Separation of Diastereomers and Enantiomers by Capillary Zone

Aug 25, 1999 - Corresponding authors phone: +41-1-823 40 84; e-mail: roland. ... performed using the HP3DCE system (Hewlett-Packard) with a built-in ...
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Environ. Sci. Technol. 1999, 33, 3462-3468

Sulfonic and Oxanilic Acid Metabolites of Acetanilide Herbicides: Separation of Diastereomers and Enantiomers by Capillary Zone Electrophoresis and Identification by 1H NMR Spectroscopy D I A N A S . A G A , †,‡ S I E G R U N H E B E R L E , † DANIEL RENTSCH,§ R O L A N D H A N Y , * ,§ A N D STEPHAN R. MU ¨ L L E R * ,† Swiss Federal Institute for Environmental Science and Technology (EAWAG), CH-8600 Du ¨ bendorf, Switzerland, and Swiss Federal Laboratories for Materials Testing and Research (EMPA), CH-8600 Du ¨ bendorf, Switzerland.

Biological degradation of acetanilide herbicides in soil results in the formation of the ethanesulfonic acid (ESA) and oxanilic acid (OXA) derivatives. These molecules exist in two (alachlor), four (acetochlor), and eight (metolachlor) stereoisomeric forms. Due to the hindered s-cis/s-trans isomerization of the amide bond, most of the diastereomers of the ESA and OXA compounds were separable by capillary zone electrophoresis (CZE). Using γ-cyclodextrin as chiral selector in CZE, complete separation of all four isomers of enantiomerically enriched (5S)-metolachlor OXA was achieved. The enantiomers of acetochlor ESA, acetochlor OXA, and racemic metolachlor OXA were partially separated. The proton nuclear magnetic resonance (1H NMR) spectra showed separated signals for all diastereomers in achiral environment and enantiomers with γ-cyclodextrin as chiral selector. The assignment of these signals to the corresponding absolute configurations was used for the identification of observed CZE peaks. Rate constants for the amide s-cis/s-trans isomerization were evaluated. The time scales for this isomerization at room temperature are seconds for the parent herbicides and several hours for the metabolites. Rotation of the phenyl ring with respect to the amide moiety was not observed. CZE allows the determination and quantification of the different isomers of ESA and OXA metabolites and will facilitate the investigation of the probable stereoselective degradation of acetanilide herbicides in the environment.

Introduction Acetanilide herbicides (see Figure 1) are among the most widely used pesticides in the United States both for crop and * Corresponding authors phone: +41-1-823 40 84; e-mail: roland. [email protected]; fax: +41-1-823 40 12; +41-1-8235460; e-mail: [email protected]. † EAWAG. ‡ Present address: University of Nebraska at Kearney, Kearney, NE 68849. § EMPA. 3462

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FIGURE 1. Structures of the acetanilide derivatives and numbering of the protons used for the assignment of the 1H NMR signals. noncrop use (1). The most important compounds in this class of herbicides are alachlor, acetochlor, and metolachlor, all of which are selective herbicides used to control specific annual grasses and broadleaf weed. Previous studies (2-4) have shown that biological dechlorination of these herbicides via glutathione conjugation leads to the formation of ethanesulfonic acid (ESA) and oxanilic acid (OXA) derivatives. These metabolites are highly polar and thus leacheable into the groundwater. Recent studies (5-7) show that these metabolites occur at higher concentrations and are more frequently detected in surface and groundwater than their parent compounds. A common feature in the chemical structure of acetanilide herbicides is the presence of hindered rotation about the phenyl-N bond (8-10). Indeed, the rotation of the phenyl ring with respect to the amide moiety is known to be very slow. On the basis of molecular mechanics calculations stable perpendicular configurations between the phenyl ring and the N-CdO moiety have been proposed (9, 10). For metolachlor, the rotational energy barrier was determined to be Ea ) 154.3 kJ/mol (isomerization half-lives of 50.6 h at 128 °C and 3 h at 154 °C) (11). From these data, an isomerization rate constant of less than 10-12 s-1 at room temperature can be estimated. This means that rotation of the phenyl ring does not occur at environmetally relevant temperatures. The rotations about the amide bonds are also hindered (8, 9); however, quantitative data are not available for the herbicides and their metabolites. For slow N-CO bond isomerization, two experimentally separable stereoisomers of alachlor (metabolites) are expected, namely s-cis- and s-trans-alachlor, where the carbonyl group is in the cis or trans position with respect to the phenyl ring. Because of the unsymmetrical substitution of the aromatic ring, leading to aR and aS atropisomers, four stereoisomers of acetochlor 10.1021/es990288w CCC: $18.00

