Determination of Acetanilide Degradates in Ground and Surface

Chapter 15

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Determination of Acetanilide Degradates in Ground and Surface Waters by Direct Aqueous Injection LC/MS/MS John D. Fuhrman and J. Mark Allan Environmental Sciences Technology Center, Monsanto Company, 800 North Lindbergh Boulevard, St. Louis, M O 63167

Acetanilide herbicides are widely used throughout the United States to control annual grasses in corn, soybeans and other crops. The herbicides studied, include the acetanilides; acetochlor, alachlor, metolachlor, propachlor and the acetamide; dimethenamid. The biological degradation of these herbicides produces a myriad of polar metabolites the most prominent of which are the ethanesulfonic acids (ESA) and oxanilic acids (OX) moieties. These degradates have been found in surface waters from agricultural runoff and in shallow groundwater due to leaching through vulnerable soils. Several less significant degradates have recently been identified and incorporated into a single multi-residue methodology. A high throughput method has been developed for the analysis of fourteen soil degradates of the specified herbicides. The method uses direct aqueous injection (DAI) liquid chromatography / mass spectrometry / mass spectrometry (LC/MS/MS) to analyze these materials without any sample pretreatment or concentration. MS/MS provides a very specific and highly sensitive mode of detection using TurboIonSpray in the negative ion mode. Thirteen of the fourteen degradates were validated at the 0.05 μg/L level in three different water matrices, raw surface water, finished surface water (drinking water) and groundwater.

256

© 2003 American Chemical Society

In Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS; Ferrer, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

257


Introduction Acetochlor, alachlor, and propachlor are soil-applied herbicides manufactured by Monsanto for pre-emergence and early post-emergence control of annual grasses and broadleaf weeds in crops. These chloroacetanilides degrade readily and extensively in soil, mainly through displacement of chlorine followed by further metabolism to numerous degradation products (1-3). In aerobic soil studies conducted with alachlor, acetochlor, and propachlor, the most abundant metabolites have been identified as the water-soluble oxanilic, sulfonic, and sulfinylacetic acids. Public environmental concerns and government regulatory requirements continue to prompt the need for reliable methods to determine residues of these herbicide metabolites. The objective of this study was to develop and validate a multi-residue confirmatory method for the major soil dégradâtes of acetochlor, alachlor, metolachlor, propachlor, and dimethenamid in water from ground and surface water sources. This multi-residue method includes the major chloroacetanilide and chloroacetamide soil dégradâtes and thereby ensures accurate mass spectral resolution, identification and quantification of these dégradâtes. The development of methods for chloroacetanilide soil dégradâtes is challenging due to the separation and detection of numerous analytes of similar chemical structure. Additionally, dégradâtes exist as rotational isomers due to restricted rotation at the amide bond or the bond to the aromatic ring, when the ring is asymmetrically substituted (4). These rotamers, due to their restricted rotation about the amide bond, generally interconvert rapidly, but in some cases separate into two distinct peaks during HPLC analysis. Several methods have been developed for the analysis of the most common chloroacetanilide dégradâtes, the ethanesulfonic and oxanilic acids of acetochlor, alachlor and metolachlor, in environmental waters (5-8). This methodology has expanded the number of analytes to fourteen and includes other known dégradâtes such as the sulfinylacetic acids, alachlor secondary oxanilic acid and dégradâtes from the herbicides dimethenamid and propachlor. The method presented here was designed for rapid analysis of water samples by direct aqueous injection reversed-phase liquid chromatography tandem mass spectrometry (LC/MS/MS). No pretreatment or concentration of the sample is necessary prior to analysis. The method may be used to analyze for a single degradate or any combination of the fourteen dégradâtes. The acetanilide degradate structures are presented in Figure 1 and the acetamide degradate structures are in Figure 2.


' CH20CH2CH3 Ν

COCH S0 H


2

3

CH2CH3

Acetochlor - ESA

CH2OCH3

OCH S0 H

COCH SOCH COOH 2

2

2

3

CH2CH3

Alachlor - ESA CH2CH3

DH CH3 2

CH OCH 2

H

3

Ν

\ j T \

COCOOH

^COCOOH

CH2CH3

CH2CH3

Alachlor-OX

Alachlor - sOX

.CH2CH3 CH2OCH3

CHCH3CH2OCH3

Ν N

COCH S0 H

\:OCH SOCH COOH 2

CH2CH3

Alachlor-S A A

2

2

3

CH2CH3 Metolachlor-ESA

Figure 1: Acetanilide Degradate Structures



Propachlor - ESA

Propachlor - S A A

Figure 1: Acetanilide Degradate Structures (cont)

Dimethenamid - ESA

Dimethenamid - OX

Figure 2. Acetamide Degradate Structures


260

Methods

Reagents Acetonitrile, methanol and reagent water were Optima® grade from Fisher Scientific. Absolute ethanol was obtained from Aaper Alcohol and Chemical Company. Glacial acetic acid was obtained from J.T. Baker. Water for mobile phase preparation was from a Milli-Q water purification system.

