EPA Method 535:
Detection of Degradates of Chloroacetanilides and other
Acetamide Herbicides in Water by LC/MS/MS
Christopher Borton
AB SCIEX Golden, Colorado
Overview
Described here is the analysis of Chloroacetanilide and
Acetamide degradates in drinking water using Liquid
Chromatography Tandem Mass Spectrometry, LC/MS/MS. This
analysis follows U.S. Environmental Protection Agencies’
Method 535 guidelines for sample preparation and analysis. The
method used two Multiple Reaction Monitoring (MRM) transitions
per analyte for both quantitation and confirmation. Monitoring a
second MRM transition for each target compound adds an
additional order of confidence when looking at dirty matrices,
therefore, the possibility of false positive detection is reduced.
Detection limits in drinking water were determined to be 0.002 to
0.004 μg/L using established guidelines.
Introduction
The EPA has established Method 535 for the analysis of
ethanesulfonic acid (ESA) and oxanilic acid (OA) degradates of
chloroacetanilide herbicides in drinking water and surface water.
Chloroacetanilide herbicides are extensively used for weed
control on crops throughout the US. In this method degradates of
Alachlor, Acetochlor and Metolachlor are monitored and a
Lowest Concentration Minimum Reporting Level (LCMRL) of
0.012 to 0.014 μg/L was validated for all compounds.
Chromatography has also been set up to include the degradates
of Dimethenamid, Flufenacet, and Propachlor in future work. The
surrogate and internal standard used for this method were
Dimethachlor ESA and Butachlor ESA respectively.
Despite using a specific MS/MS scan, Alachlor ESA and
Acetochlor ESA are structural isomers (Figure 1) that have
similar product ions of m/z 80 and 121. For this reason,
consistent chromatographic resolution is necessary and was
achieved using a shallow stepping gradient on a Restek Ultra
C18 column (Figure 2).
Results showed consistent performance for both standards and
samples over several days of work. From the product ion spectra
in Figure 1, there are several unique product ions for both
Alachlor ESA, m/z 158, 160, 176, and Acetochlor ESA, m/z 144,
146, 162. These product ions are not as sensitive as m/z 80 and
121, therefore they were not used. As an alternative to the
method described below, using the unique product ions for both
Alachlor ESA and Acetochlor ESA would eliminate the need for
baseline chromatographic separation. It is important to note that
using product ions other than m/z 80 and 121 will result in loss of
sensitivity.
Both matrix effects, such as ion enhancement and ion
suppression, due to high total organic carbon (TOC) were a real
concern during method development. Surrogate recoveries,
matrix spikes, MDLs, and internal standard recoveries were
monitored but did not indicate any interference using the method
below with drinking water.
p1
-EPI (314.05) Charge (+0) FT (400): Exp 2, 15.939 to 16.119 min from Sampl...
Max. 3.0e5 cps.
x 5.0
O
3.0e5
2.5e5
314
O
OH
80
H3C
S
O
N
50 psi, Ion source temperature: 500°C, Collision gas: Medium,
Interface heater: On, Vertical probe position: 2, Horizontal probe
position: 5, Dwell time: 50 ms.
O
2.0e5
160
121
1.5e5
H3C
CH3
1.0e5
5.0e4
176
77
149
0.0
80
100
120
140
160
180
200
220
240
m/z, amu
-EPI (314.05) Charge (+0) FT (400): Exp 2, 16.300 to 16.411 min from Sampl...
260
280
300
320
Max. 3.8e5 cps.
x 5.0
3.8e5
3.5e5
O
OH
S
H3C
3.0e5
O
N
O
121
2.5e5
Table 1. LC gradient
CH3
H3C
2.0e5
1.5e5
314
O
80
144
77
1.0e5
Time (min)
80
100
120
140
160
180
200
m/z, amu
220
240
260
280
300
XIC of -MRM (19 pairs): 356.1/80.0 amu from Sample 6 (50.0 ng/mL Standard) ...
