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High-performance liquid chromatographic determination of sulfonylureas in soil and water

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High-performance liquid chromatographic determination of sulfonylureas in soil and water
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  ELSEVIER Journal of Chromatography A, 692 (1995) 27-37 JOURNAL OF CHROMATOGRAPHY A High-performance liquid chromatographic determination of sulfonylureas in soil and water Guido C. Galletti a, Alessandra Bonetti b, Giovanni Dinelli b'* lstituto di Microbiologia e Tecnologia Agraria e Forestale, Universita di Reggio Calabria, P.zza S. Francesco 4, 1-89061 Gallina, Reggio Calabria, Italy bDipartimento di Agronomia, Universita di Bologna, via F. Re 8, 1-40126 Bologna, Italy Abstract Isocratic and gradient conditions for the separation of four sulfonylurea herbicides, namely chlorsulfuron, metsulfuron, chlorimuron and thifensulfuron, by reversed-phase high-performance liquid chromatography (HPLC) on C 6 and C~8 columns were established. Liquid-liquid (LL) and solid-phase extraction (SPE) procedures for the extraction and concentration of the herbicides from water and soil samples were tested. LL and SPE recoveries, HPLC detection limits and repeatability and dependence of the capacity factor on mobile phase composition are discussed. Typical chromatograms are shown. 1. Introduction Sulfonylureas are a class of herbicides char- acterized by low application rates (typically in the range 10-100 g ha -1) and low toxicity to mammals. Such molecules are formed by three moieties, generally (i) a monosubstituted ben- zene ring, (ii) a diazinic or triazinic ring with various substituents and (iii) a sulfonylurea bridge (Fig. 1). In some instances, a disubsti- tuted benzene, a thiophene, a pyridine or a non-aromatic moiety is present as moiety (i) (Fig. 1). Various methods have been published for the determination of sulfonylureas. The most recent papers include bioassay [1], enzyme immuno- assay [2], gas chromatography [3,4], capillary electrophoresis [5] and high-performance liquid * Corresponding author. chromatography (HPLC) [6,7]. Each has its own advantages and disadvantages. Bioassays reach very low detection limits (0.1 ppb), but are aspecific. Immunoassays show similar sensitivity and reduce sample work-up and analysis time, but are expensive and not yet commercially available. Two recent papers showed an elegant way to form stable N,N'- dimethyl derivatives of sulfonylureas in soil and water using diazomethane in ethyl acetate [3,4]. However, gas chromatographic analysis contrasts with the low volatility and thermal instability of underivatized and monomethylated sup fonylureas, and is uncommon for such a class of herbicides. Capillary electrophoresis has been used to determine chlorsulfuron and metsulfuron in tap water [5], but has yet to be applied to soil, its main problem being the low sample load- ability. HPLC is the most commonly adopted method 0021-9673/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD1 0021-9673(94)00738-1  28 G.C. Galletti et al. / J. Chromatogr. A 692 (1995) 27-37 O OCH 3 [ SO2NHCONH N CH 3 O OCH 3 v SO2NHCON N CH 3 OCH O OCH ~ ~OCH2CH3 N//~ 4 ~ SO2NHCONH//~ N//J..CI Fig. 1. Structures of sulfonylureas: ] = thifensulfuron; 2 = metsulfuron; 3 = chlorsulfuron; 4 = chlorimuron. products containing different sulfonylureic active ingredients have appeared on market. Surpris- ingly, most HPLC publications have dealt with the simultaneous determination of one or two sulfonylureas only, aimed at improving the de- tection limit rather than increasing the number of detected molecules. Prior to research on sulfonylurea degradation in soil, we decided to test some extraction and HPLC conditions in an attempt to increase the number of sulfonylureas detectable in one run. Four sulfonylureas (Fig. 1) were selected among those commercially available. Three compounds, chlorsulfuron, metsulfuron and chlorimuron, are monosubstituted benzenic sulfonylureas. Thifen- sulfuron is characterized by a monosubstituted thiophene instead of a benzene ring. The former compounds are characterized by long residence times in soil. The latter is representative of a category of sulfonylureas more easily degraded in soil. Such molecules are of less environmental impact, but are more elusive, because they decompose faster. 