Reference Standards A l l fourteen acetanilide and acetamide dégradâtes used in this method were obtained from the Standards Reference Officer at Monsanto Company (Saint Louis, Missouri, USA). All compounds were either synthesized internally or custom synthesized by external contractors. A l l dégradâtes were used as received without further purification and were certified and issued as their respective sodium salts. Purity of the reference compounds should generally be greater than 95%, but in no instance less than 90%. All degradate solution concentrations were prepared purity corrected as the free acid. A list of the reference compounds used in this study is in Table I.

Stock Solutions Stock solutions of the individual dégradâtes were prepared in absolute ethanol at nominal concentrations of 1000 μg/mL (weight adjusted for purity of the free acid). Mixed stock solutions for the fourteen dégradâtes were prepared to facilitate calibration and fortification. A mixed degradate stock solution at 1.0 μg/mL was prepared from the individual stock solutions. From this mixed stock solution serial dilutions at 100.0 μg/L and 10.0 μg/L were prepared in absolute ethanol. Working solutions were prepared in reagent water in order to fortify control matrices to determine analytical accuracy and to calibrate the response of the analyte in the mass spectrometer. All standard solutions (stock, fortified, and calibration) were stored refrigerated (2-10°C) in clean amber glass bottles with Teflon-lined screw caps. The stock solutions are adequate to prepare the fortification and calibration standards in the range of 0.010-20.0 μg/L of each analyte.

261

Table I: Reference Compounds


Degradate Name - TUPAC and Common [(ethox3miethyl)(2-ethyl-6-methylphenyl) amino]-2oxoacetic acid, (acetochlor oxanilic acid) 2-[(ethoxymethyl)(2-ethyl-6-methylphenyl)amino]2-oxoethanesulfonic acid, (acetochlor sulfonic acid) {2-[(ethoxymethyl)(2-ethyl-6methylphenyl)armno]-2-oxoethylsulfinylacetic acid, (acetochlor sulfinyl-acetic acid) [(2,6-diethylphenyl)(methoxymethyl)amino]oxoacetic acid, (alachlor oxanilic acid) 2-[(2,6-diethylphenyl)(methox)miethyl)amiûo]-2oxoethanesulfonic acid, (alachlor sulfonic acid) (2,6-diethylphenyl)amino-2-oxoacetic acid, (alachlor sec-oxanilic acid) {2-[(methoxymethyl)(2,6-diethylphenyl)amino]-2oxoethyl} sulfinylacetic acid, (alachlor sulfinylacetic acid) [(2,4-dimethyl-3-thienyl)(2-methoxy-lmethylethyl)amino]oxoacetate, (dimethenamid oxanilic acid) 2-[(2,4-dimethyl-3-thienyl)(2-methoxy-lmethylethyl)amino]-2-oxoethanesulfonate, (dimethenamid sulfonic acid) 2-[(2-ethyl-6-methylphenyl)-(2-methoxy-1 methylethyl)amino]-2-oxoacetic acid, (metolachlor oxanilic acid) 2- [(2-ethyl-6-methylphenyl)(2-methoxy-1 methylethyl)amino]-2-oxoethanesulfonic acid, (metolachlor sulfonic acid) [( 1 -methylethyl)phenylamino]oxoacetic acid, (propachlor oxanilic acid) 2-[( 1 -methylethyl)phenylamino]-2-oxoethane sulfonic acid, (propachlor sulfonic acid) [[(methylethyl) phenylamino] acetyl] sulfinylacetic acid, (propachlor sulfinylacetic acid)

AcOX

CAS Registry No. 194992-44-4

AcESA

187022-11-3

AcSAA

NA

AlOX

140939-14-6

A1ESA

140939-15-7

Al-sOX

NA

A1SAA

NA

DimOX

NA

DimESA

205939-58-8

MeOX

152019-73-3

MeESA

171118-09-5

ProOX

NA

ProESA

NA

ProSAA

NA

Acronym

(NA = not available)


262


Fortification Solutions In order to estimate the analytical accuracy of the method within a given set of water samples, it was necessary to fortify control water samples with known amounts of each degradate. Fortification solutions were prepared by serial dilution from the mixed stock solutions into the appropriate control water. The fortification samples were prepared at 0.025, 0.05, 0.10, 0.25, 0.50, 1.0 and 2.0 μg/L by serial dilution from the mixed stock solutions.

