Analysis of Chloroacetanilide and Acetamide Herbicide Degradates in Drinking Water by UPLC-MS/MS

This application note demonstrates the analysis of chloroacetanilide and acetamide herbicide degradates in drinking water by UPLC-MS/MS.

Chloroacetanillide and acetamide herbicides are widely used in the U.S. for the control of broadleaf and annual weeds on crops such as corn and soybeans.¹ These herbicides are thought to be degraded by microbial action to the oxanilic acid (OA) and ethanesulfonic acid (ESA) metabolites of the parent compounds. The OA and ESA metabolites are more water soluble and therefore more mobile than the parent herbicides. Several studies by the United States Geological Survey (USGS) and others have reported higher occurrences of the OA and ESA metabolites than the parent herbicides in both ground and surface water.^2,3 The health effects of these metabolites is unknown and limits in drinking water have not been established.

U.S. EPA Method 535 was developed for the determination of OA and ESA metabolites of alachlor, acetochlor, metolachlor, propachlor, flufenacet and dimetheamid in drinking water.⁴

Six of the metabolites analyzed by EPA method 535 are among the 26 contaminants proposed to be regulated in second round of the Unregulated Contaminant Monitoring Rule (UCMR2). Alachlor OA, Alachlor ESA, Acetochlor OA, Acetochlor ESA, Metolachlor ESA and Metolachlor OA are on the screening survey list. Contaminants on the screening survey list will be monitored by approximately 1,100 public water systems (PWSs) during a 12 month period between July 2007 and June 2010. The results obtained from UCMR2 monitoring will be used to determine the occurrence of contaminants in drinking water, the population being exposed to these contaminants and the level of exposure. When the study is completed in 2010, this information can then be used by the EPA to determine if these contaminants should be regulated in the future.⁵

U.S. EPA Method 535 Description

A 250 mL water sample is extracted using a graphitized carbon SPE cartridge, dried and made up to 1 mL with 5 mM ammonium acetate. The samples are injected on a C₁₈ column at 65 °C and analyzed by LC-MS/MS in negative ion mode. The analyte concentrations are determined by use of a chromatographic internal standard.

The HPLC-tandem quadrupole MS method described in EPA method 535 has a 35 minute runtime and the HPLC-ion trap MS method has a 60 minute runtime. Two of the analytes, Alachlor ESA and Acetochlor ESA are structural isomers and are monitored using the same MRM transition (314 > 80). Therefore, chromatographic separation is required. The method states that these analytes must be separated chromatographically with a resolution (Rs) of 1.0 or greater.

This note describes an UltraPerformance Liquid Chromatography (UPLC) and tandem mass spectrometry (MS/MS) method for the analysis. The advantages of using UPLC rather than HPLC include a 2.5 to 4x increase in throughput due to decreased runtimes, improved resolution of Alachlor ESA and Acetachlor ESA and increased sensitivity of 2 to 4x.

Software

Data was acquired with Waters MassLynx Software and processed with Waters QuanLynx Application Manager.

Figure 1 shows the chromatogram obtained using a traditional HPLC-MS/MS system and the gradient specified in the EPA 535 method. The first eluting compound is Propachlor OA with a retention time of 9 minutes. The internal standard, Butachlor ESA elutes last at 30 minutes. The total runtime for the HPLC method is 35 minutes and run times of up to 60 minutes using the ion trap MS instrument are reported in the EPA method. Figure 2 illustrates the improved chromatography with respect to run time when the analysis is carried out by UPLC-MS/MS. The total runtime is shortened to 14 minutes.

Crucial to this method is the chromatographic separation of the structural isomers, Alachlor ESA and Acetochlor ESA. Figure 3 shows the spectra of these compounds. The two most intense product ions for both compounds are m/z 80 and m/z 121. Dissimilar product ions m/z 176 for Alachlor ESA and m/z 146 for Acetochlor ESA are approximately 15% the intensity of the product ion at m/z 80. Therefore, the use of these dissimilar product ions would greatly reduce the sensitivity if used for quantitation.

Even though the UPLC-MS/MS method results in 2.5x increase in throughput, resolution is not compromised. In fact, the use of 1.7 μm particles in UPLC columns results in higher efficiencies. Figure 4 shows the resolution of Alachlor ESA and Acetochlor ESA on both UPLC-MS/MS and HPLC-MS/MS. The resolution obtained on the UPLC-MS/MS is superior to that obtained on the HPLC-MS/MS system and well surpasses the method requirement of Rs > 1 for these compounds.

Improvements of 2 to 4x in sensitivity were achieved using UPLC versus HPLC. Table 2 lists the signal to noise values obtained for a 0.05 μg/L standard of the six herbicide metabolites proposed to be monitored under UCMR2.

The QuanLynx Application Manager was used to construct calibration curves using the internal standard technique. The calibration curves were linear with r² >0.995 in the range 0.05–1.0 μg/L. This satisfies the EPA method requirement of five calibration concentrations spanning a 20-fold concentration range. The calibration curve obtained for Acetochlor OA is shown in Figure 5.

The use of UPLC-MS/MS for the analysis of the herbicide metabolites in EPA method 535 has many benefits over traditional HPLC-MS/MS. The advantages include a 2.5 to 4x reduction in total runtime, improved resolution for the structural isomers Acetochlor ESA and Alachlor ESA and a 2 to 4x increase in sensitivity.

1. Ferrer, I.; Thurman, E.M.; Barcelo, D.; Anal. Chem. 1997, 69, 4547–4553.

2. Phillips, P.J.; Eckhardt, D.A.; Rosenmann, L.; 2000, Pesticides and Their Metabolites in Three Public Water-Supply Reservoir Systems, Western New York, 1998-99: U.S. Geological Survey Open-File Report 99–4278.

3. Koplin, D.W.; Thurman, A; Goolsby, D.A.; Environ. Sci. Technol. 1996, 30, 335–340.

4. EPA Document #: EPA/600/R-05/053

5. http://www.epa.gov/safewater/ucmr/ucmr2/