Environ. Sci. Technol. 2002, 36, 5411-5420
Selective Trace Analysis of Sulfonylurea Herbicides in Water and Soil Samples Based on Solid-Phase Extraction Using a Molecularly Imprinted Polymer QING-ZHI ZHU,† PETRA DEGELMANN, REINHARD NIESSNER, AND DIETMAR KNOPP* Institute of Hydrochemistry and Chemical Balneology, Technical University of Munich, Marchioninistrasse 17, D-81377 Munich, Germany
A molecularly imprinted polymer (MIP) was synthesized using the herbicide metsulfuron-methyl (MSM) as a template, 2-(trifluoromethyl)acrylic acid as a functional monomer, divinylbenzene as a cross-linker, and dichloromethane as a porogen. This polymer was used as a solid-phase extraction material for the quantitative enrichment of five sulfonylureas (nicosulfuron, thifensulfuron-methyl, metsulfuron-methyl, sulfometuron-methyl, and chlorsulfuron) in natural water and soil samples and off-line coupled to a reversed-phase HPLC/diode array detection (HPLC/DAD). Washing solvent was optimized in terms of kind and volume for removing the matrix constituents nonspecifically adsorbed on the MIP. It has been shown that the nonspecific binding ability of the sulfonylureas to the polymer largely increased along with increasing the concentration of Ca2+ ions in the water sample, whereas complexation of divalent ions with EDTA eliminated this interference completely. The stability of MIP was tested by consecutive percolation of water sample, and it was shown that the performance of the MIP did not vary even after 200 enrichment and desorption cycles. Recoveries of the five sulfonylureas extracted from 1 L of tap water and surface water samples such as river water and rainwater at a 50 ng/L spike level were not lower than 96%. The recoveries of sulfonylureas extracted from 10-g soil sample at the 50 µg/ kg level were in the range of 71-139%. Depending on the particular compound, the limit of detection varied from 2 to 14 ng/L in water and from 5 to 12 µg/kg in soil samples. The MIP was also compared with a commercially available C-18 column and an immunoaffinity support with encapsulated polyclonal anti-MSM antibodies in solgel glass.
Introduction Sulfonylureas (SUs) are a class of herbicides introduced in 1982 by Dupont Agricultural Products. They efficiently control broad-leafed weeds and some grasses in cereals and are widely used for a variety of crops because of their high * Corresponding author phone: +49 89 7095 7994; fax: +49 89 7095 7999; e-mail:
[email protected]. † On leave from Department of Chemistry, Xiamen University, Xiamen 361005, China. 10.1021/es0207908 CCC: $22.00 Published on Web 11/13/2002
2002 American Chemical Society
herbicidal activity and low toxicity for mammals. As compared with other herbicides, SUs have much lower use ranges (10100 g of active ingredient/ha) and are more rapidly degraded in soil (1, 2). Therefore, the concentration of these herbicides usually found in environmental soil and water are very low (ppt or ppb range). On the other hand, the structures of all SUs are very similar, and they may be present as a mixture of several compounds. For these reasons and because of their chemical and thermal instability, simultaneous monitoring of a series of these herbicides in environmental samples is a particularly challenging problem. Various methods have been proposed for the determination of SUs, such as gas chromatography/mass spectrometry (GC/MS) (3), high-performance liquid chromatography (HPLC) (4, 5), HPLC/MS (6-9), supercritical fluid chromatography (10), immunoassay (11, 12), bioassay (13), capillary electrophoresis (14, 15) and photochemically induced fluorescence (16). However, only a few of these methods with a limit of detection (LOD) in the ppt range meet regulatory requirements and are useful for screening. Recently, several publications reported that the SUs in water or soil could be quantified at nanograms per liter levels by using HPLC/ electrospray (ES)-MS method (17-19). However, the HPLC/ MS instrumentation is fairly expensive and rarely available in a common environmental laboratory presently. Therefore, sufficiently selective and sensitive analytical methods based on inexpensive instrumentation, such as HPLC with UV detection, are highly desirable for routine monitoring of SUs in water and soil samples. Because the UV detection often lacks the required sensitivity, a preconcentration step should be involved before HPLC/UV detection. Generally, the trace analysis of complex samples (e.g., environmental and biological samples) needs a pretreatment step in order to reduce the matrix interference and enrich the analytes. This is often performed by solid-phase extraction (SPE). This technique is more rapid, simple, and economical than the traditional liquid-liquid extraction. To date, several materials such as C18, graphitized carbon blacks (GCBs, referred to Carbograph 1 and Carbograph 4), and polystyrene divinylbenzene resin (RP-102 cartridge) have been shown to be valuable sorbent materials for sample enrichment of various SUs in water (2, 4, 17). These sorbents retain the analytes primarily by anion-exchange or reversed-phase adsorption and are thus rather nonspecific in nature. By use of SPE, the detectability of trace analytes can be greatly enhanced by applying large sample volume, but the high matrix load may also give rise to the partial coextraction of interfering substances with similar polarity because of the very limited selectivity of the sorbent materials. Therefore, an increase in sensitivity may not be obtained. Moreover, the high matrix loads would inevitably affect the performance of the extraction sorbent and result in frequent exchange of the SPE material. So, increasing the selectivity of sorbent in the extraction of analytes from large sample volume and developing new efficient cleanup techniques are highly attractive for monitoring trace analytes in complex samples. A high selectivity may be obtained using immunoaffinity columns, which rely upon reversible and highly selective antigen-antibody interactions. This technique has