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Separation of sulfonylurea metabolites in water by capillary electrophoresis

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Separation of sulfonylurea metabolites in water by capillary electrophoresis
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  ELSEVIER Journal of Chromatography A, 700 (1995) 195-200 JOURNAL OF CHROMATOGRAPHY A Separation of sulfonylurea metabolites in water by capillary electrophoresis Giovanni Dinelli*, Alberto Vicari, Alessandra Bonetti Dipartimento di Agronomia, Universit~ di Bologna, via F. Re 6/8, 1-40126 Bologna, Italy Abstract The potential of capillary electrophoresis (CE) for the separation and detection of the metabolites of nine sulfonylurea herbicides in aqueous solution was evaluated. A relationship between the structure of the sul- fonylureas tested and the metabolites formed was found: the non-o-benzene-substituted sulfonylurea rimsulfuron gave only one metabolite, whereas the other eight, o-benzene-substituted, sulfonylureas gave 4-6 metabolites. CE was confirmed to be a very efficient separation technique, suitable for the determination of sulfonylurea herbicides and their metabolites formed during hydrolysis. I. Introduction Sulfonylurea herbicides are widely used to control weeds in agricultural crops such as wheat, maize, soybean, sugarbeet and rice. The first sulfonylurea, chlorsulfuron, was marketed in USA in 1982. Worldwide, nineteen sulfonylurea herbicides had been commercialized by 1994, and five more are being developed. This rapid increase is due to their activity at low application rates (2-60 g ha -~) and their low mammalian toxicity [1]. In recent years, because of the increased herbicide degradation product analysis require- ments for pesticide registration, considerable interest has been generated concerning the de- tection and separation of herbicide metabolites. The most important degradation pathways of sulfonylureas are chemical hydrolysis and micro- bial breakdown [2,3]. Although several experi- ments have shown that sulfonylureas degrade to * Corresponding author. 0021-9673/95/$09.50 © 1995 Elsevier Science B.V. All rights SSDI 0021-9673(94)01168-0 many compounds in water and soil [4-6], not many methods for the separation of sulfonylurea metabolites have been described. Sabadie and Bastide [5,6] and Harvey et al. [7], using HPLC, separated several hydrolytic metabolites of chlor- sulfuron, metsulfuron and sulfometuron. Shalaby et al. [8] described the use of LC-MS thermo- spray for nicosulfuron and rimsulfuron and a major metabolite of each in soil. However, little information is available on the detection and separation of metabolites formed during the hydrolysis of most sulfonylureas. Because hy- drolysis is a major pathway of degradation of sulfonylureas, investigations on metabolites formed during hydrolysis should provide basic information on their general behaviour. A simple and reproducible method was needed for the determination of sulfonylurea metabolites in water samples. In a previous paper [9], we reported the detection and sepa- ration of the hydrolytic breakdown products of metsulfuron by capillary electrophoresis (CE) and their structural identification by GC-MS. reserved  196 G. Dinelli et al. / J. Chromatogr. A 700 1995) 195-200 The objectives of this study were to confirm the potential of CE for the detection and separation of the breakdown products of nine sulfonylureas in aqueous solution and to evaluate the dynamics of metabolite formation for each sulfonylurea. 2. Experimental 2.1. Reagents Reagents for CE analysis were supplied by Sigma (St. Louis, MO, USA). All solvents used were of pesticide-free grade. The nine sul- fonylureas studied were chlorsulfuron, metsul- furon, triasulfuron, ethametsulfuron, CGA 152'005, tribenuron, bensulfuron, chlorimuron and rimsulfuron. They were extracted from com- mercial products by Soxhlet extraction with freshly redistilled dichloromethane for 3 h. After drying with anhydrous sodium sulfate, dichloro- methane was distilled off in a rotary evaporator. The residual sulfonylureas were subjected to nuclear NMR, IR and MS analyses to confirm their identity and were used in subsequent ex- periments without further purification, as re- ported by Galletti et al. [10]. 