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One Stop Shop for EPA Method 535

Reliably Analyze Acetamide Herbicide Degradates by LC/MS/MS


By Jason Thomas, Innovations Chemist, Julie Kowalski, Innovations Chemist, Christopher Borton*, Field Application Specialist
  • Full package: reference standards, SPE cartridges, and HPLC columns.
  • Chromatographic resolution of alachlor ESA and acetochlor ESA isomers.
  • Meet method requirements, with superior sensitivity.

Acetamide herbicides are used in large quantities to suppress weed growth in corn and soybean fields. However, due to the polar nature of ethanesulfonic acid (ESA) and oxanilic acid (OA) degradation products, contamination of drinking water sources is a concern. EPA Method 535 is designed to monitor drinking water for ESA and OA breakdown products of these herbicides. Chromatographic analysis is extremely important for this method because two analytes, alachlor ESA and acetochlor ESA, are isomers that share the same mass spectral multiple reaction monitoring (MRM) transitions, and thus must be separated chromatographically.

Resolution of all Method 535 analytes, including alachlor ESA and acetochlor ESA isomers, can easily be accomplished using Restek's full line of Method 535 products, which includes reference standards, solid phase extraction (SPE) cartridges, and HPLC columns that meet method guidelines. In the procedure shown here, 6mL CarboPrep 90 SPE cartridges were used for sample preparation, both to help extend the lifetime of the analytical column as well as to prevent matrix enhancement or suppression. LC/MS/MS analysis was performed on an Ultra C18 column coupled to an Applied Biosystems API 3200 LC/MS/MS system equipped with a TurboIonSpray probe for the Turbo V source.

Resolution of all target analytes, including alachlor ESA and acetochlor ESA isomers, can easily be achieved.

Consistent chromatographic resolution of 3.5 or greater for alachlor ESA and acetochlor ESA was easily achieved as shown in Figure 1. Surrogate recoveries, matrix spikes, minimum detection limits, and internal standard recoveries produced consistently acceptable results at low concentrations and showed no interferences from the drinking water matrix. The method reporting limits (MRL) listed in Table I are based on seven replicate fortified blanks prepared at the proposed MRL of 0.013µg/L in drinking water. An LCMRL of 0.012 to 0.014µg/L was established and validated with a calculated detection limit of 0.004µg/L or less. Precision and accuracy were demonstrated using four replicate fortified blanks at 0.2µg/L; recovery and RSD values easily met method requirements (Table II). All analytes were detected in laboratory blanks at ≤ 1/3 MRL values, demonstrating low system background levels.

The optimized method developed here shows superior sensitivity for the ESA and OA degradates of chloroacetanilide herbicides alachlor, acetochlor, and metolachlor, as well as reliable resolution between isomers alachlor ESA and acetochlor ESA. This method is simplified by Restek's suite of Method 535 products. All of the reference materials, sample preparation products, and HPLC columns needed are now available from a single source, to facilitate successful Method 535 analysis.

*Applied Biosystems

Figure 1  Easily resolve Method 535 herbicide degradates on an Ultra C18 HPLC column.

PeakstR (min)Quantifier MRMQualifier MRM
1.Dimethachlor ESA7.9300/80300/121
2.Alachlor OA14.5264/160264/158
3.Acetochlor OA14.8264/146264/144
4.Alachlor ESA16.1314/80314/121
5.Metolachlor OA16.4278/206278/174
6.Acetochlor ESA16.4314/80314/121
7.Metolachlor ESA16.9328/80328/121
8.Butachlor ESA19.7356/80356/121
Easily Resolve Method 535 Herbicide Degradates on an Ultra C18 Column
LC_EV0477
ColumnUltra C18 (cat.# 9174312)
Dimensions:100 mm x 2.1 mm ID
Particle Size:3 µm
Pore Size:100 Å
Temp.:ambient
Sampleacetamide herbicides
Diluent:5mM ammonium acetate in water
Conc.:50 ng/mL
Inj. Vol.:25 µL
Mobile Phase
A:5mM ammonium acetate
B:methanol
Time (min)%B
0.0020
4.030
10.030
15.050
17.085
18.085
18.120
28.020
Flow:0.250 mL/min
DetectorAPI 3200 LC/MS/MS system
Ion Source:TurboIonSpray
Ion Mode:ESI-
Ion Spray Voltage:-4.5 kV
Curtain Gas:25 psi (172.4 kPa)
Gas 1:50 psi (344.7 kPa)
Gas 2:50 psi (344.7 kPa)
Source Temp.:500 °C
Dwell Time:50 ms
InstrumentApplied Biosystems/MDS Sciex LC-MS/MS System
NotesCollision gas: medium/5
Interface heater: on
Vertical probe position: 2

Table I  Reliably achieve detection limits of 0.004µg/L or less.

Analyte

LCMRL
(µg/L)

Standard Deviation

Calculated Detection
Limit in Matrix (µg/L)

alachlor OA 0.013 0.28 0.003
acetochlor OA 0.014 0.27 0.003
alachlor ESA 0.013 0.18 0.002
metolachlor OA 0.013 0.21 0.003
acetochlor ESA 0.012 0.29 0.004
metolachlor ESA 0.012 0.18 0.002

Seven matrix spikes prepared at 0.013µg/L (proposed MRL).

Table II  Outstanding accuracy and precision using Ultra C18 HPLC columns.

Analyte

Average Recovery (%)

%RSD

dimethachlor ESA 100.19.2
metolachlor OA 95.0 8.5
metolachlor ESA 94.8 8.9
alachlor OA 96.6 8.5
acetochlor OA 97.0 8.9
alachlor ESA 92.5 8.6
acetochlor ESA 94.3 8.0

Four lab fortified blanks spiked at 0.2µg/L.
Method requirements: average recovery ±30% of the true value, %RSD ≤20%.

References

*C. Borton. EPA Method 535; Detection of Degradates of Chloroacetanides and other Acetamide Herbicides in Water by LC/MS/MS. Applied Biosystems, Foster City, CA, 2008.

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