Assessment of Novel Diazinon Immunoassays for Water Analysis

Química, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain ... Environmental Science & Technology 2005 39 (8), 2786...
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Environ. Sci. Technol. 2004, 38, 1115-1123

Assessment of Novel Diazinon Immunoassays for Water Analysis E V A M . B R U N , M A R T A G A R C EÄ S - G A R C IÄ A , E S T E F A N IÄ A E S C U IÄ N , S E R G I M O R A I S , ROSA PUCHADES,* AND AÄ N G E L M A Q U I E I R A * Departamento de Quı´mica, Universidad Polite´cnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain

Diazinon is a broad organophosphate insecticide used in agricultural and other treatments, resulting in widespread water contamination. The development of easy-to-use screening immunoanalytical methods is an interesting tool to study environmental pollution impact. Two novel strategies for diazinon hapten synthesis are addressed. One of them attaches the spacer arm to the oxygen atom of the diazinon aromatic ring. The other one retains the diazinon basic structure linking the spacer to an aromatic carbon. A total of eight diazinon haptens were synthesized, demonstrating that they are suitable for immunoreagent (protein conjugates and polyclonal antibodies) production. The optimized ELISA is based on conjugate-coated format and had a detection limit of 0.40 µg/L, showing little or no cross-reactivity to similar tested compounds. The immunoassays were used as a tool to quantify diazinon in natural waters. Results are in agreement with those given by GC-MS reference method. Mean recoveries ranging between 99% and 105% confirm the potential of our approach to determine diazinon in samples without purification or preconcentration steps, being applied as a screening method for field monitoring of diazinon in river waters.

Introduction Diazinon (O,O-diethyl O-2-isopropyl-6-methylpyrimidin-4yl phosphorothioate) is a nonsystemic widely used organophosphorus insecticide (1). As a result of its widespread agricultural and domestic use, diazinon residues have been found in homes, crops, commodities, and urban storm and surface waters (2-4). If diazinon is released to water, it may sorb moderately to sediments. Diazinon persistence in soil, combined with its relatively high water solubility (log Kow ) 3.81) and resistance to degradation, poses potential concerns for transport to watercourses. Most concerns are also linked to relatively high toxicity of diazinon. Lethal concentrations range from 300-400 mg/kg for mammals to 3 and 40 mg/kg in birds with high toxicity to fish and bees. Several methods have been reported for the determination of diazinon using a variety of techniques, including gas chromatography, high performance liquid chromatography, mass spectroscopy and cholinesterase screening tests (58). Although, the chromatographic techniques provide a low level of detection for diazinon, samples must be extensively processed (extraction and cleanup) before the analysis. Presently, there is a need for acceptable, rapid, reliable, * Corresponding author phone: +34-96-387 73 42; fax: +34-96387 93 49; e-mail: [email protected] (A.M.) or rpuchades@ qim.upv.es (R.P.). 10.1021/es034892p CCC: $27.50 Published on Web 01/07/2004

