Environ. Sci. Technol. 1995,29, 2023-2030
Environmental Behavior of Acetamide Pesticide Stereoisomers. 1 Stereo- and Enantioselective Determination Using Chiral High-Resolution Gas I
High-Performance Liquid Chromatography HANS-RUDOLF B U S E R * A N D MARKUS D. MULLER Swiss Federal Research Station, CH-8820 Wadenswil, Switzerland
The chromatographic separation of enantiomers and diastereomers of some acetamide pesticides was investigated using achiral and chiral high-resolution gas c hromatog ra p hy/mass spectrometry ( HRGC/ MS) and chiral high-performance liquid chromatography (HPLC). The compounds studied were alachlor, a c eto c hIo r, meta Iaxy I, m eto Ia c hIo r, and dim ethe na mid and represent important herbicides or fungicides. Whereas alachlor is achiral, all other compounds are axial- and/or C-chiral and consist of two or four stereoisomers (enantiomers and diastereomers). Since these stereoisomers have different biological activities, stereo- and enantioselective determinations of these compounds in environmental studies seem important but have never been reported. In our study, chiral HRGC using a P-cyclodextrin derivative showed varied resolution of diastereomers and/or enantiomers; achiral HRGC showed no resolution of diastereomers. Resolution of C-chiral enantiomers was easier than resolution of axial-chiral enantiomers (atropisomers). The analysis of different batches of metolachlor revealed a varied diastereomeric composition. Chiral HPLC using modified cellulose and phenylglycine columns also showed some isomer resolution and was used for the isolation of fractions enriched with one or the other stereoisomer. The assignment of the (+)-and (-)-enantiomers was achieved for acetochlor and metalaxyl using chiral HPLC in combination with chiroptical detection. A novel thermal equilibration procedure allowed distinction among axial-chiral and C-chiral enantiomers.
0013-936X/95/0929-2023$09.00/0
1995 American Chemical Society
Introduction The group of acetamide pesticides (some also known as chloroacetamides) includes a considerable number of herbicides and fungicides for the control of weeds and fungi in crops. The compounds have the general formula given in Chart 1. The desired activity is largely depending on the acyl moiety (substituent a) and is often either herbicidal (generallya = CH2Cl) or fungicidal (generallya = CH20CH3 or heterocyclic) (I). The compounds are widely used to control annual grasses and certain broadleafweeds in corn, soybeans, and peanuts and to control phytopathogenic fungi (Peronosporules)in potatoes, sugar beets, and other crops (1-3). Alachlor and metolachlor, two of these compounds, are among the most popular pesticides used in the United States (4). Varied substitution ofthe aromatic moiety (hinderedrotation about the phenyl-nitrogen bond) andlor presence of an asymmetrically substituted C-atom in the alkyl moiety lead to stereoisomerism and chirality in these compounds. Acetamide pesticides have a moderate water solubility and are rapidly absorbed into plants (5). In susceptible plants, the herbicides act by inhibition of protein synthesis, e.g., by reaction of the activated C1 atom ofthe chloroacetyl group with reactive sites in proteins, whereas insensitive plants rapidly inactivate these herbicides via gluthathion conjugation (6'). In sensitive fungi, the structurally related fungicides have effects on RNA synthesis (7). As these reactions all involve various chiral structures, some stereoselectivity in the biological activity of these compounds is expected. Similarily, the degradation in soil is mainly microbiologically mediated (8)and may also be stereoselective. Thus, transformation reactions of these compounds in biological systems, and probably in the environment, can be expected to show enantioselectivity. This is in contrast to abiotic processes (chemical, photochemical, transport, and distribution processes), which are nonenantioselective (9-11). Stereoisomers of certain acetamide pesticides showed largely different herbicidal or fungicidal activity,sometimes with completely different biological properties for an antipode (12-141, Whereas the biological activities of such stereoisomers have been described, the differences in side effects and the fate and dynamics in the environment have hardly ever found attention. This lack of information is of special concern in situations where residue levels approach those showing adverse effects on certain organisms. This is the case with some of these pesticides detected in European and United States rivers at concentrations approaching EC50 values for freshwater algae species (1518). One of the reasons for this lack of information is the lack of suitable analytical techniques to determine residue levels in a stereo- and enantioselective way. In this paper, we discuss analytical aspects for the stereoand enantioselective determination of acetamide pesticides, and we describe the use of chiral high-resolution gas chromatography (HRGC) and chiral high-performance liquid chromatography (HPLC). In the following paper (191, we present the first results on the application of these techniques to environmental samples. The compounds -Nstudied were alachlor [2-chloro-N-(2,6-diethylphenyll (methoxymethyl)acetamidel, acetochlor [2-chloro-N-(2-
VOL. 29, NO. 8, 1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY I 2 0 2 3
CHART 1