Calibration Standards The calibration standards were prepared at 0.010, 0.025, 0.05, 0.10, 0.25, 0.50, 1.0, 2.0 and 5.0 μg/L by serial dilution from the mixed stock solutions. Fisher Optima® grade reagent water was used as the matrix for all calibration standards.

Instrumentation An LC/MS/MS was used for separation and quantitation of dégradâtes. Using multiple reaction monitoring (MRM), in the negative ion electrospray ionization mode (-ESI) the LC/MS/MS gives superior specificity and sensitivity when compared to conventional LC/MS techniques. The improved specificity eliminates interferences typically found in LC/MS or LC/UV (ultraviolet detection) analyses. The Sciex Analyst software, ν ΐ . ΐ β , provided complete control of the mass spectrometer as well as data acquisition and processing. Two ions were monitored for each degradate, one transition (deprotonated molecule) ion and one quantitation (fragment) ion. The transition and quantitation ions for the dégradâtes are listed in Table II.

HPLC Description and Conditions The dégradâtes were chromatographed on a Zorbax StableBond C column, 50 mm χ 4.6 mm χ 3.5 μ, in combination with a Zorbax StableBond C guard column, 12.5 mm χ 4.6 mm χ 5 μ. The liquid chromatograph was a Hewlett Packard 1100 system, including a binary pump, degasser, column heater and autosampler. The column was maintained at 70°C to minimize or eliminate chromatographic separation of the rotational isomers. A solvent gradient was 8

8


263 Table II. Multiple Reaction Monitoring Ion Selection


Degradate acetochlor oxanilic acid acetochlor ethanesulfonic acid acetochlor sulfinylacetic acid alachlor oxanilic acid alachlor s-oxanilic acid alachlor ethanesulfonic acid alachlor sulfinylacetic acid dimethenamid oxanilic acid dimethenamid ethanesulfonic acid metolachlor oxanilic acid metolachlor ethanesulfonic acid propachlor oxanilic acid propachlor ethanesulfonic acid propachlor sulfinylacetic acid

MRM Transition Ion (Daltons) 264 314 340 264 220 314 340 270 320 278 328 206 256 282

Quantitation Ion (Daltons) 146 162 146 160 148 176 160 198 121 206 121 134 121 134

used comprising a mixture of mobile phase A: 95:5 water: methanol (with 0.2% acetic acid) and mobile phase B: 50:50 acetonitrile: methanol (with 0.2% acetic acid). Initial conditions were 95:5 A:B, to 50:50 A:B at 3 minutes, to 30:70 A:B at 6.5 minutes and hold to 7.5 minutes. Re-equilibration to 95:5 required an additional 2.5 minutes. The flow rate was 700 μ ι / minute and the column effluent was split approximately 14:1 at the ion source (-50 \£L / minute of flow to the ion source). The injection volume was 100 μΐ.. To minimize source contamination the column flow was diverted to waste for 3.5 minutes following injection.

Mass Spectrometer Description and Conditions An Applied BioSystems ΡΕ Sciex API-3000 tandem mass spectrometer using Analyst software, ν 1.1 β was used in this study. The API-3000 was coupled to the HP 1100 LC system through a TurboIonSpray ion source. A Valco, Model EHMA, electrically actuated 6 port switching valve was used to divert the column flow from the ion source prior to and following elution of the analytes of interest. The MS was operated in the negative ion mode with a



264 TurboIonSpray voltage of 4.2 kilovolts. The TurboIonSpray gas flow was 6 L/minute (nitrogen) at a temperature of 350°C. Nitrogen was used for both the curtain and nebulizer gases. From injection to injection the total run time was 10 minutes. The M R M experiments did not require chromatographic separation of the dégradâtes. Compounds were identified based on their molecular weight and specific quantitation ion. Therefore, other L C conditions, columns, gradient, and injection volumes may be used provided there is adequate sensitivity, specificity and the chromatographic quality is not compromised.

Sample Preparation Sample preparation was not necessary for direct aqueous injection. The samples were transferred directly to 2 mL autosampler vials for analysis. No preconcentration, sample cleanup or (in most cases)filtrationwas required prior to analysis.

