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Acetanilide Herbicide Degradation Products By LC/MS

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Abstract: Acetanilide herbicides are frequently applied in the U.S. on crops (corn, soybeans, popcorn, etc.) to control broadleaf and annual weeds. The acetanilide and acetamide herbicides currently registered for use in the U.S. are alachlor, acetochlor, metolachlor, propachlor, flufenacet and dimethenamid. Acetanilide degradation products are generally more water soluble and mobile than the parent herbicide, thus there is greater potential for these degradates to be found in ground waters and surface waters. The most common acetanilide degradation products are the ethanesulfonic acid (ESA) and oxanilic acid (OA) derivatives of the parent herbicides. The ESA and OA degradates of alachlor, metolachlor, and acetochlor have been reported in U.S. Midwestern surface and ground waters at typical concentrations of 0.1- 20 �g/L [1-5].

High priority has been given to this research project in EPA's Office of Research and Development because acetanilide degradation products are identified on the 1998 Drinking Water Contaminant Candidate List (CCL) [6]. Specifically, analytical methodology is needed to gather occurrence data on the acetanilide degradation products. While several methods have been reported in the literature [3,7], these methods do not address issues specific to analyzing compounds in drinking water, such as preservatives and internal and surrogate standards. In addition, the reported methods do not contain all 12 target analytes (the ESA and OA of the six U.S. registered acetanilide/acetamide herbicides). The objective of this research is to develop an accurate and precise analytical method to detect and quantitate the 12 ESA and OA acetanilide pesticide degradates in drinking water matrices. This will include two steps: 1) evaluating the capability of solid phase extraction (SPE) techniques to concentrate the acetanilide ESA and OA degradates from drinking water, and 2) evaluating the capability of liquid chromatography/mass spectrometry (LC/MS) techniques to separate and detect ESA and OA acetanilide herbicide degradates in the concentrated sample extracts. Future occurrence data gathered with this developed method can then be used in determining whether to study the health effects of the acetanilide herbicide degradation products, and ultimately whether to regulate these compounds or remove them from the CCL.

Approach

LC/MS: Since the EPA methodology developed will be used to determine the occurrence of acetanilide degradates in drinking water, positive confirmation by LC/MS with negative ion electrospray ionization (ESI) is desired. However, two structural isomer pairs, alachlor ESA/alachlor ESA and alachlor OA/acetochlor OA present a difficult challenge. Not only are alachlor ESA and acetochlor ESA not separated on conventional LC columns, they also have the same molecular weight. LC/MS typically only provides a protonated molecule [M+H]+ or deprotonated molecule [M-H]-; therefore, alachlor ESA and acetochlor ESA would be indistinguishable by LC/MS. Alachlor OA and acetochlor OA would also be indistinguishable by LC/MS (without fragmentation) for the same reason. In-source CAD (collisionally activated dissociation) on LC/MS instruments or tandem mass spectrometry (MS/MS) has been used to produce unique product ions for the ESA and OA degradates of alachlor and acetochlor [7,8]. However, the in-source CAD and MS/MS fragment ions unique to acetochlor ESA and alachlor ESA are not very abundant, thus compromising sensitivity. To overcome this problem, chromatographic approaches, such as mobile phase modifiers and column heating, were investigated to achieve separation of the structural isomers.

SPE: Another challenge in methods development is the ability to concentrate the target analytes from water using a suitable SPE sorbent. The goal is to find an SPE procedure that, combined with LC/MS analysis, will produce a method that will meet our data quality objectives of 70-130% mean recovery (% of true value) and <30% relative standard deviation (RSD). Polystyrenedivinylbenzene or C18 SPE sorbents are most commonly used to extract the ESA and OA degradates of acetochlor, alachlor, metolachlor and dimethenamid ESA from water [2,7,9]. However, there were no literature reports indicating recoveries of propachlor or flufenacet degradates. Thus, experiments were conducted on all 12 target analytes to determine recoveries on C18 SPE cartridges using a procedure similar to Thurman [2], as well as on other types of SPE sorbents, such as C18OH and carbon.

Internal and Surrogate Standards: Internal standards (IS) and surrogates (SURR) are an important part of MS methods development research in order to achieve the highest quality data. Ideally, the IS and SURR should be similar in properties and structure to the target analytes and perform similarly in the extraction and detection as the target analytes. Unfortunately, obtaining an IS with these similarities is not always achievable. A number of acids were evaluated for use as potential internal standards and surrogates, but many were not chromatographically suitable or did not mimic the target analytes in the extraction process.

Preservatives: EPA drinking water regulatory methods typically use preservatives to prevent microbial degradation (e.g., acid, copper(II) sulfate, DZU, trinitro) and to dechlorinate (e.g., sodium sulfite, trizma) the sample. Microbial degradation of the target analytes cannot be predicted in all types of matrices containing various types of microbiological contaminants, thus an anti-microbial agent is desirable. While chlorine may not adversely affect the stability of acetanilide degradates, it can interfere in the solid phase extraction, thus the residual chlorine should be removed. A number of preservative combinations were investigated based on research conducted by Winslow and colleagues [10] (e.g., copper(II) sulfate/trizma, DZU/trizma, trisnitro/trizma, hydrochloric acid (pH=2)/sodium sulfite).

Results

The methods development for these acetanilide ESA and OA degradates is nearing completion. Chromatographic separation of alachlor ESA and acetachlor ESA has been achieved using a 5 mM ammonium acetate/methanol gradient and heating the analytical column to 65�5�C [11]. SPE extraction of the target analytes from drinking water using carbon sorbents produced typical recoveries of 85-115% and relative standard deviations <12%. Studies were conducted to determine the effect of water quality parameters, such as humic/fulvic material and hardness, on the recovery of these analytes. Humic/fulvic material, measured as total organic carbon (TOC), is retained on the carbon sorbent causing low recoveries (<70%) for propachlor OA at TOC levels above 5 mg/L. Many of the preservative combinations tested interfered in the extraction process. Ammonium chloride is currently being evaluated as a potential dechlorination agent, but no anti-microbial agent, which does not affect the analyte recoveries, has been found. Holding time studies will be performed to ensure the target analytes will survive typical shipping conditions and times with the chosen preservative. Currently, 4-phenoxybenzoic acid (PBA) is the internal standard and butachlor ESA and dimethachlor ESA are the surrogate standards. The internal calibration using PBA is linear with r2>0.995 for all target analytes. Butachlor and dimethachlor are not registered for use in the U.S., thus the potential for environmental contamination of their ESA degradates is minimal. Finally, the minimum reporting level (MRL) will be determined and is expected to be = 0.1 �g/L.

Disclaimer: This work has been conducted by the United States Environmental Protection Agency. It has been subjected to Agency review and approved for publication. Mention of trade names or commercial products does not constitute endorsement of recommendation for use.
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Citation:Shoemaker, J. A. Acetanilide Herbicide Degradation Products By LC/MS. Presented at American Water Works Association Water Quality Technology Conference, Philadelphia, PA, November 2-6, 2003.
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Contact: Mary P. O'Bryant - (919)-541-4871 or obriant.mary@epa.gov
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Division: Microbiological & Chemical Exposure Assessment Division
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Branch: Chemical Exposure Research Branch
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Product Type: Abstrct/Oral
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Presented: 11/02/2003
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Related Entries:
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Bullet Item Analytical Method Development for Alachlor Esa and Other Acetanilide Pesticide Transformation Products
spacer Relationship Reason:   A Project of the Product
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Last Updated on Monday, October 22, 2007
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