 1999 American Chemical Society Published on Web 08/25/1999

FIGURE 2. Amide bond isomerization and perpendicular arrangement between the phenyl ring and the N-CdO amide plane of acetanilide derivatives. then occur (s-cis,aR; s-cis,aS; s-trans,aR; s-trans,aS). For metolachlor, eight stereoisomers exist, as a result of the additional chiral carbon atom (see Figures 1 and 2). The stereochemistry of the compounds plays an important role in their biological activity and degradation pathways. In general, only certain enantiomeric forms of acetanilides have herbicidal activity. The degradation of these herbicides has been shown to be stereoselective and/or enantioselective (12). The racemic metolachlor herbicide formulation is currently being replaced by the enantiomerically enriched (5S)-metolachlor because the 5R-isomer does not have herbicidal activity. Acetanilide herbicides were separated and quantified directly by gas chromatography-mass spectrometry (GCMS) (10). The sulfonic acid and oxanilic acid metabolites, however, need to be derivatized for GC-MS analysis, otherwise analytical methods such as high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE) have to be used. For the separation of isomers of the ESA and OXA compounds, CZE is especially well-suited because the experiments can be performed at environmentally relevant temperatures, in contrast to GC-MS, where elution temperatures are well above 100 °C. Most importantly, CZE offers high-efficiency separation, easily adjustable parameters, and a wide selection of commercially available chiral selectors for the separation of enantiomers. As diastereomers can be distinguished by their different 1H chemical shifts and spin-spin coupling constants, the use of NMR is highly informative for the elucidation of the composition of diastereomeric mixtures. Addition of chiral shift reagents separates the NMR resonance lines from enantiomeric pairs of molecules (13), and compositions can be determined without the need to separate individual components from a mixture. In this paper, CZE was used to separate and quantify the different diastereomers and enantiomers of alachlor, acetochlor, and metolachlor sulfonic acids and oxanilic acids, respectively. 1H NMR spectroscopy was used to identify the different stereoisomers in achiral and chiral environment, to quantify the s-cis/s-trans amide isomerization rate constants, and to assign the isomers separated by CZE. For comparison purposes, the parent compounds were also studied by NMR. The paper begins with the structurally simplest compounds, alachlor and its metabolites, followed by acetochlor and metolachlor and their metabolites.

Materials and Methods Chemicals. γ-Cyclodextrin was purchased from Fluka (Buchs, Switzerland). Standards of alachlor ESA, alachlor OXA, and acetochlor OXA were obtained from Monsanto Chemical Co. (St. Louis, MO). Racemic and enriched standards of (5S)metolachlor, metolachlor ESA, and metolachlor OXA were donated by Novartis AG (Basel, Switzerland). Acetochlor ESA was synthesized using the method described previously (4, 14). Capillary Zone Electrophoresis. CZE separations were performed using the HP3DCE system (Hewlett-Packard) with