been applied for the determination of several organic pollutants in complex environmental samples (20, 21), and the extracts obtained were almost completely free of interferent matrix components. Unfortunately, the difficulty and high cost in obtaining polyclonal and monoclonal antibodies and the poor stability of the antibodies in organic solvents hinder a largescale use of these sorbents. A rather new rapidly growing VOL. 36, NO. 24, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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trend in SPE technique is the design and use of synthetic antibody mimics, such as molecularly imprinted polymers (MIPs). Molecular imprinting is an increasingly applied technique that allows the formation of selective recognition sites in a stable polymer matrix. In this technique, polymerizable functional monomers are prearranged around a template molecule by noncovalent or covalent interactions prior to initiation of polymerization. A rigid, highly cross-linked macroporous polymer is formed that contains sites complementary to the template molecule both in shape and in the arrangement of functional groups. After removal of the template molecule by extraction, the MIPs may then be used as an artificial receptor to selectively rebind the template from a mixture of chemical species. The advantages of the MIPs are their ease of preparation, stability, high selectivity, and high affinity constants. MIPs have been exploited in a number of applications including their use as separation materials, as antibody mimics in binding assay systems, and as recognition elements in biosensors for assay of various analytes (22-26). Recently, because of their compatibility with organic solvents, MIPs have attracted considerable attention as SPE sorbents for the cleanup and preconcentration of target analytes prior to determination. To date, molecularly imprinted solid-phase extraction (MISPE) has been applied to determine drugs (27, 28), nicotine (29), triazine (30-32), nitrophenol (33), bentazone (34), and chlorinated phenoxyacids (35). Recent developments in MISPE have been reviewed by several authors (36, 37). However, the use of MIPs as separation materials for enriching SUs from environmental samples has not been reported so far. In this paper, a MIP was synthesized using MSM as the template molecule, 2-(trifluoromethyl)acrylic acid as the functional monomer, and divinylbenzene as cross-linker and evaluated as a selective sorbent in SPE coupled off-line to a reversed-phase HPLC to selectively enrich five SUs from a large volume of drinking and surface water samples as well as soil. A comparison of the MIP with conventional sorbent such as C-18 and a natural polyclonal antibody against MSM encapsulated in sol-gel glass was also discussed. The major advantages of this method are that MIP shows high selectivity and affinity to the target analytes and is very stable for a real environmental application. These advantages make the MISPE successfully avoid the problems in both conventional nonspecific SPE and immunoaffinity SPE. To our knowledge, MIPs against any of the SU compounds have not been prepared before, except we reported on a MIP rather selective for MSM as based on binding characteristics of selected SUs in organic solvent (38). The present study is the first work describing a method for the determination of trace SUs in real environmental samples with MISPE enrichment.
Experimental Section Reagents and Chemicals. Metsulfuron-methyl (99.5%) (MSM), thifensulfuron-methyl (96.5%) (TSM), sulfometuron-methyl (10 ng/µL in water) (SMM), tribenuron-methyl (98.5%), bensulfuron-methyl (93%), triflusulfuron-methyl (95.5%), chlorsulfuron (99.5%) (CS), rimsulfuron (99.5%), chlorimuron-ethyl (99.0%) and triasulfuron (99.0%) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Amidosulfuron, nicosulfuron (NS), prosulfuron, primisulfuronmethyl, and cinosulfuron were a gift from Dr. Fo¨ry (CibaGeigy, Basel, Switzerland). Stock standard solutions of SUs (1.0 mg/mL) were prepared in acetonitrile and stored at 4 °C. Divinylbenzene (DVB, technical grade, 80%) (treated with basic alumina before use) and acetonitrile were purchased from Aldrich (Steinheim, Germany). 2-(Trifluoromethyl)acrylic acid (TFMAA) was purchased from Acros Organics 5412
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(Geel, Belgium). 2,2′-Azobisisobutyronitrile (AIBN), methanol, dichloromethane (distilled before use), trifluoroacetic acid (TFA), and ethylenedinitrilotetraacetic acid disodium salt (EDTA) were purchased from Merck (Darmstadt, Germany). Humic acid sodium salt was purchased from Carl Roth GmbH (Karlsruhe, Germany). All the solvents were of HPLC quality. Ultrapure water used for sample preparation was obtained by reversed osmosis including UV treatment (Milli-RO 5 Plus, Milli-Q185 Plus, Millipore, Eschborn, Germany). Preparation of MIP. For the preparation of MSM imprinted polymer, 120 mg (0.32 mmol) of template (MSM) and 225 mg (1.6 mmol) of functional monomer TFMAA were dissolved in 2.0 mL of CH2Cl2 in a 4-mL screw-capped glass vial. A 1.14-mL sample of cross-linker DVB (6.4 mmol because of the presence of 20% ethylvinylbenzene in the commercial DVB) and 10 mg of initiator AIBN were then added to the above solution. The solution was cooled on ice and sparged with nitrogen for 5 min, and the glass vial was sealed under nitrogen and then placed in a thermoblock TB1 (Biometra, Go¨ttingen, Germany) at 60 °C for 24 h. The resultant hard polymer monolith was crushed, ground, and wet-sieved with water. The particle size fraction of 32-64 µm was collected. These particles were then sonicated in MeOH/acetic acid solution (9:1, v/v) for 15 min, followed by centrifugation to remove solvent. This procedure was repeated several times until the template could not be detected (λmax ) 230 nm) in the extraction solvent. Then, the particles were sonicated again in MeOH three times for 15 min per cycle. Finally, the solvent was removed by centrifugation, and the particles were dried under