Detector Calibration A calibration curve was generated for every set of samples. The standards were placed among the analytical samples for each set. The first and last sample in each analytical sample set was a standard. The calibration curve was generated by plotting the response of each analyte in a calibration standard against its concentration. Least squares estimates of the data points was used to define the calibration curve. Linear, exponential, or quadratic calibration curves may be used. An example calibration curve for propachlor oxanilic acid, a 1/x weighted linear fit of response versus concentration, is in Figure 3.

Quantitation Criteria Each individual standard, control and environmental sample was analyzed one time only. Analyte calibration was performed for each chromatographic set using a multi-point calibration curve. The complete chromatographic set containing calibration standards, control, fortified control, and fortified field samples was arranged such that the set began and ended with a calibration standard (i.e., control, fortified, and fortified field samples are bracketed by calibration standards) with the standards evenly distributed throughout the set. The minimum correlation coefficient for the linearity of the analyte calibration


265 curve is 0.99. The average analytical recovery of fortified samples for each analyte in each set should range between 70-110% of the amount fortified. Each chromatographic set shall contain at least one laboratory control sample and one fortified laboratory control sample.


ProOx : "Linear" Regression ("1 / x" weighting): y = 1.74e+005 χ + -288 (r = 0.9999)

Conœntration, ng/mL

Figure 3. Propachlor Oxanilic Acid Calibration Curve

Results and Discussion During this study, control finished surface water, raw surface water, and ground water samples were fortified with known concentrations of the fourteen dégradâtes, at 0.05 μg/L, 0.1 μg/L, 0.25 μg/L, 0.50 μg/L, and 1.0 μ ^ , and carried through the analytical procedure. Results confirm that reliable quantitation in these water matrices can be achieved by direct aqueous injection LC/MS/MS at these low part per billion levels. Five replicates each, in ground water, raw surface water and finished surface water, were analyzed by the method described herein. No significant



Time, min

4.8

5.0

5.2 5.4 5 Ï ' 5 . 8 Time, min

8.Ô

6.2

6^

5.Ô

Ô.2 6.4 51 Time, min

Γ8

0$

1000

2000

5 2~ 5.4 5$~ Time, min

~TS

Ο

Sample Name: "FWval08081 23" Sample ID: "std 0.05 reag w" Peak Name: "AI-sOx" Massles): "220.0/148.0 amu" Comment: "01-27-R-1" Annotation: "Finished Surface Water" 4.60 9000

4.8

Sample Name: "FWval08081 23" Sample ID: "std 0.05 reag w" Peak Name: "AcOx" Mass(ês): "264.0/146.0 amu" Comment: "01-27-R-1" Annotation: "Finished Surface Water"

6.2 ~6.4

6.2 6.4

Figure 4: Example Ion Chromatograms - 0.05 μg/L Standard Continued on next page.

4.6

Sample Name: "FWval08081 23" Sample ID: "std 0.05 reag w" Peak Name: "AcSAA" Masses): "340.0/146.0 amu" Comment: "01-27-R-1" Annotation: "Finished Surface Water" 4.85

4.r 5.6

Sample Name: TWval08081 23" Sample ID: "std 0.05 reag w** Peak Name: "AcESA" Masses): "314.0/162.0 amu" Comment: "01-27-R-1 " Annotation: "Finished Surface Water"


In Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS; Ferrer, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003. Time, min

Sample Name: "FWva108081 23" Sample ID: "std 0.05 reag w" Peak Name: "DimESA" Mass(es): "320.1/121.0 amu" Comment: "01-27-R-1" Annotation: "Finished Surface Water*

Sample Name: "FWval08081 23" Sample ID: "std 0.05 reag w" Peak Name: "AISAA" Massles): "340.0/160.0 amu" Comment: "01-27-R-1" Annotation: "Finished Surface Water"

Time, min

Sample Name: "FWval08081 23" Sample ID: "std 0.05 reag w" Peak Name: "AlOx" Mass(es): "264.0/160.0 amu" Comment: "01-27-R-1" Annotation: "Finished Surface Water"

Sample Name: "FWval08081 23" Sample ID: "std 0.05 reag W Peak Name: "AIESA" Massles): "314.0/176.0 amu" Comment: "01-27-R-1" Annotation: "Finished Surface Water"



4.6

4.8

5.0

5.2 5.4 6. Time, min

5.8

6.0~ 6.2 ~6.4

0

43 43 53 Ο

53 5 F Î 8

Time, min

4U

5ÛU

5^ Time, 5^ min53

53

6X5

6.0 6^2" 6~4~

3 3 4 T Ô Time, min

Sample Name: "FWval08081 23" Sample ID: "std 0.05 reag w" Peak Name: "ProESA" Masses): "256.0/121.0 amu" Comment: "01-27-R-1" Annotation: "Finished Surface Water"

4Ί5

Sample Name: "FWval08081 23" Sample ID: "std 0.05 reag w" Peak Name: "MeESA" Mass(es): "328.0/121.0 amu" Comment: "01-27-R-1" Annotation: "Finished Surface Water" 5.18

63

6^

Figure 4: Example Ion Chromatograms - 0.05 μg/L Standard (cont.) Continued on next page.