Achiral HRGC was carried out with a 22-m mixed-phase SilarlOclOV1701 (SILOV and a 60-m OV1701 fused silica General Stnicture of Acetamide Pesticide9 (0.25 mm i.d.) HRGC column. The columns were temperature programmed as slow as 0.5 "Clmin for optimum resolution. This resulted in retention times as long as 150 min (elutiontemperatures, 149- 172 "C). Chiral HRGC was carried out with a 20-m OV1701-BSCD fused silica column (0.25mm i.d) (BSCD = tert-butyldimethylsily1lyl-B-cyclodextrin; relative amount, ra, 50%) (20). This column was temperature programmed as follows: 70 "C, 2 min isothermal, 20 "Clmin to 120 "C, then at 3 "Clmin to 230 "C. On-column injection (1-2pL) at 70 "C was used. Initially, a chiral 20-m OV1701-GDEX (GDEX = permethylated y-cyclodextrin; ra, 20%),a chiral20-m OV61-PMCD (PMCD a The angle a between the aromatic and the amide plane is =90° (see text). R and R' are CH3- and/or CzHs-. In dimeth= permethylateda-cyclodextrin; ra, 30%),and a chiral 30-m enamid, phenyl is replaced by 2,4-dimethylthien-3-yl. PS086-DMPCD (DMPCD= dimethyl-pentyl-P-CD,courtesy M. Galli, Legnano, Italy; ref 21) columns were also used. ethyl-6-methylphenyl)-N- (ethoxymethyl)acetamide], met Chiral High-Performance Liquid Chromatography alaxyl [N-(2,6-dimethylphenyl)-N-(methoxyacety1)alanine (HPLC) and Fractionation of Acetamide Compounds. Two methyl ester], metolachlor [2-chloro-N-(2-ethyl-6-meth- chromatographic systems were operated at room temperylphenyl)-N-(2-methoxy-l-methyIethyl)acetamide], and ature: (I) a chiral reversed-phase system consisting of a dimethenamid [2-chloro-N-(2,4-dimethylthien-3-yl)-N-(2250 x 4 mm column with a cellulose carbamate on silica methoxyl- 1-methylethyl)acetamidel(for structures,see Char& (ChiralcelOD-R;Daicel Chemical Industries, Tokyo,Japan) 2). Whereas alachlor is achiral and shows no stereoisomoperated with 0.8 mLlmin 40% acetonitrile/60% water to erism, all other compounds are axial- andlor C-chiral, resolve acetochlor, metalaxyl, and metolachlor and (11) a consisting of up to four stereoisomers. Stereo- andlor chiral normal-phase system consisting of a 250 x 4 mm enantioselective analyses of these compounds in environPirkle type column (Sumipax OA-2000; Sumitomo, Osaka, mental applications pose some difficulties and so far have Japan) operated with 0.8 mL/min n-heptanelO.l% isonever been reported. propanol to resolve dimethenamid. A Spectra-Physics (San Jose, CA) Model 8800 pump was used. Samples (5-20 pL Experimental Section of 0.1-0.5% solutions in methanol or heptane, respecCompounds Investigated. Technical standards (purities, tively) were injected via a Rheodyne (Cotati, CA) Model 299%) of alachlor (Monsanto, St. Louis, MO), acetochlor 7125 injector and a 20-pL loop. Detection was either by a (ICUStauffer,Femhurst, UK, or Monsanto, St. Louis, MO), Knauer (Berlin,Germany) variable wavelength W detector metalaxyl and metolachlor (Ciba-Geigy,Basle, Switzerland), set at 254 nm or with a IBZ (Hannover, FRG) polarimetric and dimethenamid (Sandoz, Basel, Switzerland) were detector with a 300-pLcell. Optical rotation was monitored obtained. These materials were dissolved in ethyl acetate using both polychromatic and monochromatic light (inat concentrations of 1-10 nglpL for analysis by HRGC or terference filter, 589 f 15 nm) as in a previous study (22). at 0.1-2% for analysis by HPLC. Two batches of metoFor optical rotation, more concentrated sample solutions lachlor, dated 1975and 1988,and two formulated products, (2%)were analyzed to improve signal-to-noise ratios. dated 1993 and 1994, were analyzed. The formulations, a Fractions were collected at the outlet of the Wdetector wettable powder and a liquid (33% active ingredient, ai), while observing the absorbance, taking a time lag of ~ 1 6 were extracted with methylene chloride or ethyl acetate. In s between detection and elution into account. With case of the wettable powder, water was added prior to unresolved peaks, peak cutting was carried out as recently extraction. After centrifugation, an aliquot of the clear described (22). The presence of the proper analytes in these organic phase was dried over sodium sulfate and directly fractions was determined by full-scan GUMS; enantiomeric analyzed after suitable dilution with ethyl acetate. or stereoisomeric composition was determined from the various SIM analyses using chiral HRGC. Subsamples of HRGC Mass Spectrometric Analysis. A VG Tribrid the fractions collected from the reversed-phase HPLC double-focussing magnetic sector mass spectrometer (MS) system were prepared by mixing 50pL of eluent with 50 mg instrument was used. The ion source was operated in the of sodium sulfate and diluting with 500 pL of toluene; the electron ionization mode (EI, 70 eV, 180 "C). Full-scan mass spectra (mlz 35-435; 1.16 slscan; resolution M l A M fractions collected from the normal-phase system were = 500) were recorded. Analysis was also carried out with dilutedwith cyclohexaneand then used for GUMS analysis. selected ion monitoring (SIM) for optimum isomer/ Microscale Thermal Equilibration of Atropisomers (Topomerization). Samples including HPLC fractionswith enantiomer separation (fastercycle times; 0.5 slscan) using the ions mlz 237 (alachlor;M' - CH30H),223 (acetochlor; amounts as low as 10 ng of a compound were brought to dryness, redissolved in 10 pL of n-tetradecane, and placed M + - C2H50H),206 (metalaxyl;M + - COCH20CH3),238 (metolachlor; M" - CHZOCH~), and 230 (dimethenamid: in 100-pLmicropipets (80 x 1 mm). The micropipets were M + - CHzOCHs). A lock mass of mlz 207.033 from the flame sealed (air not removed) and heated in a GC oven for silicone bleed of the HRGC columns was used in SIM. 60 min at 200 "C. After being cooled, the micropipets were Enantiomeric or stereoisomeric ratios (ER values) were opened, and the solutions were analyzed by chiral HRGC defined as ER = pllp2 wherebypl and p2are the peak areas after suitable dilution with ethyl acetate. ER values (peak of the enantiomers (stereoisomers) determined in SIM area ratios) were compared to the untreated samples. analyses: enantiomeric resolution ( R values) was defined Relative peak areas among the five acetamides were not as previously reported (20). changed upon thermal equilibration when achiral HRGC
b
I
------J
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ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 8,1995
CHART 2
Structures of Five Acetamide Pesticides StudieP
alachlor
as-acetoc hlor
1‘S-rnetalaxyl
I
I
CH3
Q.43
cH3
aS,1 ‘S- dimethenamid
aS,I’S- metolachlor
aThe isomers with 1’s- or a S,l’S-configuration are shown (where applicable). The asterisks mark the axial- and C-chiral elements. CHART 3
Structures of Two Enantiomers of Acetochlor (Top Row) and Four Stereoisomers of Metolachlor (Bottom row)
v aR-
as-
a33 aS,l‘S.
