution on to the analytical column.The instrument system configuration is shown in Figure 1,and the instrument operating conditions are shown inTables 1 and 2. The quantitation and qualifier ions, fragmenta-tion voltages, and collision energies for each compound wereoptimised using Mass Optimizer.
SPE system:• Binary pump SL (opt. Quaternary pump)• G1329A autosampler with 900 µL head• 6-port 2-pos valve• 12-port 6-pos valve• 12-port stream selection valve
RRLC system:• Binary pump SL • Well plate sampler SL• Column department SL
PreColumn no. 6
PreColumn no. 1 CSV valve“Channel no. 1” position
Valve no. 1“Load” position
waste-water
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3
4
56
2
2
3
3
4
4
5
5
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1
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34
5 6 78
9
1011121
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Figure 1. Online SPE LC/MS system configuration.
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Table 1. Online SPE Conditions Table 2. LC and MS Instrument Conditions
Mobile phase A: 1% Formic acidB: Acetonitrile
SPE cartridge PLRP-S 10 × 2 mm, 15–25 µm
Temperature Ambient
Flow (load) 1 mL/min
Gradient program Time Gradient (%B) Flow rate (mL/min)
0.0 0 1.03.0 0 1.03.5 0 0.54.5 100 0.518.0 100 0.518.2 100 1.025.0 100 1.025.5 0 1.0
Injector program Command:DRAW def. amount from sample 750 µL speed 800VALVE mainpassWAIT 1.5 minutesEJECT def. amount into seat, max. speedDRAW def. amount from sample 750 µL speed 800VALVE mainpassWAIT 1.5 minutesREMOTE start pulseEJECT def. amount into seat, max. speed
LC conditions
Analytical column Agilent ZORBAX C-18 Eclipse Plus, 2.1 mm × 150 mm, 3.5 µm (p/n 959763-902)
Column temperature 60 °C
Injection volume Injection program, 2 × 750 µL, for a total of 1.5 mL
Mobile phase A = 0.1% Acetic acidB = Acetonitrile
Run time 29.0 minutes
Flow rate 0.25 mL/min
Gradient program Time (min) Gradient (%B)0 151.00 151.01 252.00 2517.00 7018.00 10020.00 10021.00 15
MS conditions
Acquisition parameters ESI mode, pos/neg ionization; MRM (7 time segments)
Gas temperature 250 °C
Drying gas 8 L/min Nitrogen
Nebulizer pressure 40 psig
Vcap voltage 3,000 V
Sample Preparation From each sample source, 50 mL were added to a 60-mLamber bottle. If the samples contained particulates, they were filtered through Whatman GF filter paper before measuring 50 mL. To each sample were added 250 µL offormic acid and 50 µL working internal standard solution. Thebottles were shaken and 2 mL removed and placed in a 2-mLlabeled vial for analysis.
Analysis ParametersThe Triple Quadrupole LC/MS multiple reaction monitoring(MRM) acquisition parameters are shown in Table 3.
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Table 3. Agilent 6410 Triple Quadrupole LC/MS MRM Acquisition Parameters
Retention time (min) Compound
Precursor ion (m/z)
Product ion (m/z)
Dwell (msec)
Fragmentor voltage
Collision energy(V) Polarity
5.387 Clopyralid 194 148 100 65 19 Pos
192 146 100 65 19 Pos
6.103 Picloram 243 197 100 75 18 Pos
241 195 100 75 18 Pos
6.576 Imazapyr 262.2 234.3 50 130 14 Pos
217.2 50 130 17 Pos
8.933 Dicamba 221 177 100 60 0 Neg
219 175 100 60 0 Neg
9.455 Benazolin 242 198 70 100 0 Neg
170 70 100 8 Neg
9.782 Fluoroxypyr 255 197 75 100 8 Neg
253 195 75 100 8 Neg
11.361 Bentazone 239 197 25 130 20 Neg
132 25 130 25 Neg
12.379 2, 4-D 221 163 35 80 15 Neg
219 161 35 80 15 Neg
12.426 Bromoxynil 276 81 75 110 35 Neg
274 79 75 110 35 Neg
12.613 MCPA 201 143 35 100 15 Neg
199 141 35 100 15 Neg
12.63 DCPAA (internal standard)
205 161 35 50 0 Neg
202.9 159 35 50 0 Neg
13.476 Triclopyr 256 198 200 60 5 Neg
254 196 200 60 5 Neg
14.114 Ioxynil 369.8 214.9 50 120 30 Neg
126.9 50 120 35 Neg
14.223 Dichlorprop 235 163 50 80 10 Neg
233 161 50 80 10 Neg
14.359 2,4,5-T 254.9 196.9 50 80 10 Neg
252.9 194.9 50 80 10 Neg
14.371 MCPP 215 143 50 80 20 Neg
213 141 50 80 20 Neg
15.319 2,4-DB 249 163 75 80 10 Neg
247 161 75 80 10 Neg
15.466 MCPB 229 143 75 105 2 Neg
227 141 75 105 2 Neg
19.636 PCP 266.9 266.9 50 126 0 Neg
264.9 264.9 50 126 0 Neg
262.9 262.9 50 126 0 Neg
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Results and Discussion
Analysis of Small Sample VolumesUsing online SPE sample preparation enables the use of a1.5-mL sample volume in standard 2-mL autosampler vials,which, in turn, enables the use of the 100-position sampletray and ample throughput. Figure 2 shows a representativetotal ion chromatogram (TIC) for a 0.10 µg/L standard, using a1.5-mL sample. Example EICs of a few of the herbicides andpentachlorophenol (PCP) at 0.10 µg/L are shown in Figure 3.