a built-in UV-diode-array detector. Separations were carried out with fused silica 50 µm i.d. capillaries with a bubble cell at the detection window (Hewlett-Packard). The background electrolyte optimized for the chiral separation of the ESA and OXA compounds was composed of citrate (30 mM in water, pH 5.5), with 2% (% w/vol) γ-cyclodextrin. Electrophoresis was conducted at an applied voltage of 30 kV. The capillary column (effective length 56 cm) was thermostated at 20 °C, unless otherwise specified. The analysis program consisted of preconditioning of the capillary by rinsing with NaOH (0.1 M for common CZE and 1 M for CZE with γ-cyclodextrin) for 1 min, followed by a 4 min wash with water and a 5 min wash with background electrolyte. Sample injection was carried out by pressure injection for 2 s at 50 mbar. The detection wavelength was 210 nm. NMR Spectroscopy. 1H NMR spectra were recorded on a Bruker ASX-400 NMR spectrometer using a 5 mm probe for inverse experiments with a z-gradient. All experiments were performed at ambient temperature or under temperature-controlled conditions at 303 K for 1H-1H nuclear Overhauser effect (NOE) experiments, T1 measurements (by inversion recovery), and saturation transfer experiments. NOE presaturation delays of 3 s for alachlor, alachlor ESA, acetochlor, and acetochlor ESA were applied. For metolachlor and metolachlor ESA, the recently developed DPFGSE NOE pulse sequence (15) was used. Saturation transfer experiments (16) were performed with CW irradiation (67 dB) of selected resonance lines with a presaturation delay of 20 s (>5T1). The 1H chemical shifts were assigned by homodecoupling experiments and 1H-1H chemical shift-correlated spectroscopy (17) where necessary. Experiments in D2O were performed with 30 mM citric acid to simulate the experimental conditions from electrophoresis. The numbering for the 1H chemical shift assignment of the acetanilide derivatives (Supporting Information) is given in Figure 1. Due to overlapping resonance lines, the signal pattern of the aromatic and the methylene protons attached to the phenyl ring (phenyl-CH2-CH3) were not evaluated in detail. The diastereomeric ratios were evaluated via signal integration from NMR spectra taken at least 16 h after preparing the corresponding solution.

Results and Discussion Alachlor and Its Metabolites. The restricted rotation about the amide bond of alachlor ESA and alachlor OXA generates stable s-cis and s-trans diastereomers at 12 and 20 °C which could be separated using CZE (see Table 1). For illustration, electropherograms of alachlor ESA are shown in Figure 3. The first eluting peak corresponds to the s-cis configuration (see below). Since the separation mechanism in CZE is based on the differences in the volume-to-charge ratios of the analytes, s-cis-alachlor ESA has a smaller molecular diameter relative to the s-trans component (population ratio 52:48, see Table 1). As evident from the electropherograms taken at 30 and 55 °C (see Figure 3), rapid conversion of the isomers occurs at elevated temperatures, where separation is no longer possible. VOL. 33, NO. 19, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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73 71 not evaluated not evaluated not evaluated not evaluated no separation no separation

s-trans

alachlor alachlor ESA acetochlor

H-(6) f H-(4) H-(6) f H-(4) H-(8) f H-(5)

acetochlor ESA

H-(8) f H-(5)

H-(6) f H-(2), H-(3) H-(6) f H-(2) H-(8) f H-(2), H-(4) H-(4) f H-(5), H-(8) H-(8) f H-(2), H-(4) H-(4) f H-(8)

TABLE 3. Amide Bond Isomerization Rate Constants (s-1) and Proton Relaxation Times CH2-Cl, T1 (s), of Alachlor, Acetochlor, and Their ESA Derivatives T1

1.5 8 7 18 14

CDCl3 DMSO-d6 CDCl3 DMSO-d6 D2O D2O D2O D2O racemic metolachlor

racemic metolachlor ESA (5S)-metolachlor ESA racemic metolachlor OXA (5S)-Metolachlor OXA

C6D6 D2O D2O acetochlor acetochlor ESA acetochlor OXA

(5S)-metolachlor

CDCl3 D2O D2O alachlor alachlor ESA alachlor OXA

1

1.5 5 5 8 12

2

b ) s-trans 94 46 25 b ) s-trans,aR/aS 92 54 33 b ) s-cis,aR,5S; s-cis,aS,5R

c ) s-trans,aR,5R; s-trans,aS,5S 33 33 49 48 33 34 22 32

d ) s-trans,aR,5S; s-trans,aS,5R 67 64 51 49 54 54 52 42

k(s-cisfs-trans)

a ) s-cis 6 54 75 a ) s-cis,aR/aS 8 46 67 a ) s-cis,aR,5R; s-cis,aS,5S

diastereomer ratios NMR 9

NMR solvent

TABLE 1. Ratios between Diastereomers of Acetanilide Derivatives in Solution 3464

s-cis

a The carbonyl group is in the cis or trans position with respect to the phenyl ring.