-
I
cH3
aR,1‘S-
cH3
was used. This indicates negligible decomposition of the compounds under these reaction conditions. When analyzed by chiral HRGC, the relative peak areas of stereoisomers may change due to topomerization (interconversion of atropisomers) to statistical (1:l) or near 1:l ratios.
Results and Discussion Structural Considerations. The pesticides selected include compounds with different elements of chirality. The compounds were alachlor, acetochlor, metalaxyl, metolachlor, and dimethenamid (for structures, see Chart 2) and show (except alachlor) axial- and/or C-chirality (23). C-chirality results from the presence of an asymmetrically substituted C-atom in the alkyl moiety (substituent b in metalaxyl, metolachlor, and dimethenamid), and axialchirality results from the hindered rotation about the phenyl-nitrogen bond and a suitable asymmetric substitution of the phenyl ring (acetochlor and metolachlor) or another asymmetric moiety (dimethenamid). Semiempirical MNDO calculations (courtesyF. Miiller-Plathe,Swiss Federal Institute of Technology, Zurich, Switzerland) on model compounds (see below) indicated an =90° angle (a, Chart 1) between the aromatic and the amide plane (241, as depicted in Charts 2 and 3. This is in agreement with X-ray structure analysis on related compounds (12, 13). The partial double-bond character of the amid (N-CO) bond is not expected to lead to additional, conformationally
aR. 1‘R-
CH3
as, 1 ‘R-
stable stereoisomers [s-nuns(Z) isomer preferred energy barrier of rotation in substituted amides, 66-88 kJM-l, see ref 231. The compounds are briefly described below. Alachlor has no asymmetrically substituted C-atom and a symmetric phenyl substitution (see Chart 2). This compound is achiral and shows no stereoisomerism. Acetochlor is axial-chiral due to the asymmetric phenyl substitution and consists of two enantiomeric atropisomers with aS- and ati-configuration as shown in Chart 3 (aS-and ati- are used as sterochemical descriptors for the chiral axis) in the technical product expected to be present in a 1:l ratio. Metalaxyl is C-chiral due to the presence of the asymmetrically substituted C-atom in the alkyl moiety. No atropisomerism exists due to the symmetric phenyl substitution. Metalaxyl thus consists of a single pair of enantiomers with 1’s- and 1’R-configuration, expected to be formed in a 1:l ratio if synthesized from racemic materials. The two enantiomers were previously isolated, and the absolute configurations are known (14). The enantiomer with 1’s-configuration showed dextrorotation, and the enantiomer with 1’R-configurationshowed levorotation. The 1’R-(-)-enantiomer has a higher fungicidal activitythan the antipode (14). Thus, the fungicidal activity of technical metalaxyl is based mainly on the presence of the 1’R-(-1 -enantiomer. Metolachlor and dimethenamid are both C-chiral and axial-chiral due to the presence of the asymmetrically VOL. 29, NO. 8 , 1995 /ENVIRONMENTAL SCIENCE 81 TECHNOLOGY
2026
100
90
8oall 70
AC
OM
60 50 40
30 20
10
0 20 I o 0
\
I
-
22:oo
24100
26100
TI&
+
+
FIGURE 1. El mass and SIM chromatograms (nJz223 230,upper panels; dz206 237 + 238. lower panels) showing elution of acetamide pesticides on (a.c) the achiral SlLOV and (b,d) the chiral OV1701-BSCD HRGC columns. Note the elution of single peaks from the echiral column. Note the marginal resolution of acetochlor into the atropisomerson the chiral column. Note the resolution of metalaxyl, metolachlor, and dimethenamid into t w o or three isomers on the chiral HRGC column. Abbreviations: AL, alachlor; AC, acetochlor; MX, metalaxyl; ME, metolachlor; DM, dimethenamid. Retention time in minutes.