vacuum. As a blank, a nonimprinted polymer was simultaneously prepared in the same way but without the addition of the template. During the polymer grinding, a small amount of dust particles is generated. Therefore, the use of gloves and carrying out this procedure within the ventilation hood is recommended. MISPE Cartridges Preparation, Washing, and Elution Procedures. A slurry of 100 mg of MIP in 1.0 mL of MeOH was placed into an empty glass SPE cartridge (3-mL cartridge from J. T. Baker, Deventer, Holland). PTFE frits (porosity 10 µm, Merck) were placed above and below the sorbent bed. Before the water samples were processed, the cartridge was preconditioned with 1.0 mL of MeOH and 2.0 mL of LCgrade water. As a control, a blank SPE column was also prepared in the same manner but with the blank polymer. For condition optimum experiments, only standard solutions of SUs were used. A 1.0-mL sample of 0.2 µg/mL sulfonylurea standard solution was passed through at a flow rate of ∼1 mL/min, the cartridge was dried by forcing nitrogen through it for 20 min, and then it was washed with 2.0 mL of a CH2Cl2/ACN solution (93:7, v/v). The analytes retained in the cartridge were eluted with 2.0 mL of CH2Cl2/MeOH (90:10, v/v). Both the washing and elution fractions were collected and dried using a gentle stream of nitrogen, the residues were redissolved in 1.0 mL of ACN/water solution (32:68, v/v), acidified with TFA at 3 mmol/L concentration level, and analyzed by HPLC/DAD. HPLC. Analysis was performed on a Shimadzu LC system equipped with a SCL-6A controller, two LC-6A pumps, photodiode array UV-visible detector SPD-M6A, and CTO10A column oven (Shimadzu, Duisburg, Germany). Chromatographic separations were carried out with a Bischoff C18 column 250 × 4.6 mm i.d. (3.0-µm particle size). Injection was performed with a model 7125 injector (Rheodyne, Cotati, CA) equipped with a 100-µL sample loop. Analysis was carried out using a gradient solvent program. The initial composition of the mobile phase was 32% ACN and 68% water; both solvents contained 3.0 mmol/L TFA. For separation of 14 SUs, the initial mobile phase composition of 32% ACN was increased linearly to 62% in 40 min. To clean the column,
the amount of ACN was increased from 62% to 90% within 5 min and kept constant for 3 min. The initial mobile phase composition was restored, and the column was equilibrated for 10 min. One the other hand, for only separation of five selected SUs (NS, TSM, MSM, SMM and CS) because their retention times are lower than 20 min, a gradient was programmed to linearly increase the amount of ACN from 32% to 50% in 24 min and from 50% to 90% in 5 min. The flow rate was 0.8 mL/min, and the column temperature was 25 °C. The UV detector was set at 227 nm wavelength. Data were acquired and evaluated by using the CSW v.1.7 package (Date Apex, Prague, Czech Republic). Peak areas were used for quantification. Calibration curve of each of the SUs was used to calculate the recoveries of the analytes. Water Sample Preparation and MISPE Extraction. Surface water was collected from two rivers in south Bavaria (Isar and Windach). Rainwater was collected at the area of Grosshadern in Munich. Both surface water samples were filtered using glass fiber filter MN 85/90 BF (Macherey-Nagel, Du ¨ ren, Germany) to remove particles larger than 0.5 µm, were collected in brown bottles, and were kept at 4 °C in the dark until analysis. The drinking water sample was collected from the tap in the laboratory and extracted unfiltered. Before being spiked with analytes, the free Ca2+ ions in the water samples were complexed by the addition of EDTA at a 9.3 g/L level. For recovery studies, surface water and drinking water samples were spiked with each of the SUs at a 50 ng/L concentration level. The MISPE cartridge was attached to vacuum manifold apparatus (model AI 6000, Analytical International, Harbor City, CA), which was connected to a water vacuum pump. Prior to sample application, the cartridge was conditioned with 2.0 mL of MeOH and then 5 mL of LC-grade water. A total of 1.0 L of each sample was forced to pass through the MISPE cartridge at a flow rate of ∼10 mL/min by negative pressure. After the sample was passed through the cartridge, the cartridge was dried with a nitrogen stream for 20 min. The washing step was carried out with 2.0 mL of a CH2Cl2/ ACN solution (93:7, v/v). The analytes were then eluted with 2.0 mL of a CH2Cl2/MeOH solution (90:10, v/v). Both the washing and elution fractions were collected and dried using a gentle stream of nitrogen, and the residue was reconstituted with 0.5 mL of ACN/water solution (32:68, v/v) acidified with TFA at 3 mmol/L concentration level and analyzed by HPLC/ DAD. As a control, the sample extraction was simultaneously applied on a blank polymer SPE cartridge in the same manner. Soil Sample Preparation and MISPE Extraction. The soil used was kindly provided by the Bayerische Landesanstalt fu ¨ r Bodenkultur und Pflanzenbau, Munich. The sample was ground in a mortar to a fine powder before use. Freshly spiked soil sample was prepared by weighing 10.0 g of soil into a flask followed by spraying of 0.5 mL of spiking solution (1.0 µg/mL of each of the five SUs). The spiked sample was allowed to stand overnight before extraction. Fortification was made at 50 µg/kg. The extraction of SUs from soil sample was performed according to Dinelli’s method (4) with a minor modification. A 20-mL sample of sodium hydrogencarbonate solution (0.1 M, pH 7.8) was added to 10.0 g of spiked soil sample, and the suspension was shaken for 1 h. The slurry was then centrifuged at 5000 rpm for 10 min. The extraction procedure was repeated three times, and the liquid extracts were combined. After the addition of 3 g of EDTA, the extract was passed through the MISPE cartridge. The washing, elution, and analytical procedures were the same as described above. A blank soil sample, without SUs, was also extracted and analyzed. Furthermore, a blank polymer SPE cartridge was simultaneously applied in the same manner.