1000

2000

3000

4000

5000

6000

Sample Name: "FWval08081 23" Sample ID: "std 0.05 reag w" Peak Name: "MeOx" Masses): "278.0/206.0 amu" Comment: "01-27-R-1" Annotation: "Finished Surface Water" 5.21 6995

0

1000

2000

3000

4000

5000

6000

7000

Sample Name: "FWval08081 23" Sample ID: "std 0.05 reag w" Peak Name: "DimOx" Massues): "269.9/198.0 amu" Comment: "01-27-R-1 " Annotation: "Finished Surface Water" 4.59 8000



200

400

600

800

1000

1200

1400

1600

3.6

33

415

Time, min

0

500

1000

1500

2000

2500

2849

4.04

Sample Name: "FWval08081_23" Sample ID: "std 0.05 reag w" Peak Name: "ProSAA" Mass(es): "282.0/134.0 amu" Comment: "01-27-R-1" Annotation: "Finished Surface Water

Figure 4. Continued.

T2

Sample Name: "FWval08081 23" Sample ID: "std 0.05 reag w" Peak Name: "ProOx" Massfës): "206.0/134.0 amu" Comment: "01-27-R-1 " Annotation: "Finished Surface Water" 3.94 1928 1800


271 Table ΠΙ. Percent Recovery ± RSD: All Matrices


Degradate

AcESA AcOX AcSAA AIESA AlOX AISAA AI-sOX ProESA ProOX ProSAA MeESA MeOX DimESA DimOX

0.05 pg/L

97.1% ± 94.6% ± 101.7% ± 87.0% ± 52.1% ± 99.1% ± 98.9% ± 100.1% ± 102.9% ± 96.0% ± 97.9% ± 108.0% ± 94.2% ± 101.4% ±

8.6% 4.2% 9.9% 5.3% 6.7% 7.6% 4.9% 3.8% 8.1% 3.9% 9.9% 4.8% 6.2% 3.3%

0.10 Mg/L

93.4% ± 87.6% ± 92.0% ± 99.2% ± 69.5% ± 97.5% ± 93.0% ± 94.3% ± 95.4% ± 94.0% ± 92.6% ± 104.4% ± 96.3% ± 96.1% ±

9.5% 3.2% 11.0% 9.3% 5.5% 5.6% 4.6% 5.6% 4.1% 4.7% 8.2% 5.0% 10.4% 3.9%

0.25 pg/L

105.3% ± 99.5% ± 104.5% ± 100.5% ± 95.0% ± 106.2% ± 101.1% ± 103.3% ± 103.4% ± 103.5% ± 104.8% ± 106.9% ± 106.0% ± 104.2% ±

6.6% 2.6% 6.9% 9.1% 4.2% 4.7% 3.0% 4.1% 3.6% 2.5% 6.7% 4.3% 4.7% 4.0%

secondary fragment ions m/z 176 and 162, respectively. While this provides confirmatory identification of the dégradâtes, the secondary fragment ions are much less intense than the m/z 121 ion resulting in approximately ten fold poorer sensitivity for these compounds. Reconstructed ion chromatograms of all 14 degradate standards, at the 0.05 μg/L level (LOQ) are presented in Figure 4. Ion suppression and ion enhancement effects are often found in MS/MS methods where there is limited analyte resolution and co-elution of both known and unknown components. To test the method for these matrix effects, a series of standard addition experiments were conducted. Real environmental samples covering all three water matrices from approximately 50 different sites were analyzed as received. Aliquots of the samples were also spiked with known and variable amounts (0.10 to 2.0 μg/L) of mixed degradate fortification solution and reanalyzed. The individual degradate results from both samples were compared and the percent spike recovery calculated. At all sites and for all dégradâtes, the spike recoveries were well within the 70%-110% range. This was further evidence there were no matrix induced suppression or enhancement effects in the data. The standard addition summary included only results at or above the LOQ of 0.05μg/L, the level at which recovery and variability met or exceeded the quantitation criteria. The environmental samples were analyzed in a previous monitoring study and were selected because they generally had known residues of several of the common dégradâtes.


Determination of Acetanilide Degradates in Ground and Surface

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