4.8\ESI TIC MRM (”->”)ahspe031004.d
×105
4.6
4.4
2 3 3 4 4 5 5 6 6 7
4.2
4.0
3.8
3.6
3.4
3.2
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
03 4 5 6 7 8 9 10 11 12
Acquisition time (min)
Coun
ts
13 14 15 16 17 18 19 20
Figure 2. Total ion chromatogram for a 0.10 µg/L standard of 17 herbicides as well as the positions of the time segments.
Sensitivity and Accurate QuantificationThis method enables detection of these herbicides at concen-trations at and below 0.010 µg/L, as shown in Figure 4.Calibration curves were constructed using four concentra-tions of standard from 0.02 to 0.20 µg/L. All coefficients ofcorrelation (R2) were greater than 0.999. Figure 5 shows threerepresentative calibration curves.
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Figure 3. EICs of the transitions for 0.10 µg/L standards of picloram (top two traces in top panel) andclopyralid (bottom two traces in top panel); benazolin (top two traces in middle panel) anddicamba (bottom two traces in middle panel); pentachlorophenol (bottom panel, all three traces).
4.5 2 3 3 4 4 5 5 6 6 7
Coun
ts (×
103 )
Coun
ts (×
103 )
Coun
ts (×
103 )
Coun
ts (×
103 )
3.52.51.50.5
54321
2.01.61.20.80.4
3.5
0.5
3 4 5 6 7 8 9 10Acquisition time (min)
11 12 13 14 15 16 17 18 19 20
2.5
1.5
1.1
Coun
ts (×
104 )
Coun
ts (×
102 )
Coun
ts (×
104 )
Coun
ts (×
104 )
0.90.70.50.30.1
3
2
1
1.00.80.60.40.2
1.41.8
0.2
3 4 5 6 7 8 9 10Acquisition time (min)
11 12 13 14 15 16 17 18 19 20
1.00.6
Coun
ts (×
105 )
Coun
ts (×
105 )
Coun
ts (×
105 )
1.20.80.60.40.2
1.82.2
1.41.00.60.2
0.70.91.1
0.13 4 5 6 7 8 9 10
Acquisition time (min)11 12 13 14 15 16 17 18 19 20
0.50.3
2 3 3 4 4 5 5 6 6 7
2 3 3 4 4 5 5 6 6 7
2 3 3 4 4 5 5 6 6 7
2 3 3 4 4 5 5 6 6 7
2 3 3 4 4 5 5 6 6 7
2 3 3 4 4 5 5 6 6 7
2 3 3 4 4 5 5 6 6 7
2 3 3 4 4 5 5 6 6 19.061
7.640
8.233
8.234
9.271
9.269
7.252
5.261
4.170
3.624
3.9224.287
5.170
6.766 7.316
6.9546.340
5.823
4.022
6.9406.363
5.851
4.025
8.979
8.113
19.061
19.061
17.637
20.01218.506
2 3 3 4 4 5 5 6 6 7
2 3 3 4 4 5 5 6 6 7
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567 2 3 3 4 4 5 5 6 6 7
Coun
ts (×
102 )
Coun
ts (×
102 )
Coun
ts (×
102 )
Coun
ts (×
102 )
4621
654321
4
3
2
1
6
2
3 4 5 6 7 8 9 10Acquisition time (min)
11 12 13 14 15 16 17 18 19 20
4
1.0
Coun
ts (×
103 )
Coun
ts (×
102 )
Coun
ts (×
104 )
Coun
ts (×
103 )
0.80.60.40.2
4
3
2
1
1.21.00.80.60.40.2
1.62.0
0.4
3 4 5 6 7 8 9 10Acquisition time (min)
11 12 13 14 15 16 17 18 19 20
1.20.8
Coun
ts (×
104 )
Coun
ts (×
104 )
Coun
ts (×
103 )
1.20.80.60.40.2
1.6
2.0
1.2
0.8
0.4
5
7
9
13 4 5 6 7 8 9 10
Acquisition time (min)11 12 13 14 15 16 17 18 19 20
3
2 3 3 4 4 5 5 6 6 7
2 3 3 4 4 5 5 6 6 7
2 3 3 4 4 5 5 6 6 7
2 3 3 4 4 5 5 6 6 7
2 3 3 4 4 5 5 6 6 7
2 3 3 4 4 5 5 6 6 7
2 3 3 4 4 5 5 6 6 7
2 3 3 4 4 5 5 6 6 19.553
7.649
8.291
8.205
10.433
9.272
9.295
9.605
7.252
5.164
3.806
3.172
3.815
5.183
6.934
5.634
3.111
3.7814.079
6.9206.282
5.675
4.041
4.660
9.806
8.131
19.551
18.74618.049
19.554
17.629
20.25718.497
18.03216.404
2 3 3 4 4 5 5 6 6 7
2 3 3 4 4 5 5 6 6 7
Figure 4. EICs of the transitions for 0.010 µg/L standards of picloram (top two traces in top panel) andclopyralid (bottom two traces in top panel); benazolin (top two traces in middle panel) anddicamba (bottom two traces in middle panel); pentachlorophenol (bottom panel, all three traces).