Results and Discussion Binding Characteristics of the MIP, Washing, and Elution Conditions. The binding characteristics of the MIP presently used in this work were previously evaluated by our group on the basis of equilibrium binding experiments in organic solvents (38). In this previous work, it was shown that the MIP exhibited high selectivity and affinity to MSM in dichloromethane, the solvent was used as porogen, and the binding equilibrium between MIP and MSM was almost established within 1 min. On the other hand, the nonspecific binding of various SU herbicides on the polymer was very low. These characteristics of the MIP, namely, fast adsorption-desorption kinetics in the host-guest interactions and low nonspecific adsorption, are very important because they can directly affect the application of the MIP as SPE material. It is well-known that the molecular recognition principle of most of MIPs is based on the hydrogen binding between the target and the polymer functional groups, which often occurs in aprotic and low polar organic solvents, normally the one used as porogen in the polymerization process. In such system, specific hydrogen bonds are stabilized, and nonspecific hydrophobic interactions are suppressed. Therefore, the direct application of MIPs to the specific extraction of analytes from water samples is generally not possible because the MIPs often lack the selective binding ability in aqueous media (31, 33). In the present work, a washing step is necessary to remove the nonspecifically adsorbed substances from the MISPE column. After disrupting the nonspecific interaction between the MIP and interferences by a washing step, the specifically bound analytes were retained, and then the analytes could be quantitatively recovered in the following elution step. For optimizing the conditions of the washing step, a standard solution of MSM was applied to the MISPE and blank polymer cartridges. After passage of MSM solution, both the MISPE and the blank polymer columns were submitted to a washing step, which was carried out with 2 mL of either toluene, chloroform, dichloromethane, acetonitrile, or methanol. Next, the cartridges were eluted with 2 mL of methanol. Both the washing and elution fractions of the solvent were collected and analyzed; the results are shown in Figure 1. It can be seen that the low polar organic solvents (toluene and chloroform) cannot disrupt the nonspecific binding between the polymer and MSM because almost all of the MSM was still retained on the blank column after it was washed by using 2 mL of either toluene or chloroform. On the contrary, the MSM nonspecifically adsorbed on the blank polymer can be efficiently removed by high polar solvents (methanol and actonitrile); however, the specific interaction between the analyte and MIP was also suppressed by the use of these polar solvents in the washing step. On the other hand, when using dichloromethane as washing solvent, a different result was observed. About 40% of the amount of MSM loaded on the blank cartridge was washed off by using 2 mL of this solvent. The MSM was still selectively retained on the MIP cartridge after the washing step and then quantitatively eluted by methanol. However, a large amount of MSM (about 60%) was still nonspecifically adsorbed on the blank cartridge after washing with 2 mL of dichloromethane. To check the efficiency of dichloromethane as the washing solvent further, larger volumes of this solvent were tested. The results showed that the amount of MSM washed off from the blank cartridge increased along with increasing the volume of dichloromethane, but it almost reached constant (about 70%) when the volume of dichloromethane was higher than 5 mL. Even when 10 mL of dichloromethane was applied to the blank cartridge, only about 80% of MSM was removed from the blank cartridge. Although the MSM was still selectively retained on the MIP cartridge at this time, the large volume of washing solvent VOL. 36, NO. 24, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Recovery of MSM in the washing (shaded bars) and elution (open bars) fractions after loading 1.0 mL of 0.2 µg/mL MSM solution onto the blank polymer (a) and MISPE (b) cartridges. Washing step: 2 mL of each of the solvents in the figure. Elution step: 2 mL of MeOH. on the blank cartridge was completely removed after the washing step, whereas the specific binding of analyte on the MIP column was still retained. For this reason, 7% of ACN in dichloromethane was selected as the washing solvent for all further experiments. The effect of the washing solvent volume on MISPE extraction was investigated. The results showed that the optimum volume of the washing solvent was 1.5-3.0 mL. In the present work, a volume of 2.0 mL was chosen.
FIGURE 2. Recovery of MSM in the washing and elution fractions after preconcentration of 1.0 mL of 0.2 µg/mL MSM solution on blank polymer and MISPE cartridges in dependence on the ACN concentration in the washing solution. Blank polymer: washing (0), elution (9). MISPE: washing (O), elution (b). Washing step: 2 mL of washing solvent (ACN/CH2Cl2 mixture). Elution step: 2 mL of MeOH. would result in long handling time, and more important, the 20% nonspecific adsorption on the polymers would inevitably affect the performance of the MIP cartridge for selectively enriching the MSM and other analytes from the complex samples. The mixtures of dichloromethane with different concentration of ACN were tested as washing solvent because of the high ability of ACN for disrupting the nonspecific interaction of MSM and the polymer and its relative low polarity as compared with methanol. Figure 2 shows the recoveries of MSM in the washing and elution fractions after preconcentration on the blank and MIP cartridges by using 2 mL of each of the washing solvents. According to the figure, the amount of MSM removed from the blank cartridge increased along with increasing the amount of ACN in dichloromethane, and it was totally washed off the blank cartridge when the concentration of ACN in dichloromethane is higher than 6% (v/v). On the contrary, MSM could be selectively and efficiently retained on the MIP cartridge when the amount of ACN in dichloromethane is lower than 8%, but if the concentration of ACN is higher than this value, the specific interaction of MSM and MIP was obviously disrupted. Namely, when the concentration of ACN in dichloromethane was in the range of 6-8%, the analyte nonspecifically loaded 5414
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For the elution solvent, 2 mL of methanol efficiently elutes all the analytes from MISPE cartridge after the washing step. However, we know that the imprinted polymers often show different swelling properties in different solvents because of the different solvation properties of solvents for the MIP. The various degree of swelling in different solvents may considerably change the morphology of the polymer network and the size, shape, and relative position of the functional groups, and this may affect the stability of the polymer. For this reason and because the MIP was prepared by using dichloromethane as porogen, the possibility of using a mixture of dichloromethane and methanol as elution solvent was tested. It was found that the solvent of dichloromethane containing 5% methanol can efficiently elute all the analytes from the MIP cartridge. Therefore, an elution solvent of dichloromethane containing 10% methanol was chosen in this work to ensure the recovery. Specificity of the MIP. The specificity of the imprinted polymer was evaluated by comparison of the binding characteristic of MSM and other related SUs to the polymer. Fourteen different SUs were selected for this study, and their structures are listed in Table 1. A total of 1.0 mL of a mixture of 0.2 µg/mL of each sulfonylurea was applied to the MIP and blank polymer cartridges, and then the compounds in both the washing and elution fractions were analyzed by HPLC/DAD. Figure 3 shows the chromatograms of SUs in standard solution, washing solutions, and elution fractions. It can be seen that almost all of the SUs were completely removed from the blank column after the washing step, except for NS, which was partly retained on the blank cartridge because of its nonspecific interaction with the polymer. However, a different result was observed for the MISPE cartridge. Besides the template MSM, the sulfonylureas such as NS, TSM, and SMM were still totally retained on the MISPE column after the washing step. The recoveries of these compounds were higher than 95%. In addition, CS (55%), triasulfuron (25%), and prosulfuron (14%) were also partly enriched by the MIP. The left SUs cannot be recognized by the MIP and are completely separated from the target analytes