2.2. Water samples fortification The solutions were prepared using distilled water. Duplicate 50-ml water samples, each containing 25 mg 1-1 of each sulfonylurea in 10 mM NaHCO3, were buffered to pH 4 with 0.1 M HCI. The solutions were kept in the dark at 55°C in closed vials. Samples of 1 ml were taken from each vial at different times during a 10-h period and stored at -12°C until analysed. 2.3. Capillary electrophoresis Aqueous samples were analysed directly by CE. Separation of herbicides and metabolites were performed using the micellar electrokinetic capillary chromatography with a P/ACE system from Beckman (Palo Alto, CA, USA). Sepa- rations were effected with a fused-silica capillary 50 cm long (from injection point to detector) × 75 /xm I.D. at a constant temperature of 25°C. The applied voltage was 25 kV, with an injection pressure of 3.44.103 Pa for 10 s, corresponding to an injection volume of 60 nl. The electrolyte buffer was 50 mM sodium borate-22 mM sodi- um dodecylsulfate-10% (v/v) methanol (pH 8.0). The separation efficiency was measured by the number of theoretical plates (N) according to the equation N= 5.54 tg/W) 2, where t a is the retention time of a compound and w is the peak width at half-height [11]. Peak area was used for residue quantification. The degradation rate of each sulfonylurea was determined by linear re- gression of the natural logarithm of percentage of parent herbicide remaining against time and the slope of each line was compared with analy- sis of variance. 3. Results and discussion The structures and molecular masses of the sulfonylureas studied are reported in Fig. 1. Three moieties characterize the general struc- ture: an aryl group, the sulfonylurea bridge and a nitrogen-containing heterocycle. Chlorsul- furon, metsulfuron, triasulfuron, ethametsul- furon, CGA 152'005 and tribenuron have a triazinic heterocycle group and are o-benzene- substituted; bensulfuron and chlorimuron have a diazinic heterocycle group and are o-benzene- substituted; and rimsulfuron has a diazinic heterocycle group but is not o-benzene-substi- tuted. The electropherograms of chlorsulfuron at three sampling times (0, 2 and 10 h after incuba- tion) are shown in Fig. 2, as an example of sulfonylurea hydrolysis. Chlorsulfuron degrada- tion in water led to the formation of six metabo- lites, numbered according to their increasing retention times. The electropherograms evidence the effectiveness of the separation, as suggested by the column efficiency, ranging between 50 000 and 163 000 theoretical plates. The retention times of each parent herbicide and their metabolites are shown in Table 1. The metabolites of each sulfonylurea were arbitrarily numbered according to their increasing retention  G. DineUi et al. / J. Chromatogr. A 700 1995) 195-200 197 CIt 3 N ~/~ SO2NHCONH/~ N//J~ OCH3 CHLORSULFURON M 357 CH 3 c°2cH CH 3 TRIBENURON M 395 CH 3 f~ CO2CH3 N/~N SO2NHCONH N- OCH METSULFURON METHYL M 381 f• O2CH3 N ~-~ H3 ~/J~ CH2SO2NHCONH/J~ N//[~- OCH3 BENSULFURON M 410 f~ OCH2CH2CI N/~3N ~/~x SO2NHCONH/J~ N//~ OCH3 TRIASULFURON M 401 CI f~ CO2CH2CH N~N ~/~ SO2NHCONH/~ N//J~ OCH3 CHLORIMURON M 414 OCH2CH ~SO2NHCONH ~ N//~ NHCH ETHAMETSULFURON M 410 ~CH2CH 2CF N~3N ~ SO2NIiCONH/~ N//~ OCH3 CGA 152'005 M 419 f~l ~SO2CH2CH3 N~3 so2 co N o H 3 RIMSULFURON M 431 Fig. 1. Structures and molecular mass of the sulfonylureas tudied, times. The number of metabolites formed during hydrolysis appeared to be related to the structure of the sulfonylurea. The sulfonylureas character- ized by a triazinic heterocycle and o-benzene substitution decomposed in water, leading to at least six possible degradation products, with the exception of ethametsulfuron and tribenuron, which gave five metabolites. The concentration curves of the parent herbicides and their metabo- lites (Fig. 3) indicate that metabolites 2 and 4 of chlorsulfuron, metsulfuron, triasulfuron and CGA 152'005 appear with a delay