substituted C-atom in the alkyl moietyand the asymmetric phenyl or 2,4-dimethylthien-3-y1moiety. Both compounds thus consist of four stereoisomers with aS,l’S-, aR,l’S-, aR,l’R-, and aS,l’R-configuration, as outlined for metolachlor in Chart 3. The stereoisomers with aS,l’S- and aR,1’R-configuration have an enantiomeric relationship (both chiral elements reversed) and constitute one pair of enantiomers (pair 1). Similarily, the stereoisomers with aR,l’S- and aS,l’R-configuration are enantiomeric and constitute another pair of enantiomers (pair 2). The two pairs of enantiomers have a diastereomeric relationship. The four stereoisomers of metolachlor were individually synthesized (12). Both stereoisomerswith aS-configuration showed dextrorotation, and both stereoisomers with aRconfiguration showed levorotation. The stereoisomers with aS,l’S- and aR,1’s-configuration had highest herbicidal activity (12). Metolachlor also showed some fungicidal activity with the stereoisomers having 1’R-configuration, showing higher activity than the stereoisomers with 1’sconfiguration, as was observed for metalaxyl. Apparently, the 1’s-configuration in metolachlor is important for the herbicidal activity, and the exact atropisomerism is less important. It can be speculated that for dimethenamid the 1’s-stereoisomers also have higher herbicidal activity, since metolachlor and dimethenamid have the same acyl and alkoxyalkyl moieties. Whereas the chemical and physical properties of enantiomers are identical (except rotation of polarized light and reaction in a chiral environment) (2.3, diastereomers may have different properties. For metolachlor and dimethenamid, the properties of the enantiomers of one pair may thus differ from the properties of the enantiomers of the other pair, and the two pairs need not be formed in equal amounts in a synthesis. Therefore, the four stereoisomers of metolachlorand dimethenamid may be present in a ratio differing from 1:l:l:l. However, the enantiomers in each 2026
ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 8. 1995
diastereomeric pair should still be in a 1:l ratio, with zero net optical rotation, if synthesized from racemic materials. Presently, we have no information on the actual diastereomeric composition of metolachlor and dimethenamid. Thermal Equilibration of Atropisomers. Atropisomerism depends on the hindered rotation about the phenylnitrogen bond in these compounds. Energy barriers to rotation higher than 100-120 kJ M-l make atropisomers conformationally stable at ambient temperatures (half-lives, 7 > 30 d at 25 “C). A thermal treatment, however, can enforce interconversion via coplanar transition states. In the case of metolachlor, NMR measurements indicated halflives of 50.6 and 3 h for the interconversion of one stereoisomer to the 1:l mixture of atropisomers at 128 and 154 “C, respectively (12). From these data, and using eq 1, the energy barrier to rotation (E,) was calculated as 154.3 (h13) kJ M-I
In ki = In A - E,/(RT)
(1)
whereby ki is the rate constant of interconversion (h-l; ki = In 2/71, A is a constant (frequency factor), R is the gas constant of 8.3144 J M-’ K-I, and T is the absolute temperature (K). Using eq 1, t was calculated to 2.3 min for a temperature of 200 “C. Assuming similar or lower energy barriers for other acetamides, heating to 200 “C should thus rapidly interconvert one axial-chiral stereoisomer to a mixture of both atropisomers, whereas this treatment is not expected to affect C-chiral stereoisomers. A thermal treatment for 60 min at 200 “C is expected to lead to ’95% interconversion ( t= 4.32~)for atropisomers with 7 13.9 min and thus energy barriers of ‘161 kJ M-l, as calculated from eq 1. Semiempiricalquantum chemistry (MNDO calculations) on model compounds [a = H, CH2C1;b = CH3,CH(CH3)21 indicated energy barriers of rotation for metolachlor and
acetochlor of 100 kJ M-l, for alachlor of 105 kJ M-l, for metalaxyl of 95 kJM-l, and for dimethenamid of 70 kJM-l (24. Although for metolachlor the calculatedvalue deviated significantly from the experimental value (154 kJM-l, see above), the data suggest a similar energy barrier for acetochlor but a substantially (30kJ M-9 lower barrier for dimethenamid. Such a lower barrier would lead to much faster interconversion. Assuming the same mechanism for the interconversionof these species, the half-lives expected for dimethenamid isomers according to eq 1 are estimated to be 2 s at 154 "C (=5000x shorter than for metolachlor), 1 h at 85 "C, and x3 months at 25 "C. After thermal equilibration, atropisomers should be present inastatistical (1:l)or near 1:l ratio. For acetochlor, this means that an individual stereoisomer (such as as-)is enantiomerized to its antipode (such as aR-)and finally transformed to a racemic (or near racemic) mixture. For metolachlor (two elements of chirality),thermal equilibration of a stereoisomer (such as aS,l'S-) will not lead to its antipode but to a diastereomer (such as