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Figure 5. Representative calibration curves from 0.02 to 0.20 µg/L for picloram (top), dicamba (middle)and pentachlorophenol (bottom). All R2 values were >0.999.
4.0y = 1463.8016*x2 +190.9364*xR2 = 0.99990547
Picloram
Dicamba
PCP
×10-1
3.83.63.43.23.02.82.62.42.22.0
Rel
ativ
e re
spon
ses
1.81.61.41.21.00.80.60.40.2
0
0 2 4 6 8 10Relative concentration (×10-4)
12 14 16 18 20
y = 23806.9748*x2 + 678.8077*xR2 = 0.99982362
Rel
ativ
e re
spon
ses
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
-0.1
0 2 4 6 8 10Relative concentration (×10-4)
12 14 16 18 20
y = 139669.3201*x2 + 5252.5566*xR2 = 0.99990095
Rel
ativ
e re
spon
ses
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0 2 4 6 8 10Relative concentration (×10-4)
12 14 16 18 20
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Method ValidationValidation of the method was carried out on 11 sets of spikedduplicates, blanks, and AQC samples using surface, borehole,and treated water sources spiked at 0.10 µg/L with the stan-dard mix of the 17 pesticides plus PCP. Most method limits ofdetection (LODs) were well below 0.01 µg/L. All of the recov-eries were between 86 and 125%, with the majority fallingbetween 95 and 104% (Table 4). The Aquacheck Test is a pro-ficiency testing scheme performed by LGC Standards, a UKASaccredited international provider of proficiency testing (PT)services. It showed excellent correlation with the assignedvalues, which must have a Z score within ±2 to pass the test(Table 5). In addition, the method meets the performancerequirements set by the UK Drinking Water Inspectorate forstandard deviation, bias, recovery, and total error (data notshown).
Conclusions
An online SPE method accredited by the UKAS has beendeveloped on the 6410 Triple Quadrupole LC/MS for theanalysis of 17 chlorinated phenoxy acid herbicides and pen-tachlorophenol that reduces the required sample size by afactor of almost one thousand versus the previous GC/MSmethod. Even so, it delivers ~12 part per trillion (ppt) LODs,as well as recoveries >95% for most pesticides. In addition toa reduction in sample volume, this method provides fasterresults at lower cost. The solid phase extraction cartridgesare reuseable, and less solvent is used for extraction. Finally,the results are more reproducible because the system is fullyautomated and less prone to extractor error.
For More Information
These data represent typical results. For more information onour products and services, visit our Web site atwww.agilent.com/chem.
Table 4. Validation Data for the Online SPE Method
Table 5. Aquacheck Test Data for the Online SPE Method
*LOD = Limit of Detection (three times the standard deviation of the lowstandard for each pesticide)
*Z Score is a measure of correlation with the assigned values, and must valuewithin ±2 to pass the proficiency test.
% Recovery
Compound Surface water BoreholeTreated water LOD* (µg/L)
Clorpyralid 86.29 97.34 86.62 0.012
Picloram 99.58 96.74 93.33 0.009
Imazapyr 124.87 96.92 96.72 0.009
Dicamba 98.94 95.36 93.64 0.003
Benazolin 92.54 96.71 96.13 0.003
Fluroxypyr 92.50 97.26 96.96 0.003
Bentazone 97.59 97.58 96.50 0.003
2,4-D 98.48 96.67 97.47 0.003
Bromoxynil 95.84 95.00 96.75 0.003
MCPA 98.88 95.97 96.47 0.003
Triclopyr 98.22 95.78 96.15 0.003
Ioxynil 100.03 98.65 99.87 0.003
Dichlorprop 100.08 97.01 97.79 0.003
2,4,5-T 102.60 99.07 99.42 0.003
MCPP 101.86 98.29 98.45 0.003
2,4-DB 98.21 97.73 97.30 0.003
MCPB 99.45 98.18 98.83 0.003
PCP 103.68 98.22 98.08 0.006
CompoundAquacheck assignedconcentration (ng/L)
Online SPE method result (ng/L) Z score*
Dicamba 115.6 116.7 0.09
Bentazone 85.9 81.85 -0.47
2,4-D 84.4 88.7 0.51
Bromoxynil 118.3 115.15 -0.27
MCPA 83.8 80.95 -0.34
Triclopyr 44.4 41.2 -0.64
Ioxynil 103 94.05 -0.87
Dichlorprop 54.1 54.95 0.17
MCPP 53 54.45 0.27
2,4-DB 32.8 28.35 -0.89
MCPB 42.6 39.55 -0.61
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