of 1-3 h with respect to other metabolites, suggesting that they are secondary by-products of sulfonylurea hy- drolysis. The same pattern was observed for the metabolite 3 of ethametsulfuron and metabolite 2 of tribenuron. Chlorimuron and bensulfuron, sulfonylureas characterized by a diazinic  198 G. Dinelli et al. / J. Chromatogr. A 700 1995) 195-200 0.02 a.u. t a.i. a.i. b 3 _ I 0.002 a.u. I1 t 4 5 c :3 0.002 a.u. T it 4 58 TIME min) Fig. 2. Electropherograms of chlorsulfuron samples in aque- ous solution. Samples in (a), (b) and (c) correspond to sampling times of 0, 2 and 10 h after incubation, respectively. The parent herbicide is indicated by a.i. and metabolites are arbitrarily numbered according to their increasing retention times. Separation conditions as reported under Experimen- tal. heterocycle and o-benzene substitution, de- graded in water to form four metabolites (Fig. 3). The non-o-benzene-substituted sulfonylurea rimsulfuron gave only one metabolite (Fig. 3). Hydrolysis of all the sulfonylureas followed first-order kinetics, as regression analysis of the natural logarithm of herbicide remaining yielded significant linear determination coefficients for all nine sulfonylureas (Table 2). Analysis of variance showed differences in the degradation rate constants (k) among the sulfonylureas. Tri- benuron, the sulfonylurea most susceptible to hydrolysis, had a half-life of 0.37 h, whereas those for triasulfuron, ethametsulfuron and chlorimuron, the least susceptible, were >2 h. The seven times longer half-life of chlorimuron than tribenuron confirms the large differences in degradation rate within this class of herbicides, as observed in other work [1,12]. It is also clear that many sulfonylureas are extremely labile in aqueous solution and most of them undergo various transformation reactions to generate a complex mixture of decomposition products. This study revealed that sulfonylureas and their degradation products may be simultaneous- ly detected and separated by CE. The ease and efficiency of the method make it suitable for the analysis of large numbers of water samples for sulfonylurea metabolites and parent herbicides. However, the nature of the degradation products was not investigated in this work. Further studies are necessary to identify their structures and to Table 1 Retention times (min ±S.D., n = 16) of sulfonylureas and their metabolites Sulfonylurea Parent Metabolite No. herbicide 1 2 3 4 5 6 Chlorsulfuron 4.26 ± 0.11 3.19 ± 0.09 3.32 ± 0.08 3.55 ± 0.09 4.33 ± 0.10 4.62 ± 0.12 Metsulfuron 4.45 --- 0.07 3.36 ± 0.06 3.50 ± 0.04 3.54 ± 0.06 4.50 ± 0.08 4.64 ± 0.09 Triasulfuron 4.51 ± 0.09 3.36 --- 0.06 3.51 ± 0.03 3.61 - 0.04 4.73 ± 0.06 5.01 -+ 0.08 CGA 512'005 4.57 ± 0.10 3.38 -+ 0.06 3.55 ± 0.07 4.14 -+ 0.09 4.67 ± 0.08 4.77 -+ 0.11 Ethametsulfuron 4.62 ± 0.05 3.26 --- 0.08 3.62 ± 0.03 4.70 -+ 0.05 4.93 ± 0.06 5.20 --- 0.05 Tribenuron 4.31 ± 0.03 3.19 ± 0.02 3.39 ± 0.04 3.49 ± 0.05 4.68 ± 0.03 4.81 ± 0.04 Bensulfuron 4.39 ± 0.08 3.47 ± 0.09 3.63 ± 0.07 4.56 -+ 0.08 4.82 ± 0.09 - Chlorimuron 4.96 ± 0.05 3.99 --- 0.08 4.31 ± 0.02 5.53 ± 0.04 5.87 --- 0.03 - Rimsulfuron 4.59 - 0.03 6.10 ± 0.07 .... 4.78 ± 0.11 4.92 ± 0.05 5.37 --+ 0.06 4.95 -+ 0.09  o o chlorsulfuron ~o . x x, x% O. 0 2 4 --w-- chlorsuluron • 1 ---o--- 2 • 3 J. 4 • -<P-- #5 • 6 8 10 t% ---~-- me~luron metsulfuron • ' 8 2 #6 10' O~ O, 0 2 4 6 8 10 . ---W'-- oga ~ ,~, CGA 152 005 • :~ ~,,,, etllametsulfuron ..... 1°thametsulfur°n ~o ' , .4'3 20 1 ' - ,3r~ 5 ~ 14 k'"'X' x% I0 10 '~' X,, O' 0 ) 2 4 6 8 10 0 2 4 6 8 10 0' I0' ).g.~ '0 , bensulfuron ---.~-. bo~ul,ron ,,,. chlorimuron , 8 #1 20 ~'~ ~X. .I. #3 X . . . M #4 k. ~K, 10 ~', 2 4 6 8 10 0 2 4 - - - N-- - chlodmuron • 1 • 2 • ¢ 3 #4 ; 8 10 0 I0' ), i rimsulfuron I0 ~:" 0 x "":- m .... . ........ ,- 0 2 4 6 8 I trlasulfuron ....~.. ~s,,uron • 1 8 2 • J. #4 2 4 6 8 10 trlbenuron - - - M.-. tdbenuron ;~ : #3 .' #4 -- T i i | i 2 4 6 8 10 -- - le-- dmsuluron • 1 I • Time after incubation h) :ig. 3. Hydrolysis of the nine sulfonylureas and formation of degradation products.
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