27 29

52 72 a ) s-cis,aR/aS

48 28 b ) s-trans,aR/aS not evaluated 43 57 70 30 a ) s-cis,aR/aS,5R/5S b ) ) s-trans,aR/aS,5R/5S

b ) s-trans not evaluated a ) s-cis

diastereomer ratios CZE

TABLE 2. Significant NOE Enhancements Required for the Assignment of the s-cis and the s-trans Configuration of the Amide Bond (see Figures 1 and 2)a

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alachlor alachlor ESA acetochlor acetochlor ESA

0.17

k(s-transfs-cis) 0.01

(9.9 ( 0.2) × 10-5 0.38

12.1 ( 0.1 × 10-5 0.033

(9.6 ( 0.4) × 10-5

8.3 ( 0.2 × 10-5

s-cis

s-trans

2.1

2.9

2.7

3.5

1H NOE NMR experiments allowed the identification of the alachlor ESA s-cis and s-trans diastereomers (see Table 2), and their population ratio of 54:46 (see Table 1) was used to assign the amide configurations of the two components in CZE. The changing isomeric ratio of alachlor ESA within a day, from 4:96 just after preparation of the D2O solution to its equilibrium value, was further utilized to quantify the amide isomerization rate constant. Integration of the signals from consecutive NMR measurements at distinct periods after preparing the solution revealed a rate constant on the order of 10-4 s-1 at room temperature (see Table 3) (18). This corresponds to an isomerization time scale of several hours. Since alachlor ESA (and acetochlor ESA, see below) decompose upon heating, an activation energy for the amide bond isomerization could not be determined. The assignment of the s-cis and s-trans configuration in alachlor OXA is not possible by 1H NMR experiments due to the lack of the methylene group CH2-R in this compound. The separation of the diastereomers of alachlor OXA by CZE, however, suggests a slow isomerization rate constant, as observed for alachlor ESA. Using the same argument that the smaller sized s-cis isomer will elute first in CZE, the first and second eluting peak in the electropherogram (area ratio of 72:28) were assigned to the s-cis and s-trans isomers, respectively. Two sets of alachlor OXA signals with a similar area ratio (75:25) were also obtained from 1H NMR. No CZE experiments were carried out for alachlor, due to the lack of a charge on the parent compound. However, the alachlor amide isomers and their populations were evaluated by NMR experiments (see Tables 1 and 2). Moreover, spinsaturation transfer experiments on the methylene protons CH2-Cl revealed that the s-cis/s-trans isomerization rate constant for alachlor is about 102-103 times higher (isomerization time scale of seconds) than that observed for the metabolites (see Table 3). This large difference may be explained by a solvent stabilization of the ESA and OXA isomers using water or DMSO and/or the bulkiness of the SO3- and COO- group relative to the chlorine atom. Acetochlor and Its Metabolites. The assignment of the s-cis and s-trans configurations of acetochlor and acetochlor ESA/OXA was accomplished using CZE (see Figure 4a for acetochlor ESA) and/or NMR experiments (see Tables 1 and

FIGURE 3. CZ electropherograms of alachlor ESA at different temperatures: (a) 12 °C, (b) 20 °C, (c) 30 °C, and (d) 55 °C. 2) as described for alachlor and its derivatives. As expected from the small structural difference between alachlor and acetochlor, similar rate constants for the amide bond isomerization were obtained (see Table 3). For acetochlor OXA, the possible separation of its s-cis and s-trans isomers using CZE (70:30) again indicates a slow rate of isomerization. The first eluting isomer with higher intensity can be tentatively assigned to s-cis acetochlor OXA. The asymmetric substitution of the phenyl ring in acetochlor and the stable configuration between the planes of the aromatic ring and the amide moiety lead to an additional center of chirality (see Figure 2), called atropisomerism (19). Thus, each of the s-cis and s-trans isomer consists of a pair of enantiomers, i.e., aR and aS. These enantiomers were identified with NMR spectroscopy, where baseline separations of the resonance lines H-(5) of the enantiomeric pairs of acetochlor ESA and acetochlor OXA were achieved using γ-cyclodextrin as the chiral shift reagent. In terms of separating these enantiomers by CZE using γ-cyclodextrin in the background electrolyte, only one pair of enantiomers could be resolved, thereby showing three peaks in the electropherogram (see Figure 4b). On the basis of the integrated peak area ratios (60:20:20 for acetochlor ESA), the first eluting peak in Figure 4b could be assigned to the sum of s-trans,aS and s-trans,aR, whereas the smaller peaks represent the separated s-cis,aS and s-cis,aR enantiomers. However, the elution order of the s-cis and s-trans isomers in the CZ electropherogram with γ-cyclodextrin as chiral selector was reversed. Also for acetochlor OXA, only the s-cis,aS and s-cis,aR enantiomers were separated and the elution order of components changed. Again, three peaks were obtained in the electropherogram with intensity ratios of 30:35:35, whereas for CZE without γ-cyclodextrin the area