aR,l'S-) since only the axial-chiral element is reversed but not the C-chiral element. Non-1:1 diastereomeric mixtures thus are expected to be transformed into 1:1 diastereomeric mixtures or to 1:l:l:l stereoisomericmixtures from 1:l enantiomeric mixtures. Achiral and Chiral Chromatography of Acetamides. All five acetamides showed single peaks when analyzed using achiral HRGC (see Figure la,c). The 22-m SiILOV HRGC column resolved all five compounds but showed no resolution of the diastereomers of metolachlor and dimethenamid, even when using temperature programming as slow as 0.5 "Clmin. Similarily, no isomer resolution was observed when using a 60-m OV1701 HRGC column (theoreticalplate number, 200 000).Apparently, resolution of diastereomers resulting from axial- and C-chirality is not easily achieved even when using efficient and polar HRGC columns, whereas diastereomers resulting from C-chirality alone often are easier resolved (26,27). The E1 SIM chromatograms in Figure lb,d now show some isomer resolution when these acetamides were analyzed using the chiral OV1701-BSCD HRGC column. Whereas alachlor still eluted as a single peak, metalaxyl and dimethenamid were clearly resolved into two peaks, and metolachlor resolved into three peaks. Acetochlor eluted as a broadened peak (increased peak width), indicating some marginal resolution of its enantiomers. No resolution or just marginal resolution was observed when using chiral OV1701-GDEX,OV61-PMCD, or PS086DMPCD HRGC columns although these columns resolved other chiral compounds such as a-hexachlorocyclohexane (a-HCH, data not shown). E1 mass spectra of enantiomers are expected to be identical, but in principle, mass spectra of diastereomers may differ. In Figure 2a-c, we show E1 mass spectra for the three peaks of metolachlor (M+, = 283). The mass spectra were virtually identical despite the diastereomeric relationship of some components (see below). Mass spectra of the individual acetochlor, metalaxyl, and dimethenamid stereoisomers were expectedly identical. Using HPLC, the chiral system I (ChiralcelOD-R)showed some resolution for metalaxyl, acetochlor, and metolachlor, and the chiral system I1 (Pirkle) showed some resolution for dimethenamid (see Figure 3a-e). Expectedly, alachlor still eluted as a single peak from both HPLC systems. Chiroptical measurements allowed some assignments of
FIGURE 2. El mass spectra of the three peaks for metolachlor, analyzed using the chiral OV1701-BSCD column: (a) peak 1 (PI); (b) peak 2 (~2);and (c) peek 3 (p3). Note the virtually identical mass spectra despite the diasteraomeric relationship of p2 with p1 and Pa.
levo- and dextrorotatory stereoisomers (see below). In the case of metalaxyl, the two peaks observed using the chiral OV1701-BSCDHRGC column (see Figure Id) are the 1's-(+I- and 1'R-(-)-enantiomers, present in an approximate 1:l ratio. Chiral HPLC (ChiralcelOD-R)showed good resolution (R = 5) (see Figure 3c), and chiroptical measurements indicated the (+)-enantiomer as earliereluted and the (-)-enantiomer as later-eluted. When isolates of these HPLC peaks were reanalyzed using the chiral OV1701-BSCDHRGC column, the same enantiomer elution order was observed (see Table 1). Thermal equilibration of small aliquots of these isolates and reanalysis by HRGC indicated no interconversion of one into the other enantiomer (no change in ER values), a finding consistent with the C-chiral relationship of the two enantiomers (data not shown). In case of acetochlor, a broadened peak indicated just marginal resolution of the as- and aR-enantiomers, when the chiral OV1701-BSCDHRGC columnwasused (see Figure IC). Apparently, the resolution of enantiomers with axialasymmetry is more difficult than that of enantiomers with C-asymmetry, using the present chiral selectors. Chiral HPLC (ChiralcelOD-R) also showed just marginal resolution ( R = 0.2; see Figure 3b); nevertheless, chiroptical measurements indicated the (-)-enantiomer as earlier-eluted and the (+)-enantiomeras later-eluted. When enantioenriched VOL. 29, NO. 8, 1995 I ENVIRONMENTAL SCIENCE & TECHNOLOGY
2027
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70 60
P3
50
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-24
16
d
16
~3,1
30
20 10
I
28
16
I
24
e
-16
24
40
chiral HPLC chiral HRGC Chiralcel OV1701-BSCDc OD-RC Pirkle P2 PI P1 P2 P3 P3 P2 Pl
nr nr nr nr nr nr nr nr
nr nr nr nr
P1 P2 P2 P3
a For column dimensions and operating conditions, see text; nr = not resolved bya particular system. Denotations reflect elution order from the chiral HRGC system; the sign of optical rotation indicated if known from chiroptical measurements; dia-I and dia-2 denote diastereomeric pairs of enantiomers. Peak denotations reflect stereoisomer elution order (p,= first-eluted, etc.), the same denotation for two stereoisomers implies that the two are unresolved on a particular system. x,Y Dimethenamid atropisomers are likely conformationally unstable at HRGC column temperature.