TABLE 1. Structures of Sulfonylurea Herbicides Used in This Study
TABLE 2. Recovery of Five Selected SUs after Loading of 1.0 mL of 0.2 µg/mL of Each Sulfonylurea onto the Cartridge (n ) 3) blank polymer (% ( SD) sulfonylurea
sulfonylureas
X
Y
Z
R1
R2
nicosulfurona thifensulfuron-methylb metsulfuron-methyl sulfometuron-methyl triasulfuron chlorsulfuron rimsulfurona amidosulfuronc bensulfuron-methyld tribenuron-methyl prosulfuron chlorimuron-ethyl triflusulfuron-methyle primisulfuron-methyl
N N N C N N C C C N N C N C
H H H H H H H H H CH3 H H H H
CON(CH3)2 COOCH3 COOCH3 COOCH3 OCH2CH2Cl Cl SO2CH2CH3
OCH3 OCH3 OCH3 CH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH2CF3 OCF2H
OCH3 CH3 CH3 CH3 CH3 CH3 OCH3 OCH3 OCH3 CH3 CH3 Cl N(CH3)2 OCF2H
COOCH3 COOCH3 CH2CH2CF3 COOCH2CH3 COOCH3 COOCH3
a Pyridine instead of phenyl ring. b Thiophen instead of phenyl ring. N(CH3)SO2CH3 instead of phenyl ring. d Benzyl instead of phenyl ring. e o-Methylphenyl ring instead of phenyl ring. c
FIGURE 3. Chromatograms obtained by off-line SPE of 1.0 mL of a mixture of 0.2 µg/mL of each sulfonylurea. (A) standard solution; (B) blank polymer, washing fraction; (C) blank polymer, elution fraction; (D) MIP, washing fraction; (E) MIP, elution fraction. (1) NS, (2) TSM, (3) MSM, (4) SMM, (5) triasulfuron, (6) CS, (7) rimsulfuron, (8) amidosulfuron, (9) bensulfuron-methyl, (10) tribenuron-methyl, (11) prosulfuron, (12) chlorimuron-ethyl, (13) triflusulfuron-methyl, and (14) primisulfuron-methyl. Washing step: 2.0 mL of CH2Cl2/ACN (93:7, v/v). Elution step: 2.0 mL of CH2Cl2/MeOH (90:10, v/v). even though their structure is also similar to that of MSM. These results show that the MIP exhibit highly selective binding affinity for MSM, TSM, SMM, and CS and demonstrate that the adsorption of these SUs is due to imprinted binding sites and not due to nonspecific binding, while other sulfonylurea herbicides show less or no binding. This could be easily explained by their close structural homology to MSM. From Table 1 it can be seen that there is only slight difference between the structure of MSM and those of TSM, SMM, and CS. For TSM, the structural difference is a thiophene instead of a benzene ring as in MSM. For SMM, the difference is a methyl instead of methoxy in the R1 position, and CS has a chlorine instead of a -COOCH3 group at the benzene moiety. The size of thiophene, methyl, and chlorine is smaller than that of a benzene ring, methoxy, and -COOCH3 group, respectively. Thus it would be reasonable
washing
nicosulfuron 52 ( 4.7 thifensulfuron-methyl 99 ( 2.5 metsulfuron-methyl 100 ( 4.4 sulfometuron-methyl 100 ( 3.8 chlorsulfuron 98 ( 1.6
elution
MIP (% ( SD) washing
49 ( 6.9 0 0 0 0 0 0 0 0 3.3 ( 3.0
elution 99 ( 3.3 98 ( 1.5 99 ( 2.8 97 ( 1.2 97 ( 4.1
to assume that TSM, SMM, and CS are able to fit into the specific binding sites in the polymer network. This further demonstrates that the imprinting is not only based on the interaction of the functional groups of the analyte with those binding sites in the polymer cavities but also based on the combined effect of shape and size complementarity. As for NS, its retention on the MIP is not totally based on the specific recognition but relied on the nonspecific adsorption to a large extent (Figure 3). The washing step is not efficient enough to disrupt its nonspecific interaction with the polymer. The reason for high nonspecific binding of NS to the polymer could be explained by the fact that the polymers used in this work (DVB as cross-linker) are nonpolar (or low polar) polymers, which show high nonspecific adsorption of low polar compounds. The chromatograms of SUs (Figure 3) show that the polarity of NS is much lower as compared to the other SUs, which results in its high nonspecific binding to the polymers. Because of the high recoveries of MSM, NS, TSM, SMM, and CS, these five SUs were thus selected as target analytes for all the further experiments. An interesting phenomenon was that when only the five sulfonylureas mentioned above were used as the analytes, the recovery of CS extract was significantly increased from 55% to 97% (Table 2). This could be explained by the fact that CS may cooperate with other SUs and be partly washed off the MIP cartridge together with them. Capacity and Stability of the MISPE Cartridge. To estimate the capacity of the prepared MISPE cartridge, 5.0 mL of water spiked at 0.2 and 1.0 µg/mL with each sulfonylurea was loaded onto the cartridge. After the washing and elution steps, the effluent, washing fraction, and elution fraction were analyzed by HPLC. The recoveries are summarized in Table 3. As could be observed, all the selected SUs were completely retained on the MIP cartridge when their loaded amounts were not higher than 1.0 µg. For loading with 5.0 µg of each of the SUs, no analytes were found in the effluent fraction, but 54% of TSM, 44% of MSM, 17% of SMM, and 61% of CS were removed from the column in the washing step. This indicated that the selective interaction between the analytes and the MIP was saturated under this condition. An exception was NS, which was still completely retained on the cartridge. The latter can be easily explained by the high rate of nonspecific interaction of NS to the MIP. When 5.0 µg of each of the SUs was loaded onto the column, the total amount bound to the MIP was about 15.6 µg. This capacity greatly exceeded the expected amount of SUs in environmental samples and was sufficient for the analysis of real samples. It is worthy to point out that a competition interaction with the limited binding sites in the MIP matrix would take place when the selected SUs were simultaneously present in the loading solution. From Table 3, it can be seen that the selective binding capacity of SMM to the MIP is the highest. NS did not compete with the other SUs for the limited binding sites because of its nonspecific adsorption on the polymer. Because the imprinted molecules are highly embedded in the polymer network, it is difficult to completely remove VOL. 36, NO. 24, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 3. Capacity of the MISPE Cartridgea load 1 µg of each sulfonylurea (% ( SD, n ) 3)
a
load 5 µg of each sulfonylurea (% ( SD, n ) 3)
sulfonylurea
effluent
washing
elution
effluent
washing
elution
nicosulfuron thifensulfuron-methyl metsulfuron-methyl sulfometuron-methyl chlorsulfuron
0 0 0 0 0
0 0 0 0 2 ( 3.7
96 ( 1.6 96 ( 2.3 101 ( 1.7 98 ( 2.2 95 ( 0.6
0 0 0 0 0
0 54 ( 5.4 44 ( 3.0 17 ( 2.7 61 ( 4.9
95 ( 4.6 47 ( 1.9 56 ( 4.9 82 ( 2.8 32 ( 2.3
Recoveries of SUs obtained after loading 5.0 mL of 0.2 and 1.0 µg/mL of each sulfonylurea onto the MIP column, respectively.