ratio of the first and second eluting peak is 70:30. Metolachlor and Its Metabolites. The amide bond, the unsymmetrical substitution at the phenyl ring, and the presence of a chiral C-atom in metolachlor lead to four diastereomeric pairs of enantiomers (total of eight isomers). The intensities of the NMR signals from two of the four diastereomers of metolachlor are very small in CDCl3, and the signal intensities of their methyl groups (phenyl-CH3) are of similar magnitude as the corresponding 13C satellites of the major components. This explains why they escaped attention in earlier work (11, 20). However, as evident from Table 1, four diastereomers could clearly be identified from the NMR spectra of metolachlor in DMSO-d6 and of the ESA (see Figure 5a) and OXA metabolites in D2O. Unfortunately, in CZE the four diastereomers of metolachlor ESA were not separated. However, two peaks were obtained for racemic and (5S)-metolachlor OXA (see Figure 6a, Table 1). With γ-cyclodextrin as chiral selector in the CZE background electrolyte, six peaks for racemic metolachlor OXA (see Figure 6b) and complete separation of the four expected isomers of (5S)-metolachlor OXA (see Figure 6c) were observed. In analogy to the results for the alachlor and acetochlor derivatives, we can assume that the amide s-cis isomers elute first in CZE without γ-cyclodextrin and that the chiral selector changes the elution order. However, the unambiguos assignment of the stereoconfiguration of metolachlor OXA isomers by NMR and their relation to the observed peaks in CZE is not possible due to the lack of the methylene group in the oxanilic acid derivative, as mentioned earlier. Therefore, the NMR stereochemical assignment was carried out for the structurally similar metolachlor and metolachlor ESA, with the aim of assigning the CZE peaks obtained for metolachlor OXA by analogy (see below). VOL. 33, NO. 19, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. CZ electropherograms of acetochlor ESA at 20 °C: (a) achiral environment and (b) addition of 2% w/vol γ-cyclodextrin in the background electrolyte. NOE experiments as carried out for alachlor and acetochlor showed that both diastereomeric components of metolachlor in CDCl3 (intensity ratio 33:67, see Table 1) and the two major components of metolachlor ESA correspond to the amide s-trans configuration. NOE spectra were also used for the more complicated assignment of the isomeric configurations based on the spatial arrangement of the chiral C-(5) moiety relative to the unsymmetrically substituted aromatic ring, i.e., to distinguish between the s-trans metolachlor c and d isomers as shown in Figure 7. Rotation of the chiral substituent around the N-C-(5) bond cannot be excluded, but the strong NOE enhancements from protons H-(5) onto H-(4) and H-(2) are consistent with relative configurations shown in Figure 7a,b, where H-(5) is located in the N-CdO plane. Selected data of NOE effects with substantial enhancement differences for corresponding protons of metolachlor components c and d are listed in Table 4. These allow metolachlor c to be assigned to the enantiomeric pair with the s-trans,aS,5S or s-trans,aR,5R configuration (Figure 7a) and metolachlor d to the configuration s-trans,aS,5R or s-trans,aR,5S (Figure 7b). Following the same arguments, the stereoisomers of metolachlor ESA were also assigned (see Tables 1 and 4). NMR signals of the metolachlor ESA enantiomers were then separated by addition of an equimolar amount of γ-cyclodextrin to the aqueous solution (see Figure 5b). Finally, the complete assignment of each resonance line to the absolute stereoconfiguration was possible by comparison with the corresponding NMR spectrum of (5S)-metolachlor ESA (Figure 5c). This assignment is listed in the caption for Figure 5. This assignment of stereoisomers by NMR spectroscopic methods is supported by inspection of the equilibrium 3466