HPLC isolates (front- and back-cuts) were reanalyzed using the chiral OV1701-BSCD HRGC column, a reversed elution order was revealed (see Table 1). When small aliquots of these isolates were equilibrated thermally and reanalyzed, they revealed approximate 1:l ratios, consistent with the atropisomeric relationship of the two enantiomers (see Figure 4). In the case of metolachlor, three partiallyresolved peaks of about equal intensity were observed when the 1975dated sample was analyzed using the chiral OV1701-BSCD HRGC column (see Figure IC). The approximate 1:1:1peak area ratio was independent of the rnlzvalues and of different ion transitions monitored in MS/MS experiments (data not 2028
, , , / , , , , , ,
10
Enantiomer/Stereoisomer Elution Onlets of Acetamid Pesticides Using Chiral HRGC and C h i d HPLP
P1 P2 P1 P2 P1 P2 P2 P3 Ply PIX P2" PZY
~~~
20
48
TABLE 1
(+)-acetochlor (-)-acetochlor S-(+)-metalaxyl R-( - )-rnetalaxyl (+I-metolachlor-1 (dia-1) metolachlor-2 (dia-2) metolachlor-3 (dia-2) (-)-metolachlor-4 (dia-1) (-)-dimethenamid-I (dia-I) dimethenamid-2 (dia-2) (+)-dimethenamid-3 (dia-I dimethenamid-4 (dia-2)
, , , , , , , , , , , ,
30
FIGURE3. HPLCchromatograms (UV detection)of racemicacetamide pesticides using (a-dl c h i d HPLC system I (Chiralcel)end (e) chiral HPLCsystem II(Pirkle)showing elution of (a) alachlor, (b) ecetochlor, (c) metalexyl, (d) metolachlor, and (e) dimethenamid. Note the differing isomer resolutions. For peak denotations, see text and Table 1.
compounds*
~"i i
40
ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 8 , 1 9 9 5
0 2O:OO
22:OO
24:OO
TI
20:OO
22:OO
24:OO
TI
FIGURE 4. El SIM chromatograms ( d z 238, upper panels; d z 223, lower panels) showing elution of metolachlor and acetochlor in untreated (left panels) and thermally equilibrated samples (right panels), using the OV1701-BSCD HRGC column: (a and b)Technical metolachlor end (c and d) enantioenriched (HPLC, back-cut) acetochlor. Note the changed peak area ratios for metolachlor and acetochlor after thermal equilibration due to interconversion of atropisomers. For abbreviations, see Figure 1. For peek denotations, see text and Table 1. Note the presence of signals for alachlor (AL) in chromatograms a and b.
shown). This ratio indicates that the diastereomeric pairs cannot be present in a 1:1 ratio but are more likely present in a 2:l ratio with one pair enantiomerically unresolved. When this sample was equilibrated thermally, the relative peak areas changed to an approximate 1:2:1ratio (seeFigure 4). This result indicated the stereoisomers to be present in a 1:1:1:1 ratio after thermal equilibration with two stereoisomers unresolved as peak 2. The total area of the peak envelope remained unchanged, and therefore no decomposition of metolachlor occurred during this treatment. The increase of peak 2 must arise from an interconversion of the atropisomers in peaks 1 and 3. These data indicate that peaks 1 and 3 must constitute one pair of enantiomers, and peak 2 must constitute the other, unresolved pair of enantiomers. Analysis of additional samples of technical metolachlor showed varied peak area ratios (see Figure 5a-d). Whereas the 1975-dated sample had an approximate 1:1:1peak area ratio, the ratio of the 1988-dated sample was almost 1 2 1 , and the ratios of the 1993- and 1994-datedsamples were 1:1.3:1. The results show that metolachlor has a varied stereoisomeric composition, whereas the peak area ratios p1lp3 of 1:l still indicate racemic mixtures. Chiral HPLC (Chiracel OD-R) of the 1988-datedmetolachlor showed three partially resolved peaks in an approximate 1:1:2 ratio. This indicates coelution of two stereoisomers, as was observed when using chiral HRGC. Isolates of the first two HPLC peaks and a front- and backcut of the major HPLC peak revealed a reversed elution order of the four stereoisomerswhen reanalyzed using the chiral OV1701-BSCD HRGC column (see Table 1). Chiroptical measurements indicated the first-eluted HPLC peak (metolachlor-4) to have levorotation and the last-eluted HPLC peak (metolachlor-1 or metolachlor-2) to have
:I
20
, , , , , , , ,,
j,,I_i
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FIGURE5. El SIM chromatograms (nJz238)showing partial resolution of the four stereoisomers of metolachlor in (a) technical metolachlor, 1975 (b) technical metolachlor, 198& (c) formulated metolachlor, 1993; and (d) formulated metolachlor, 1994. Note the varying stereoisomeric composition of these products. For peak denotations, see text and Table 1.
dextrorotation; the directions of rotation of the other components were inconclusive. In comparison to ref 12, the data indicate the dextrorotatory metolachlor-1 to have as-configuration (aS,l'S- or aS,1'R-1 and its levorotatory antipode, metolachlor-4, to have aR-configuration. A full assignment however was not possible. In the case of dimethenamid, two fully resolved ( R = 1.2) peaks of about equal intensity were observed when analyzed using the chiral OV1701-BSCD HRGC column (see Figure IC). We presume that the dimethenamid atropisomers are conformationally unstable at the column temperatures used and that the peaks represent the 1'Rand 1's-stereoisomers (elution order not known), respectively. Chiral HPLC (Pirkle),however, revealed three peaks in an approximate 1:2:1 ratio, indicating the presence of two unresolved stereoisomers in peak 2. The three HPLC peaks were isolated and reanalyzed using chiral HRGC (see Figure 6a-c). The HRGC chromatograms confirmed the above presumptions and indicated elution orders as listed in Table 1. A n ER value of 0.8 for the isolate of peak 2 indicated some marginal resolution of the two stereoisomers by the HPLC system. Chiroptical measurements were only partially successful. The low capacity of the HPLC system (=lo pg) and the incomplete resolution prevented proper assignment of optical rotation to all four stereoisomers. Nevertheless, the chiroptical measurements indicated the first-elutedHPLC peak (dimethenamid-1)to be levorotatory followed by a larger dextrorotatory peak envelope. No resolution of these stereoisomers was observed by HPLC when Chiralcel OD-R was used. Thermal equilibration of technical dimethenamid and its isolated HPLC fractions did not change the relative peak areas in the HRGC chromatograms significantly (see Figure Sd-0. This finding confirms that dimethenamid is resolved by HRGC according to C-chiralityalone. The 1:1 peak area ratio observed when using HRGC and the 1:2:1 ratio when using HPLC indicate a 1:l:l:l ratio of the stereoisomers in the technical product at ambient temperature.