Max. 1.5e5 cps.
19.7
1.5e5
Butachlor ESA
1.4e5
XICof -MRM (19 pairs): 314.0/79.9 amu from Sample 6 (50.0 ng/mL Standard) ...
1.3e5
3.7e4
3.5e4
1.2e5
3.0e4
XICof -MRM (19 pairs): 264.1/146.1 amu from Sample 6 (50.0 ng/mL Standard...
1.0e5
Max. 1.7e4 cps.
6.0e4
Max. 3.7e4 cps.
Alachlor OA
Metolachlor OA
1.0e4
0.0
15.6
15.8
16.0
16.2
16.4
16.6
16.8
17.0
Time, min
17.2
17.4
17.6
17.8
4000.0
2000.0
13.2 13.4 13.6 13.8 14.0 14.2 14.4 14.6 14.8 15.0 15.2 15.4 15.6 15.8 16.0
Time, min
4.0e4
3.0e4
Dimethachlor ESA
(Surrogate)
2.0e4
1.0e4
0.0
2
4
6
8
4.00
250
70
30
10.0
250
70
30
15.0
250
50
50
17.0
250
15
85
18.0
250
15
85
18.1
250
80
20
28.0
250
80
20
2.0e4
1.0e4
0.0
20
16.4
8000.0
5.0e4
80
2.5e4
1.5e4
6000.0
7.0e4
250
16.1
5000.0
8.0e4
0.00
Metolachlor ESA
Acetochlor OA
1.4e4
1.2e4
9.0e4
B (%)
Alachlor ESA
Acetochlor ESA
14.8
1.6e4
A (%)
320
Figure 1. Product Ion spectra of structural isomers Alachlor ESA (top)
and Acetochlor ESA (bottom)
1.7e4
Flow rate (µl/min)
162
5.0e4
1.1e5
An Agilent 1200 HPLC system was used consisting of a binary
pump, autosampler with thermal unit, and column oven.
Chromatographic separation was achieved on a Restek Ultra
C18 3 μm 100x2.1 mm column using mobile phases, A: 5 mM
ammonium acetate, B: methanol with the gradient in Table 1. A
25 μL injection volume was used.
10
12
14
16
Time, min
18
20
22
24
26
Figure 2. Reproducible chromatography was achieved using a gradient
on a Restek Ultra C18 3 μm 100x2.1 mm column. A 50 ng/mL initial
calibration point is shown. Sufficient baseline separation was achieved for
structural isomers Alachlor ESA and Acetochlor ESA with a consistent
resolution factor of 3.5 or greater.
Sample preparation was performed using Solid Phase Extraction
(SPE). Restek Carbora 90, 6.0 mL tube size, SPE cartridges
were used. Cartridges were conditioned with 20 mL of 10 mM
ammonium acetate/methanol and then rinsed with 30 mL
reagent water. Cartridges were not allowed to go dry at any time
during the sample loading process. After conditioning, 250 mL of
sample was prepared by adding 25-30 mg ammonium chloride
and spiked with 5 μL of a 12 μg/mL surrogate standard, mixed,
and then loaded using a vacuum manifold at a flow rate of 1015 mL/min. After loading, each cartridge was rinsed with 5 mL
reagent water and then allowed to dry using nitrogen. Cartridge
elution was performed using 15 mL of 10 mM ammonium
acetate/methanol at gravity flow. The extracts were concentrated
to dryness using a gentle stream of nitrogen in a heated water
bath, 60-70°C. Finally, 1 mL of 5 mM ammonium acetate/reagent
water and 10 μL of a 5 μg/mL internal standard solution were
added and transferred to an autosampler vial.