FIGURE 4. Stability of the MISPE. After the washing step, recoveries of the five SUs in the eluates of MIP and blank cartridges were determined (n ) 3) after the first and 200th cycle, respectively. the template prior to enrichment of the analyte. Consequently, a common problem associated with MISPE when using the target analyte as a template is the template bleeding from the polymer during the desorption procedure and its interference with the quantification. In our case, this phenomenon was only observed with fresh polymer. A washing step with 10 mL of MeOH prior to loading sample was enough to eliminate this problem. During the continuous use of MISPE cartridge, we did not observe any significant bleeding effect. Possibly the amount of released MSM was so low that it could not be detected in our system. The stability of the cartridges was tested by submitting the blank and MISPE cartridges to 200 consecutive equilibrating, loading, washing, and elution processes. The recoveries of analytes from the blank and MIP columns were analyzed before and after the stability test (Figure 4). It can be observed that, for the MIP cartridge, the recoveries of analytes were quantitative and remained constant even after hundreds of adsorption and desorption cycles. However, for the blank column, a different result was observed. The nonspecific absorbance of NS and TSM on the blank polymer increased along with the increase of the number of cycles. The recoveries of NS and TSM were obviously increased after 200 extraction cycles, especially for NS, it was totally adsorbed on the polymer. On the other hand, MSM, SMM, and CS still showed very low or no nonspecific adsorption on the polymer even after 200 cycles. Figure 4 shows that the MIP is sufficiently stable for being applied as an efficient SPE sorbent. Matrix Effects. Environmental samples such as surface water and soil commonly contain humic material, which often produce strong interference in the determination of polar compounds because of their broad range of molecular weight and intense UV/VIS absorption. In some case, it is impossible to quantify them because the target analyte signals are completely overlayed by the interfering background. Another problem caused by humic material is the contamination of the SPE column and the chromatographic system, 5416
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FIGURE 5. Effect of humic acid on the MISPE cartridge. A solution of 2.0 mL of 120 mg/L humic acid was passed through the MIP; the elution and cleaning fractions were analyzed: (A) a solution of humic acid (120 mg/mL) directly injected into HPLC, (B) elution fraction, (C) cleaning fraction, (D) blank solution. Elution step: 2.0 mL of CH2Cl2/MeOH (90:10, v/v). Cleaning step: 2.0 mL of MeOH with 2 mmol of TFA. which could significantly reduce the performance and lifetime of SPE column and separation system. To evaluate the adsorption of humic acids on the MISPE cartridge and their effect on the cartridge performance, 2.0 mL of 120 mg/L humic acid solution was loaded onto the MISPE column. After the washing and elution steps, the column was further cleaned with 2 mL of MeOH in the presence of 2 mmol of TFA. A comparison of the chromatograms obtained from elution fraction, final cleaning fraction, blank solution, and humic acid solution (120 mg/L) are shown in Figure 5. As can be observed, when a humic acid solution of 120 mg/L was directly injected into the HPLC, an intense and broad peak was obtained. The chromatographic signal of the elution fraction from the MIP was largely reduced as compared with that of humic acid solution, but its relative high blank absorption indicated that a small amount of humic acid was still retained on the cartridge after washing. The washing fraction was also analyzed by HPLC in order to estimate the amount of humic acid that was adsorbed on the MISPE cartridge. The result (data not shown) showed that the washing solvent was not efficient enough to remove humic acid from the cartridge. Its chromatogram was similar to that of the final cleaning fraction (Figure 5). These results indicated that the high content of humic acid directly passed through the cartridge and did not adsorb on the sorbent. Further study showed that the small amount of humic acid bound to the column did not diminish the specificity of the MIP. When the MISPE and blank polymer cartridges were directly applied to extract the target analytes from the real water samples, it was found that the nonspecific binding of the analytes on the polymer became so strong that the washing solvent was not able to remove the analytes from
TABLE 4. Recoveries (%), Precision, and Limits of Detection (LOD) of Sulfonylureas after MISPE of Water (Spiked at 50 ng/L) and Soil (Spiked at 50 µG/Kg) Samplesa tap water (1 L)
river water (1 L)
rainwater (1 L)
soil (10 g)
compounds
recovery
RSD
LOD
recovery
RSD
LOD
recovery
RSD
LOD
recovery
RSD
LOD
NS TSM MSM SMM CS
96 102 101 98 97
6.1 1.1 4.3 5.0 4.4
9 2 6 7 6
101 96 102 98 100
8.7 4.8 4.3 6.5 4.2
13 7 6 10 6
97 102 107 96
2.3 1.8 4.7 9.4
3 3 8 14
82 71 139 71
4.3 6.5 5.8 4.4
5 6 12 5
a RSD, n ) 3. LOD defined as three times the standard deviation calculated at the spike level considered. The unit of LOD of water sample is ng/L, and of soil sample it is µg/kg.