50 40
30 20 10
FIGURE 6. El SIM chromatograms (nJz 230) showing elution of dimethenamid stereoisomers in untreated (laR panels) and in the thermally equilibrated, enantioanriched HPLC fractions (right panels): (a and b) Pirkle peak 1, (c and d) Pirkle peak 2, and (e and f) Pirkle peak 3. Note that the peak area ratios are insignificantly changedupon thermal equilibration indicatinga C-chiral relationship. Note also that peak 2 was recovered as a non-1:1 mixture from the Pirkle column. For peak denotations, see text and Table 1. Note that dimethenamid atropisomers are likely conformationally unstable at the HRGC conditions used.
Conclusions Stereoisomerismfor these compounds results from axialand/or C-chirality and leads to one pair of enantiomers for compounds with one element of chirality (acetochlor, metalaxyl) and to two diastereomeric pairs of enantiomers for compounds with two elements of chirality (metolachlor, dimethenamid). The two elements (C-chirality and axialchirality) have different stabilities, influencing both the composition in the technical materials and the analytical requirements. The resolution of several stereoisomerswas achieved using both chiral HRGC and chiral HPLC, whereby C-chiral enantiomers were easier resolved than axial-chiral enantiomers. For instance, metalaxyl (C-chiral)was fully resolved whereas acetochlor (axial-chiral)was only marginally resolved by both techniques. For metolachlor and dimethenamid (both C- and axial-chiral), the four steieoisomers appear to be stable at ambient temperature. Chiral HPLC resolved both compounds into three peaks with one pair of isomers unresolved. However, at elevated temperatures such as those used in HRGC, dimethenamid appears to be less stable than metolachlor. Whereas metolachlor was still resolved into three peaks, just two peaks were observed for dimethenamid. It is presumed that dimethenamid atropisomers are unstable (rapid interconversion) at the HRGC column temperaturesused (70170 "C),as indicated by the lower energy barrier to rotation calculated by the MNDO calculations. As a consequence, VOL. 29, NO. 8, 1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY
2029
dimethenamid can probably be correctly analyzed for all stereoisomers by analytical methods such as chiral HPLC or capillary zone electrophoresis carried out at ambient temperatures, but likely not by chiral HRGC. Excessive temperatures during GC analysis (injection andlor elution temperatures) would also lead to topomerization of the other compounds. For instance at 250 "C, topomerization of metolachlor is expected (eq 1) to proceed with t = 3.2 s. Clearly, the use of such high temperatures is intolerable and would lead to changed atropisomer ratios. When using achiral HRGC, surprisingly no resolution for the diastereomers of metolachlor (and dimethenamid) was observed, even under optimum separation conditions (high theoretical plate numbers of the columns; slow temperature programming). Sincethere are several reports showing the resolution of diastereomers resulting from C-chirality alone (26, 27), it appears that diastereomers resulting from axial- and C-chirality are more difficult to separate. In contrast, chiral HRGC showed some resolution of stereoisomers. These analyses revealed a varied diastereomeric composition (1:l to 21) for different batches of metolachlor. This finding is of interest because these stereoisomers may have a different herbicidal activity. Thermal equilibration was used to distinguish among axial-chiral and C-chiral stereoisomers. This treatment resulted in approximate 1:1mixtures of atropisomers from samples with an excess of one or the other atropisomer whereas C-chiral enantiomers remained unaffected. By careful observation of changes in the peak ratios, a distinction among these stereoisomers was thus possible. The procedure was carried out with HPLC isolates, with technical materials (metolachlor) having a non- 1:l diastereomeric composition, and with residues (amounts in ng) from the stereo- and enantioselective degradation of these compounds in sewage sludge and soil (19). Stereoselectiveanalyses of atropisomers require special attention. Because of possible topomerization,fast analyses at the lowest possible temperatures are required. This can be achieved by using short but efficient HRGC columns operated at high carrier gas velocities and preferably using hydrogen. In our study, however, hydrogen was not used for safety reasons. Low-temperature, on-column injection should be used in these analyses. The identification of the stereoisomers of these compounds was only partially successful. For acetochlor and metalaxyl, the (+I- and (-)-enantiomers were assigned using chiral HPLC and chiroptical measurements. Enantioenriched isolates were then used for identification using chiral HRGC. For metalaxyl, the absolute configurations of these enantiomers are known (141, but for acetochlor, they are s t i l l unknown. For metolachlor and dimethenamid, each consisting of four stereoisomers, only a partial assignment was possible due to incomplete resolution in HPLC leading to loss of optical rotation with chiroptical detection. The analytical techniques outlined should be valuable for investigating the environmental behavior of these and other related compounds. In the following paper (19), the application of these techniques to environmental studies is described and preliminary results are reported.