Experimental
The method uses an AB SCIEX API 3200™ LC/MS/MS system
equipped with Turbo V™ source and Electrospray Ionization
probe. All compounds were detected using negative ionization in
Multiple Reaction Monitoring (MRM) mode using two MRM
transitions for each target compound and surrogate. The
following mass spectrometer conditions were used: Curtain
Gas™ interface: 25 psi, IS voltage: -4500 V, Gas1: 50 psi. Gas2:
Six calibration points (0.5, 1.0, 10.0, 50.0, 100.0, and
125.0 ng/mL) prepared in 5 mM ammonium acetate/reagent
water, were used for the initial calibration curve. A linear fit was
used with 1/x weighting and a correlation coefficient, r, of 0.995
or greater was achieved. All stock and primary dilution standards
were prepared in methanol and stored at 4°C.
p2
Sample Name: "Blank" Sample ID: "" File: "MDL Analysis.wiff"
Peak Name: "Alachlor OA 1" Mass(es): "264.1/160.2 amu"
Comment: "" Annotation: ""
Results and Discussion
30
20
10
0
12.5
13.0
13.5
14.0
14.5 15.0 15.5
Time, min
Sample Name: "Blank" Sample ID: "" File: "MDL Analysis.wiff"
Peak Name: "Acetochlor OA 1" Mass(es): "264.1/146.1 amu"
Comment: "" Annotation: ""
16.5
17.0
14.9
13.5
50
16.0
I n t e n s it y , c p s
40
30
20
10
0
12.5
13.0
13.5
14.0
14.5 15.0 15.5 16.0
Time, min
Sample Name: "Blank" Sample ID: "" File: "MDL Analysis.wiff"
Peak Name: "Alachlor ESA 1" Mass(es): "314.0/79.9 amu"
Comment: "" Annotation: ""
16.5
17.0
16.4
300
200
Analyte
Quantifier MRM
Qualifier MRM
MRM Ratio
Range (±20%)
264 / 160
264 / 158
0.24 – 0.36
Acetochlor OA
264 / 146
264 / 144
0.23 – 0.35
0
15.8 16.1
14.0
14.5
15.0
15.5
16.0 16.5 17.0
Time, min
Sample Name: "Blank" Sample ID: "" File: "MDL Analysis.wiff"
Peak Name: "Metolachlor OA 1" Mass(es): "278.1/206.2 amu"
Comment: "" Annotation: ""
17.5
18.0
18.5
50
I n t e n s it y , c p s
Alachlor OA
100
40
30
Alachlor ESA
314 / 80
314 / 121
0.28 – 0.42
20
10
0
Metolachlor OA
278 / 206
278 / 174
0.10 – 0.15
Acetochlor ESA
314 / 80
314 / 121
0.33 – 0.50
14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5
Time, min
Sample Name: "Blank" Sample ID: "" File: "MDL Analysis.wiff"
Peak Name: "Acetochlor ESA 1" Mass(es): "314.0/79.9 amu"
Comment: "" Annotation: ""
18.0
18.5
I n t e n s it y , c p s
16.4
300
200
Metolachlor ESA
328 / 80
328 / 121
0.32 – 0.48
100
0
14.0
300 / 80
300 / 121
0.39 – 0.59
Butachlor (IS)
356 / 80
–
–
14.5
15.0
15.5
16.0 16.5 17.0 17.5
Time, min
Sample Name: "Blank" Sample ID: "" File: "MDL Analysis.wiff"
Peak Name: "Metolachlor ESA 1" Mass(es): "328.1/79.9 amu"
Comment: "" Annotation: ""
18.0
18.5
I n t e n s it y , c p s
Dimethachlor ESA
(Surrogate)
15.8 16.1
60
40
20
0
14.5
MRM area ratios were used for confirming detection. Each ratio,
displayed in Table 2, was calculated by determining the MRM
ratio of each calibration standard and then taking the average of
all standards. A ±20% range was then applied to each unknown
sample. The Analyst Reporter automatically flagged any
unknown sample with a calculated MRM ratio outside the
established 20% range.
Once method development was completed an Initial
Demonstration of Capability (IDC) was performed. First an initial
demonstration of low system background was run by preparing a
Laboratory Reagent Blank (LRB). For each analyte, detection in
the prepared LRB needed to be < 1/3 of the MRL detection
(Figure 3).