FIGURE 6. (a) Effect of Ca2+ ions in the water sample on the nonspecific binding of a blank polymer: NS (b), TSM (O), MSM (4), SMM (2), and CS (0). (b) Recovery of each sulfonylurea mixed with 30 mg/L Ca2+ in the presence of 7.5 mmol/L EDTA.
FIGURE 7. LC/UV chromatogram obtained with eluates after loading 1 L of river water spiked with five sulfonylureas at the individual level of 50 ng/L onto the MIP (a) and blank (b) cartridges: (1) NS, (2) TSM, (3) MSM, (4) SMM, and (5) CS.
the blank polymer cartridge any more. Because humic acid did not affect the selectivity of the MIP (data not shown), this phenomenon must have been caused by the other matrix constituents. Taking into consideration that the organic compounds in the matrix normally show no or less effect on the performance of MIP, the effect of the inorganic ion Ca2+ on the selectivity of the MIP was studied because of its much higher concentration in the drinking and surface water as compared with other inorganic ions in the Munich area. This experiment was carried out by loading 1.0 mL of 0.2 µg/mL of each SU in the presence of various concentrations of Ca2+ onto the blank polymer cartridge and analyzing the washing fraction with HPLC. The results in Figure 6a show that the amount of SUs nonspecifically retained on the cartridge became significantly increased with increasing concentration of Ca2+ ions. When the concentration of Ca2+ was higher than 30 mg/L, all analytes were almost totally retained on the blank column after the washing step. This indicated that the specificity of MISPE would be completely lost in the presence of a high concentration of Ca2+. The phenomenon could be explained by the fact that Ca2+ ions could form a complex together with SUs and the monomer TFMAA in the polymer matrix. It is well known that -COOH and -NH- groups are efficient ligands for divalent metal ions such as Ca2+, Mg2+, etc. They easily can form coordinate bonds with these kinds of metal ions. In this system, Ca2+ may form coordinate bonds with the carboxyl group of TFMAA molecule and the seconday amino group of SU compounds. The analytes were bound to the polymer with Ca2+ as a “bridge”, and the washing solvent was not strong enough to disrupt the coordinate bonds between SUs, Ca2+, and TFMAA, which are much stronger than hydrogen bonds. This hypothesis could be confirmed by adding EDTA to the mixture of SUs and Ca2+ and loading this solution onto the blank polymer column. As can be observed from Figure 6b, the recoveries of TSM,
MSM, SMM, and CS in the washing fraction again were higher than 95%. This indicated that the presence of EDTA could efficiently mask free Ca2+ in the water sample and completely eliminate its interference with the specificity of the polymer. Even though the blank and MIP columns were first blocked by a high concentration of Ca2+ (higher than 70 mg/L), their specificity could be completely recovered by washing the columns with a certain concentration of EDTA solution (data not shown). It should be pointed out that the recovery of NS in the washing fraction was only about 20% after the addition of EDTA. This was not surprising because this sulfonylurea compound was shown to be nonspecifically adsorbed by other mechanisms than complexation via divalent cations. To further confirm our hypothesis mentioned above, the effect of another divalent cation (Mg2+ ions) on the selectivity of MIP was also tested. The results showed that a similar behavior was observed when Mg2+ ions were present in the SU sample (data not shown). Considering that the concentration of Ca2+ is around 1.85 mmol/L in the drinking water in the Munich area and may be depending on the drainage area higher in river water samples, the addition of 9.3 g of EDTA/L of water samples (25 mmol/L) was selected for the analysis of real samples to completely mask the free Ca2+ and other divalent cations, mainly Mg2+. The effect of the sample pH on the SPE extraction was also evaluated. The results showed that the performance of the MISPE cartridge almost remained unchanged when the pH value of the water sample was varied from 1 to 10. The recoveries of TSM, MSM, SMM, and CS were higher than 95% in this pH range; however, the recovery of NS reduced to about 60% when the pH was lower than 2. On the other hand, the nonspecific adsorption of all the SUs on the polymer increased when the pH was higher than 9. VOL. 36, NO. 24, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 8. LC/UV chromatograms obtained by extracting SUs from 100 mL of river water, spiked at the level of 1.0 µg/L, with C-18, MIP, and immunosorbent cartridges. The dashed line chromatogram of the MIP was obtained from the washing fraction: (1) NS, (2) TSM, (3) MSM, (4) SMM, (5) CS, (6) cinosulfuron, (7) triasulfuron, (8) tribenuron-methyl, (9) chlorimuron-methyl, and (10) primisulfuron-methyl. MISPE of Environmental Samples, Precision, and LOD. To demonstrate the applicability and reliability of this method for environmental application, real environmental samples, like surface water and soil samples, were selected and analyzed. Tap water, river water, and rainwater were spiked with the five SUs at the 50 ng/L concentration level and were preconcentrated by MISPE. After analysis by HPLC, the recoveries, reproducibility, and LOD of the method were calculated and summarized in Table 4. As can be seen, for analysis of TSM, MSM, SMM, and CS in the water samples, the analyte recoveries were higher than 96% and were unaffected by the nature of the aqueous matrix in which the analytes were dissolved. But for NS, it could not be detected in more complex matrixes such as rainwater and soil samples 5418
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because its signal was completely overlayed by the matrix background. The relative standard deviation (n ) 3) for quantitation was between 1.1 and 6.1% for tap water and between 1.8 and 9.4% for surface water, which is an acceptable value for real sample analysis. LOD calculated for tap water was between 2 and 9 ng/L, which is clearly lower than the legal threshold value of 100 ng/L set for pesticides in drinking water by the European Community. LODs for surface water samples were very similar and varied from 3 to 14 ng/L. A typical HPLC/UV chromatogram obtained by analyzing SUs in fortified river water is shown in Figure 7. It can be seen that none of the analytes were retained on the blank polymer column when a washing step was employed. However, with the MIP, the selected SUs were selectively extracted from the