2. Experimental 2.1. Reagents for sulfonylurea determination in soil and water [6,7]. Chromatographic techniques, including HPLC, need preliminary enrichment steps when analyte concentrations in the sample are below the minimum injectable level. Minimum con- centrations of sulfonylurea standard solutions for HPLC with UV detection are about 1 ppm. Photoconductivity detectors lower such detection limits by about one order of magnitude. Al- though sulfonylurea concentrations as low as 0.2 ppb have been determined in soil using HPLC with photoconductivity detection [7], careful control of various operating parameters is needed to optimize sensitivity and baseline stability [8-10]. In conclusion, photoconductivity detectors are not commonly adopted in HPLC. Since 1982, when the first sulfonylurea her- bicide, chlorsulfuron, became commercially available, at least sixteen different herbicide Reagents for HPLC separations and extraction were pesticide-flee and supplied by Sigma (St. Louis, MO, USA). The sample concentration column for solid-phase extraction consisted in a Bakerbond (Phillipsburg, NJ, USA) Cts (1 g, 40-/~m silica particles). Sulfonylurea commercial products were kindly provided by Professor P. Catizone and Dr. A. Vicari, Department of Agronomy, University of Bologna. 2.2. Sulfonylurea samples Chlorsulfuron, metsulfuron, chlorimuron and thifensulfuron were extracted from commercial formulates with freshly redistilled dichloro- methane in a Soxhlet extractor for 3 h. After dehydration with anhydrous sodium sulfate, di- chloromethane was distilled off in a rotary evaporator. The residual sulfonylureas were sub-  G.C. GaUetti et al. / J. Chromatogr. A 692 (1995) 27-37 29 jected to nuclear magnetic resonance, infrared and mass spectral analyses to confirm their identity and used for subsequent experiments without further purification. Sulfonylurea yields from the commercial formulates ranged from 24 to 71% (Table 1). 2.3. Standard solutions A stock standard solution at a concentration of 100 ppm was prepared by dissolving 10 mg each of the four sulfonylureas in 100 ml of methanol- water (40:60). Appropriate dilutions of this stock standard solution were made with methanol- water (40:60) to obtain working standard solu- tions of 0.25, 0.5, 1, 2, 4, 5 and 10 ppm. A similar procedure was adopted for (a) thifensulfuron, metsulfuron and chlorsulfuron in one stock solution and (b) chlorimuron in a separate stock solution, in order to run "isocratic 2 and 3" respectively (see Section 2.7). 2.4. Water sample fortification and extraction Water (100 mi) containing the four sul- fonylureas (4 ppb each) was passed through a disposable C~s column previously conditioned with methanol (5 ml) and water (5 ml). Ad- sorbed sulfonylureas were eluted with methanol (5 ml). Excess of solvent was removed in a rotary evaporator. The residue was dissolved in 0.01% HC104-methanol (1:1, 100 /xl). An aliquot of this solution was injected into the HPLC system. The whole procedure was done in triplicate. Table 1 Sulfonylurea yield (%) after duplicate Soxhlet extraction from commercial formulations Compound Declared Found Sample 1 Sample Chlorsulfuron 75 71 73 Metsulfuron 20 24 24 Chlorimuron Not declared 27 26 Thifensulfuron Not declared 66 66 2.5. Soil sample fortification The soil used for the trials was a sandy loam soil (58% sand, 15% silt, 27% clay, 1.3% or- ganic matter, pH 6.5) from Cadriano, Bologna, sieved to 3 mm. A stock standard solution containing 1 ppm of the four sulfonylureas was obtained by dissolving 10 mg of each compound in 10 1 of doubly distilled water. A 500-g amount of soil (on an oven-dry basis) was treated by uniform spraying of 25 ml of the stock standard solution to obtain a final concentration of 50 ppb. The same pro- cedure was effected for the soil samples at 20 and 10 ppb, spraying 10 and 5 ml of the stock standard solution on 500 g of soil (on an oven- dry