15.0
15.5
16.0
16.5 17.0 17.5
Time, min
18.0
18.5
19.0
14.4
14.5
800
600
400
200
0
12.2 12.5
12.9 13.2
12.5
13.0
15.0 15.3
13.5
16.2
15.8
14.0
14.5 15.0 15.5
Time, min
Sample Name: "MDL 11" Sample ID: "" File: "MDL Analysis.wiff"
Peak Name: "Acetochlor OA 1" Mass(es): "264.1/146.1 amu"
Comment: "" Annotation: ""
16.9
16.0
16.5
17.0
14.8
1000
800
600
400
200
0
14.4
12.5
13.0
13.5
16.0
16.8
14.5 15.0 15.5 16.0
Time, min
Sample Name: "MDL 11" Sample ID: "" File: "MDL Analysis.wiff"
Peak Name: "Alachlor ESA 1" Mass(es): "314.0/79.9 amu"
Comment: "" Annotation: ""
In te n s ity , c p s
Table 2. Detection in Multiple Reaction Monitoring (MRM). Two MRM
transitions were monitored for all target analytes and the surrogate. Only
1 MRM was used to monitor the internal standard (IS).
I n t e n s it y , c p s
Method development was performed using the automated
Quantitative Optimization feature of the Analyst® Software. Each
target compound, surrogate, and internal standard was infused
into the mass spectrometer at a low flow rate of 10 μL/min.
Quantitative Optimization identified the precursor ion, the most
sensitive product ions for each compound, and optimized all
compound dependant parameters automatically. Results are
shown in Table 2.
Sample Name: "MDL 11" Sample ID: "" File: "MDL Analysis.wiff"
Peak Name: "Alachlor OA 1" Mass(es): "264.1/160.2 amu"
Comment: "" Annotation: ""
14.0
16.1
16.5
17.0
16.4
2000
1500
1000
500
16.9 17.0 17.5
0
14.0 14.5 15.0 15.5 16.0 16.5 17.0
Time, min
Sample Name: "MDL 11" Sample ID: "" File: "MDL Analysis.wiff"
Peak Name: "Metolachlor OA 1" Mass(es): "278.1/206.2 amu"
Comment: "" Annotation: ""
17.5
18.0
18.5
16.3
1000
500
0
14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5
Time, min
Sample Name: "MDL 11" Sample ID: "" File: "MDL Analysis.wiff"
Peak Name: "Acetochlor ESA 1" Mass(es): "314.0/79.9 amu"
Comment: "" Annotation: ""
16.1
18.0
18.5
16.4
2000
1500
1000
500
18.8
16.9 17.0 17.5
0
14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5
Time, min
Sample Name: "MDL 11" Sample ID: "" File: "MDL Analysis.wiff"
Peak Name: "Metolachlor ESA 1" Mass(es): "328.1/79.9 amu"
Comment: "" Annotation: ""
18.0
18.5
16.9
1500
1000
500
14.6
0
14.5
16.2 16.5
15.2
15.0
15.5
16.0
17.3 17.7 17.9 18.2
16.5 17.0 17.5
Time, min
18.0
18.6
18.5
18.9 19.3
19.0
Figure 3. Extracted Ion Chromatograms (XIC) for the Laboratory Reagent
Blank (LRB) for all target analytes are displayed in the left column and
the XIC of the proposed Method Reporting Limit (MRL) are in the right
column.