Schelling, Sandoz Agro, Basel Switzerland. We thank M. Galli, Legnano, Italy for the gift of dimethyl-pentyl-B-CD, and F. Miiller-Plathe, Department of Chemistry, Swiss Federal Institute of Technology, Zurich, Switzerland, for the MNDO calculations. Furthermore, we acknowledge grants for the chiroptical detector to G. Karlaganis, Swiss Federal Office of Environment, Forests and Landscape, Bern, Switzerland.
Literature Cited (1) Worthing, C. R., Hance, R. J., Eds. ThePesticideManual, 9th ed.; British Crop Protection Council: Farnham, UK, 1991. (2) Sharp, D. S. In Herbicides: Chemistry, Degradation and Mode
of Action; Kearney, P. C., Kaufman, D. D., Eds.; Marcel Dekker Inc.: New York, 1988; Vol. 3, pp 301-333. (3) LeBaron, H.; McFarland, J. E.; Simoneaux, B. J. In Herbicides: Chemistry, Degradation and Mode of Action; Kearney, P. C., Kaufman, D. D., Eds.; Marcel Dekker Inc.: New York, 1988;Vol. 3, pp 335-382. (4) Anonymous. Environ. Sci. Technol. 1994, 28, 355A. (5) Humburg, N. E.; Colby, S . R.; Hill, E. R.; Kitchen, L. M.; Lym, R. G.; McAvoy, WJ.; Prasad, R. Herbicide Handbook of the Weed Society ofAmerica,6th ed.; Weed Society ofAmerica: Champain, IL, 1989. (6) Chesters, G.; Simsiman, G. V.; Levy, J.; Alhajjar, B. J.; Fathulla, R. N.; Harkin, J. M. Rev. Environ. Contam. Toxicol. 1989, 110, 1-74. (7) Buchenauer, H. In Chemistry of Plant Protection; Haug, G., H o h a n n , H., Eds.; Springer Verlag: New York,1990;Vol. 6, pp 234-238. (8) Montgomery, J. H.Agrochemica1DeskReference, Environmental paca; Lewis Publishers: Chelsea, MI, 1993. (9) Aberg, B. In The Chemistry and Mode ofAction of Plant Growth
Substances; Wain, R. L., Wightman, F., Eds.; Butterworths Scientific Publications: London, 1956; p 102. (10) Venis, M. A. Pestic. Sci. 1982, 13, 309-317. (11) Buser,H.R.;Muller,M.D.Environ.Sci. Technol. 1993,27,12111220. (12) Moser, H.; Rihs, G.; Sauter, H. Z. Naturforsch. 1982, 87B, 451462. (13) Eckhardt, W.;Francotte, E.; Herzog, J.; Margot, P.; Rihs, G.; Kunz, W. Pestic Sci. 1992, 36, 223-232. (14) Hubele, A.; Kunz, W.; Eckhardt, W.; Storm, E. Proceedings of the
5th IUPACCongress on Pesticide Chemistry, Miyamoto; Kearney, P. C., Ed.; Pergamon Press: NewYork, 1988;Vol. 2, pp 233-242. (15) Oehmichen, U.; Haberer, K. Vom Wasser 1986, 66, 225-241. (16) Pereira, W. E.; Rostad, C. E. Environ. Sci. Technol. 1990,24,14001406. (17) Thurmann, E. M.; Goolsby, D. A.; Meyer, M. T.; Mills, M. S.; Pomes, M. L.; Kolpin, D. A. Environ. Sci. Technol. 1992,26,24402447. (18) Squillace, P. J.; Thurman, E. M. Environ. Sci. Technol. 1992,26, 538-545. (19) Muller, M. D.; Buser, H. R.Environ. Sci. Technol. 1995,29,20312037. (20) Buser, H. R.; Muller, M. D. Anal. Chem. 1992, 64, 3168-3175. (21) Bicchi, C.; d'Amato, A.; Artuffo, G.; Manzini, V.; Galli, A,; Galli, M. J. High Resolut. Chromatogr. 1992, 15, 710-714. (22) Muller, M. D.; Buser, H. R. Anal. Chem. 1994, 66, 2155-2162. (23) Eliel, E. L.; Wilen, S. H. Stereochemistry of Organic Compounds; John Wiley and Sons: New York, 1994. (24) Muller-Plathe, F. Personal communication, 1994. (25) March, J. Advanced Organic Chemistry, 3rd ed.; John Wiley and Sons: New York, 1985; pp 82-140. (26) Papadopoulou-Mourkidou, E. In Analytical Methodsfor Pesticides and Plant Growth Regulators; Sherma, J., Ed.; Academic Press, Inc: New York, 1988; Vol. XVI, p 191. (27) Muller, M. D.; Bosshardt, H. P. J. Assoc. O@c. Anal. Chem. 1988, 71. 614-617.
Received for review November 18, 1994. Revised manuscript received April 4, 1995. Accepted April 13, 1995.@
ES940711V
Acknowledgments We acknowledge discussions and comments from H. Egli, Ciba, Basel, Switzerland, and from H. Sauer and H. P.
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@Abstractpublished in Advance ACS Abstracts, June 1, 1995.