basis). After the fortification, the soil sam- ples were mixed for 5 min in a blender and frozen at -20°C. 2.6. Soil extraction A liquid-liquid and a solid-phase extraction were employed and compared. Liquid-liquid extraction (in duplicate) of the fortified soil samples was performed according to Zahnow [11]. Briefly, the buffer for extraction was methanol-0.1 M NaOH (1:1, v/v) (pH 11). Purification of extract was performed using methylene chloride. The solvent was discarded and the aqueous phase was adjusted to pH 3-4 by adding 10% HC1 dropwise. Again, methylene chloride was added, shaken, separated from the aqueous phase and evaporated to dryness in a Rotavapor at 45°C. A 50-g amount of soil (on an oven-dry basis) for the 50 and 20 ppb levels and 100 g for the 10 ppb level were used. The dry residues after all extraction steps were dissolved in 1 ml of methanol-water (60:40). In this way, an enrichment of 50-fold was obtained for the samples at 50 and 20 ppb and 100-fold for the sample at 10 ppb. Solid-phase extraction was performed in dupli- cate according to Dinelli et al. [5]. Briefly, 100 ml of sodium hydrogencarbonate solution (0.1 M, pH 7.8) was added to 50 g of soil (50 and 20 ppb fortifications) and to 100 g of soil for the 10 ppb level. The suspension was shaken for 1 h.  30 G.C. Galletti et al. / J. Chromatogr. A 692 (1995) 27-37 The slurry was centrifuged at 12 000 rpm for 5 min. The extraction procedure was repeated twice and the liquid extracts were combined. The extracts were adjusted to pH 2.5 with 0.1 M HC1 and passed through the solid-phase extraction column. The dry residues were reconstituted with 1 ml of methanol-water (60:40), thus ob- taining an enrichment of 50-fold for the 50 and 20 ppb samples and 100-fold for the 10 ppb sample. ing the B content linearly to reach a final water:methanol ratio of 30:70, which was main- tained for 5 min before resetting. The injections volume was 20 /xl and detection was performed at 224 and 234 nm. 3. Results and discussion 3.1. Reversed-phase C 6 column 2.7. High-performance liquid chromatography The HPLC system was a Beckman (Palo Alto, CA, USA) System Gold 126 with two pumps and a Rheodyne Model 7725-i valve (20-/xl loop). A Beckman Model 168 diode array detector was used. Reversed-phase C 6 and C18 columns were tested using both isocratic and gradient elution modes. The dependence of the capacity factors on the mobile phase composition was checked for both columns. Response and retention time repeatabilities were checked for the C18 column, which was used more generally. The C 6 column was a Viospher (5 /~m, 120 × 4.6 mm I.D.) (Violet, Rome, Italy). The mobile phase was (A) 0.01% HCIO 4 in water and (B) methanol at a flow-rate of 1 ml/min. The A:B ratio was 55:45 (v/v) for isocratic elution (here- after called isocratic 1 ). For gradient eleution, the A:B ratio was varied from 60:40 to 40:60 (v/v) in 20 min, holding the final conditions for 5 min (hereafter called gradient 1 ). The Cls column was a Beckman C~s Ultra- sphere (25 cm × 4.6 mm I.D., 5 /xm particle size). Analyses were performed in isocratic and gradient modes. For the first isocratic separation (hereafter called isocratic 2 ) the mobile phase was (A) water (pH 2.5, adjusted phosphoric acid) and (B) methanol in the ratio 60:40 (v/v) at 1 a flow-rate of ml/min. For the second isocratic condition ( isocratic 3 ) the mobile phase was the same as above but in ratio 40:60. For the gradient separation, the gradient was performed by maintaining initial conditions at water (pH 2.5, adjusted with 85% phosphoric acid)-methanol (60:40) for 5 rain, then increas- This column was tested first because it was expected that the analysis times would be faster than those with C18 columns. Actually, sepa- rations of standard mixtures of the four sul- fonylureas were completed in about 25 min adopting both isocratic 1 and gradient 1 con- ditions (Fig. 2a and c). Responses (data not shown) were linear in the 0.1-2 ppm range (R z > 0.999). The retention time of chlorimuron was much longer than those of the other three compounds. This observation can be explained by considering that its structure bears a