real water sample. This result further clearly confirmed the existence of specific interactions between analytes and MIP arising from imprinting. To estimate the application of the MIP for recognizing the analytes in more complex matrixes, a soil sample was spiked with the five SUs at the individual level of 50 µg/kg and analyzed for this purpose. A total of 10 g of spiked soil sample was first submitted to an extraction step with 20 mL of 0.1 M NaHCO3, which was repeated three times. After the cleanup with MISPE cartridge, the recoveries are shown in Table 4. As can be seen, the recoveries of TSM, MSM, and CS between 71 and 82% were lower than in water. This loss of recovery was attributed mainly to the first extraction step with NaHCO3 solution. When the soil sample was first extracted with NaHCO3 solution and then the extract was spiked with analytes, the recoveries of TSM, MSM, and CS were found to be higher than 90% again (data not shown). On the other hand, the recovery of SMM was 139%, which was caused by the interference of an unknown compound retained on the MIP after the washing step. This could be proven with an unfortified blank soil sample. The washing step was not harsh enough to remove this unknown compound from the MIP column completely. Method Comparison. The MISPE cartridge proposed in this work was compared with a commercial C-18 SPE column and a specially prepared sol-gel glass immunosorbent with regard to selectivity, stability, and sensitivity. The preparation of the sol-gel glass immunosorbent cartridge was similar to the procedure proposed by our group recently (20) but used a polyclonal anti-MSM antibody. A 100-mL sample of river water was spiked with 1.0 µg/L of each sulfonylurea and analyzed (i) by extraction with MISPE, (ii) with a 0.5-g C-18 cartridge (Merck, Darmstadt, Germany), and (3) with a solgel glass immunosorbent. The C-18 cartridge was preconditioned with 5 mL of MeOH followed by 5 mL of water (4). Additionally, the water sample was acidified to pH 3 to suppress analyte dissociation (9) and sucked through the cartridge at a flow rate of 1 mL/min. After the passage of the sample, the C-18 cartridge was washed with 10 mL water and dried in a gentle stream of nitrogen. The analytes were then eluted with 5 mL of MeOH (4). Solvent removal and residue reconstitution were the same as in the MISPE procedure. Before extraction, the immunosorbent was conditioned with 5 mL of ACN/water (1:1) followed by 5 mL of water. The sample was first adjusted to a final concentration of 100 mmol/L NaCl and 5% (v/v) ACN and then passed through the immunosorbent column at a flow rate of 1-2 mL/min. After washing with 5 mL of 5% ACN, the analytes were eluted with 1 mL of 60% (v/v) ACN. Resulting LC/UV chromatograms are shown in Figure 8. It can be seen that an unknown compound A could not be separated from MSM by C-18 column without an additional cleanup step. The overlay of their peaks made a quantitation of MSM impossible (Figure 8, C-18). Comparing with MISPE, another disadvantage of the C-18 column was the lower recovery of SUs between 70% and 90%. The extraction with the sol-gel glass immunosorbent revealed that the MSM-antibody was not able to interact with NS and CS but could recognize the seven SUs (MSM, SMM, cinosulfuron, tribenuron-methyl, triasulfuron, chlorimuron-ethyl, and primisulfuron-methyl). It is interesting to note that the number of different SUs recognized by the MIP and the antibody was similar, but the pattern was different. To equitably evaluate the performance of the immunosorbent, 100 mL of river water was spiked with 1.0 µg/L of each of the eight SUs and applied to the column. The results showed that the recoveries of the analytes from the immunosorbent cartridge ranged from 33 to 73% ((5%), depending on the cross-reactivity of the antibodies (Figure 8, immunosorbent). The MIP proved to be very stable against organic solvents, high and low pH value, pressure, and
temperature; the performance of the MIP remained unchanged even after 200 extraction cycles. However, the antibody entrapped in the sol-gel glass matrix is not that robust against variable conditions of their surrounding. Depending on the sample matrix, the immunosorbent cartridge has to be changed more frequently (e.g. after about 20-30 cycles). Preparation of sol-gel glass immunoaffinity supports is easy, but the availability of suitable antibodies is a limiting factor. Whereas, comparing chromatograms of MIP and immunosorbent in Figure 8, it appears that matrix interferences are almost completely removed from the immunosorbent cartridge with a washing step. Consequently, the analytes were analyzed free of complex matrixes. On the other hand, no EDTA addition to remove interfering divalent cations is needed. In this work, the newly developed MISPE proved to be a powerful tool for the selective enrichment of five sulfonylurea herbicides from environmental samples. Its easy handling and high stability allowed reliable, rapid analysis of the analytes within complex matrix at trace level. Although some experimental conditions affected the adsorption-desorption process, once these were optimized, the MIP offered several practical advantages over other SPE materials such as C-18 and immunosorbent. Since SU herbicides include more than 20 different compounds and are often present as a mixture of several compounds, it is very important to determine a series of herbicides in a single assay in environmental analysis (multi-analyte method). Further studies will focus on the development of MIPs that can bind even more different sulfonylurea compounds with high selectivity and affinity.
Acknowledgments We acknowledge the support by the Alexander von Humboldt-Foundation (Q.-Z.Z.) and the Deutsche Forschungsgemeinschaft (Kn 348/8-1).
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Received for review June 17, 2002. Revised manuscript received September 30, 2002. Accepted October 9, 2002. ES0207908