To validate the proposed MRL, seven replicate LRBs were
spiked at a concentration of 0.013 μg/L and processed through
the sample preparation procedure above. All chromatographic
peaks for both quantifier and qualifier MRM transitions required a
signal to noise ratio of at least 3:1. Using the proposed
procedure for calculating an LCMRL in EPA Method 535 a
calculated detection limit of 0.004 μg/L or less was determined
for all analytes (Table 3).
p3
Table 3. Minimum reporting limit confirmation
LCMRL
Standard
Deviation
HRPIR
Lower PIR
Upper PIR
Alachlor OA
0.013
0.28
1.1
72.4
142.3
0.868
0.003
86.8
Acetochlor OA
0.014
0.27
1.1
74.0
141.9
0.843
0.003
84.3
Alachlor ESA
0.013
0.18
0.7
82.1
127.9
0.569
0.002
56.9
Metolachlor OA
0.013
0.21
0.8
75.7
128.2
0.651
0.003
65.1
Acetochlor ESA
0.012
0.29
1.1
62.4
134.7
0.897
0.004
89.7
Metolachlor ESA
0.012
0.18
0.7
76.1
122.5
0.576
0.002
57.6
Analyte
After the MRL was confirmed an Initial Demonstration of
Precision on Accuracy was performed. Four replicate LRBs were
fortified at a concentration of 0.2 μg/L. The Percent Relative
Standard Deviation (%RSD) for all analytes was ≤20% and the
average recovery was within ±30% of the true value. Therefore
the method satisfied the precision and accuracy requirements
(Table 4).
Table 4. Initial demonstration of precision and accuracy
Analyte
Average Recovery (%)
% RSD
Alachlor OA
96.6
8.5
Acetochlor OA
97.0
8.9
Alachlor ESA
92.5
8.6
Metolachlor OA
95.0
8.5
Acetochlor ESA
94.3
8.0
Metolachlor ESA
94.8
8.9
Dimethachlor ESA
(Surrogate)
100.1
9.2
Extract LOD Sample LOD
(µg/L)
(µg/L)
On Column
LOD (fg)
Finally the recoveries of the internal standard and surrogate
were monitored over a period of 48 hours. Samples were
analyzed consecutively over this time and the recovery and
%RSD of Dimethachlor ESA, surrogate, and Butachlor ESA,
internal standard, were calculated. The results, shown in Table
5, for the surrogate indicated that the sample preparation
efficiency is acceptable. In addition, internal standard recoveries
show that the mass spectrometer is maintaining consistent
sensitivity over long analysis times. Most importantly, results of
both QC analytes indicate that there is no ion suppression or
enhancement taking place that may affect the results of the
target analytes.
Table 5. Surrogate and internal standard recoveries were within
acceptable limits of 70-130% and 50-150% respectively.
Spike Level
(µg/L)
Average
Recovery (%)
% RSD
Dimethachlor
(ESA (Surrogate)
0.24
97.3
12.7
Butachlor ESA
0.20
87.4
23.4
Analyte
p4
Conclusion
Reference
A method following US EPA guidelines for Method 535 has been
presented. This method shows superior sensitivity for
ethanesulfonic acid and oxanilic acid degradates of
chloroacetanilide herbicides Alachlor, Acetochlor, and
Metolachlor using SPE and LC/MS/MS with an API 3200™
system. A Lowest Concentration Minimum Reporting Level
(LCMRL) of 0.012 to 0.014 μg/L was verified with a calculated
detection limit of 0.004 μg/L or less. Two MRM transitions were
used for both quantitation and confirmation of target analytes.
Surrogate and internal standard recoveries indicate that there is
no matrix interference. This method is easily transferable to any
AB SCIEX LC/MS/MS system and is available upon request.
1
U.S. Environmental Protection Agency Method 535:
“Measurement of Chloroacetanilide and other Acetamide
Herbicide Degradates in Drinking Water by Solid Phase
Extraction and Liquid Chromatography / Tandem Mass
Spectrometry (LC/MS/MS)” Version 1.1 (April 2005) J.A.
Shoemaker, M.V. Bassett
Acknowledgements
The authors would like to thank Dr. Paul Winkler at GEL
Analytics in Golden, Colorado for providing sample preparation
materials, Dr. Ali Haghani of MWH laboratories for providing
standards of all compounds for this study and Restek
Corporation for supplying the HPLC column and SPE cartridges.
For Research Use Only. Not for use in diagnostic procedures.
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