chlorine-substituted diazinic ring instead of the relatively more polar methoxy-substituted triazinic ring of the other sulfonylureas, the other features not differing substantially. Both isocratic and gradient conditions were chosen as the best compromise between rapidity of analysis and separation efficiency. This consid- eration is better explained in Fig. 3a, in which the capacity factors of the four sulfonylureas are plotted against mobile phase composition. The efficiency of the C 6 column used did not allow the separation of the four sulfonylureas with organic modifier contents in the mobile phase higher than 50%. Under such circumstances, the C 6 column was applied to the analysis of water samples only, as explained in the following section. 3.2. Reversed-phase Cls column As expected, the sulfonylurea retention times were much longer using a C18 column, the analysis of the three earlier eluting compounds requiring about 35 min. Under such conditions,  G.C. Galletti et al. / J. Chromatogr. A 692 (1995) 27-37 31 b [] f J 0 5 10 1.oo. lo- Sa. u.I [] I f 215 15 20 II i [' I I I I l 0 I0 15 20 25 7.00 '10 ~ a.Ll. I [] I 1 I 1 l 21 0 5 10 15 20 5 TIME (rain) Fig. 2. Separation of sulfonylureas on a C 6 column using isocratic 1 conditions for (a) the standard mixture and (b) a spiked water extract and gradient 1 conditions for (c) the standard mixture. Detection at 234 nm. Peak numbers: compounds as in Fig. 1. the retention time of chlorimuron was very delayed and useless for practical applications. This problem was overcome by (a) determining chlorimuron separately from the other three molecules and (b) using gradient conditions. Fig. 4a-c shows the separation of sulfonylurea stan- dard mixtures under isocratic and gradient elut- ing conditions, namely (a) isocratic 2, (b) iso- cratic 3 and (c) gradient 2. Isocratic conditions 2 and 3 were those adopted for the determination of sulfonylureas in soil in this work, as discussed in the next section. However, the capacity factors were linear and the compounds were well resolved in a range of mobile phase compositions wider than that with the C 6 column (Fig. 3b), allowing the possibility of further separations, depending on the sample nature, the sulfonylurea and the interferent concentrations. Given the more general application of the reversed-phase C18 column, the retention time and peak area repeatabilities and response linearity were checked. With regard to retention time repeatability, Table 2 shows the results obtained after ten replicate injections on the same day (intra-day) and during 1 month (inter- day). The retention time fluctuations were within a maximum of 2.8% (R.S.D.) and did not appear much larger in 1 month than in 1 day. Peak-area variability (Table 3) measured in the same way as above showed R.S.D.s in the range 3.7-8.4%, which did not differ at two detection wavelengths. Finally, the responses of all sulfonylureas were highly linear in the range' 0.25-10 ppm injected (Table 4). 3.3. Sample clean-up, sulfonylurea enrichment and analysis Water, C 6 HPLC column Table 5 shows the recoveries of sulfonylureas after triplicate solid-phase extractions of aqueous solutions at the 5 ppb level, obtaining a 103-fold enrichment. At such concentrations, i.e., 5 ppm, sulfonylureas were well above the detection limits and typical chromatograms appeared were obtained such as that shown in Fig. 2b, in which the four sulfonylureas are well separated and free from water interferences. Soil, C18 HPLC column Two different soil clean-ups were tested, one based on liquid-liquid sulfonylurea partitioning and the other on a solid-phase extraction. Sul- fonylurea recoveries as determined after dupli- cate extractions of soil samples spiked in the range 10-50 ppb are given in Tables 6 and 7. The method based on liquid-liquid partioning seemed unsatisfactory (Table 6), in that the extraction recoveries were low when the sul- fonylurea concentrations in the sample were lower than 50 ppb. The thifensulfuron recoveries were particularly low at all concentrations tested. This observation is consistent with the reported
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