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FIGURE 5. 1H NMR spectral region of the methyl groups H-(8) of metolachlor ESA in D2O at 297 K: (a) racemic mixture, (b) racemic mixture with equimolar amount of γ-cyclodextrin, and (c) enantiomerically enriched (5S)-metolachlor ESA with γ-cyclodextrin. Stereochemical configuration of metolachlor ESA isomers I-VIII from b: I ) s-trans,aS,5S, II ) s-trans,aR,5S, III ) s-trans,aR,5R, IV ) s-trans,aS,5R, V ) s-cis,aR,5R, VI ) s-cis,aS,5S, VII ) s-cis,aS,5R, VIII ) s-cis,aR,5S.

TABLE 4. NOE Enhancements Used for the Assignment of the Relative Stereoconfiguration between the Aromatic Ring and the Chiral Carbon Moiety (see Figure 7) of Metolachlor and Metolachlor ESA (stereoisomers a-d as defined in Table 1) excited 1H NMR signal H-(2c,d) H-(3c,d) H-(4c,d) H-(5c,d) H-(6c,d) H-(7c′,d′) H-(8c,d) H-(6a,b) H-(8a,b)

enhanced NMR signal of metolachlor d with intensity relative to ca H-(6d) H-(7d′) H-(7d′) H-(6d) H-(7d′) H-(8d) H-(4d) H-(4d) H-(4d) H-(4d)

0.37 1.00b 1.00b 1.38 nec 0.71 2.46 1.40 0.13 0.56

enhanced NMR signal of metolachlor ESA d with intensity relative to ca H-(6d) H-(8d)

0.46 1.00b

H-(6d) H-(7d′) H-(8d) H-(4d) H-(4d) H-(4d) H-(4d)

1.45 0.7 nec 1.91 2.95 0.54 0.70

metolachlor ESA a, b H-(4b) 1.00d H-(4a) 1.00d

a Intensity of stereoisomer c set equal to 1.00. Numbers were weighted according to the ratio between c and d from Table 1. b Intensity set equal to 1.00; no enhancement for c. c No enhancement; NOE visible for c. d Intensity set equal to 1.00; no enhancement for a or b, respectively.

constants K ) k(s-cis f s-trans)/k(s-trans f s-cis) of the amide bond isomerization from Table 1. The individual equilibrium constants for racemic mixtures and the corresponding 5Senriched compounds must be the same. Therefore, the equilibrium constants are Kc,a ≈ Kd,b ≈ 33 for the different diastereomers of racemic and (5S)-metolachlor in DMSOd6. The s-cis/s-trans equilibrium constants Kc,a and Kd,b must not necessarily be equal. However, considering the minor

FIGURE 7. Structures of metolachlor (diastereomers c and d, R ) Cl) and metolachlor ESA (diastereomers c and d, R ) SO3-) with absolute configurations (a) s-trans,aR,5R and (b) s-trans,aR,5S.

FIGURE 6. CZ electropherograms of stereoisomers of metolachlor OXA at 20 °C: (a) racemic compound in achiral environment, (b) racemic mixture with chiral selector (2% w/vol γ-cyclodextrin), and (c) enantiomerically enriched (5S)-metolachlor OXA with chiral selector. structural differences between the two stereoisomers from Figure 7, similar K values are expected. Assuming this to hold also for metolachlor ESA, the population ratio leads to Kc,a ≈ Kd,b ≈ 7. Consequently, the metolachlor ESA diasteromers a and c, as well as b and d, form pairs of s-cis/s-trans isomers, confirming the accurate assignment of the relative configurations given above. Finally, for metolachlor OXA, the amide isomerization equilibrium constants are Kc,a ≈ Kd,b ≈ 3. The tentative assignment of metolachlor OXA isomers to configurations a or b and c or d assumes an increasing relative population from a to d, as was found for metolachlor

and metolachlor ESA. The relative NMR signal intensities from diastereomers a and b correspond to the fraction of the first eluting s-cis isomers in CZE (see Figure 6a and Table 1), and the populations of c and d equal the fraction of the second eluting s-trans metolachlor OXA isomers. In Figure 6c, the peaks labeled 1, 3, 5, and 6 correspond to the absolute configurations 1, s-trans,aR,5S; 3, s-trans,aS,5S; 5, s-cis,aR,5S; and 6, s-cis,aS,5S for the four stereoisomers of (5S)metolachlor OXA. By comparison of retention times between parts b and c of Figure 6, peaks 1, 2, and 3 in Figure 6b can be assigned to metolachlor OXA in the s-trans configuration, and signals 4, 5, and 6 represent s-cis isomers. Peaks 1 and 3 correlate to the 5S-configuration, and consequently components 2 represent the sum of s-trans,aR,5R and s-trans,aS,5R. Finally, the s-cis isomers in Figure 6b overlap considerably; however, as for (5S)-metolachlor OXA and considering peak area ratios from Table 1, signal 5 can tentatively be assigned to the configuration s-cis,aR,5S, signal 4 to s-cis,aR,5R, and signal 6 to the sum of s-cis,aS,5R and s-cis,aS,5S. Analytical and Environmental Implications. The different relative populations of racemic and (5S)-metolachlor diastereomers (see Table 1) illustrate that rotation of the phenyl ring does not occur at ambient temperature; thus, the ratios are probably determined by the synthetic pathway. Population differences were also found for diastereomers of racemic and (5S)-metolachlor OXA. For metolachlor ESA, these populations seem to be coincidentally similar. It follows that the aR/aS-ratio is a marker of the original product applied. If this ratio is not affected by the metabolism, the metabolites also would be marker molecules. On the other hand, regardless of the diastereomeric composition due to the hindered amide bond rotation, the herbicides and their metabolites will reach their equilibria quite fast (days). Of interest are the changing amide s-cis/s-trans ratios observed when the analytes are dissolved from polar to nonpolar solvents (H2O or DMSO to benzene or CHCl3; data not shown). This changing preference of amide configurations may be important in the differential distribution of these compounds in soil constituents and bacterias and might influence the degradation rate and pathways of these compounds in the VOL. 33, NO. 19, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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environment. The amide s-cis/s-trans isomerization also has analytical implications, and the existence of these isomers explains for example the difficulty encountered by Pomes et al. (21) and Ferrer et al. (22) in separating acetanilide herbicides metabolites using HPLC, where broad and overlapping signals were observed at room temperature. Since the conjugation of the chloroacetanilide herbicides with the chiral compound glutathione is enzymatically mediated (23), it is possible that the different stereoconfigurations of these herbicides have different reaction rates toward glutathione. It will also be interesting to investigate whether the degradation of the herbicide-glutathione conjugates into the ESA and OXA derivatives is stereospecific, i.e., only a single stereoisomer is produced rather than a mixture. These questions are of environmental relevance considering that these herbicides are applied as mixtures of isomers and are exposed under varying environmental conditions.

Acknowledgments The authors wish to thank G. Goudsmit (EAWAG), for the evaluation of the alachlor ESA and acetochlor ESA isomerization rate constants, and Novartis, for the donation of the metolachlor standards.

Supporting Information Available 1H NMR chemical shift assignment of acetanilide derivatives

shown in Figure 1. This material is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) Gianessi, L. P.; Puffer, C. M. Use of Selected Pesticides for Agricultural Crop Production in the United States; NTIS: Springfield, VA, 1982-1985; p 490. (2) Field, J. A.; Thurman, E. M. Environ. Sci. Technol. 1996, 30, 1413. (3) Lamoureux, G. L.; Stafford, L. E.; Tanaka, F. S. J. Agric. Food Chem. 1971, 19, 346.

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Received for review March 15, 1999. Revised manuscript received June 11, 1999. Accepted July 1, 1999. ES990288W