 2004 American Chemical Society

sensitive, and cost-effective assay for determining the presence of diazinon (9). Indeed, suitably designed immunoassaybased techniques as alternative methods can meet these requirements. Only a few attempts have been made to develop sensitive and specific immunoassay for diazinon (10, 11). Most of the haptens being synthesized are based on the production of either generic or specific haptens for any organophosphorus pesticides other than diazinon (12-15). Those haptens have a O,O-dialkyl thiophosphate group common in organophosphorus pesticides. Even though the strategy used for the synthesis of haptens generates sensitive antibodies against several organophosphorus pesticides, the assays were of very low sensitivity for diazinon detection. With regard to the synthesis of diazinon haptens, in 1997 ten Hoeve et al. (10) described the synthesis of generic intermediates for preparing antibodies against diazinon making use of dioxaphosphorinan ring. This strategy led to haptens in six tedious steps capable to raise antibodies with sensitivities within the 500-1000 ng/mL range. Beasely et al. (11) followed a different chemical route, which was based on the use of a neutral 3-aminopropanol spacer arm, and successfully generated sensitive antibodies (IC50 ) 0.4 ng/ mL) against diazinon. In both cases, the synthesis of diazinon haptens was carried out through the thiophosphate moiety of the pesticide. Currently, two commercial ELISA-based plate kits are being used to determine diazinon. The one commercialized by Strategic Diagnostics Inc. is quite specific for diazinon showing a least detectable dose of 0.022 µg/L, while the one marketed by Beacon Analytical Systems Inc. shows also very good sensitivity (0.030 µg/L). The first one has already been evaluated by different authors, who concluded a positive bias in surface waters (4, 16). In contrast, the accuracy and precision of the other diazinon immunoassay kit was considered not to be satisfactory for the determination of diazinon in surface waters (17). To obtain better antibodies as well as for further improvement, two novel strategies to synthesize diazinon haptens are addressed. One of them consists on designing compounds containing only the aromatic moiety of diazinon. This approach has been previously reported for other organophosphate pesticides, like triasulfuron and azinphosmethyl, leading to excellent monoclonal antibodies (18). According to this methodology, the synthesis of fragmentary haptens, which contain the aromatic diazinon ring with a hydrocarbon spacer arm attached to the oxygen atom, could provide an easy synthetic pathway with only two steps, making it a suitable strategy to elicit polyclonal antibodies against diazinon. On the other hand, the introduction of a spacer arm as a substituent on the aromatic ring, retaining the phosphate ester, and getting compounds more similar to diazinon could be also a novel and successful strategy. So 4-hydroxy-2mercapto-6-methylpyrimidine could be a good starting moiety because it conserves the diazinon ring structure and contains a thiol group instead of an isopropyl group suitable to introduce the spacer arm by alkylation. Following this idea, alkylation of thiol group, introduction of phosphate ester, and selective hydrolysis of the spacer arm was chosen to obtain other diazinon haptens. This work reflects research on novel strategies to synthesize diazinon haptens based on hapten heterology and their use to produce polyclonal antibodies and other immunoreagents to diazinon. Furthermore, the application of VOL. 38, NO. 4, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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sensitive and selective immunoassay for the determination of diazinon in environmental water samples is also assessed.

Experimental Section Chemicals. Chemical reagents for hapten synthesis and protein conjugation purposes were purchased from FlukaAldrich Quı´mica (Madrid, Spain). Analytical-grade solvents were from Scharlab (Barcelona, Spain). Pesticide standards used for cross-reactivity studies were purchased from FlukaAldrich Quı´mica and Dr. Ehrenstorfer (Augsburg, Germany). Bovine serum albumin (BSA), ovalbumin (OVA), complete and incomplete Freund’s adjuvant, o-phenylenediamine (OPD), Tween 20, horseradish peroxidase (HRP), and peroxidase-labeled goat anti-rabbit immunoglobulins (GARHRP) were purchased from Sigma (Madrid, Spain). Instrumentation. Thin-layer chromatography (TLC) was performed on 0.25 mm, precoated silica gel 60 F254 on aluminum sheets (Merck, Darmstat, Germany). Column chromatography was carried out on silica gel (0.063-0.2 mm particle size, 70-230 mesh), also from Merck. 1H and 13C nuclear magnetic resonance (NMR) spectra were obtained with a 300 Varian spectrometer (Sunnyvale, CA). Chemical shifts values are given in parts per million (ppm) downfield from internal standard deuterium chloroform. Coupling constants are expressed in hertz (Hz); the abbreviations s, d, t, q, m, and ar represent singlet, doublet, triplet, quartet, multiplet, and aromatic, respectively. UV-Vis spectra were recorded on a Hewlett-Packard 8452 diode array spectrophotometer (Palo Alto, CA). Polystyrene 96-well microtiter plates were from Costar (Cambridge, MA), and the ELISA plate washer was from Nunc Maxisarp (Roskilde, Denmark). Well absorbances were read at 490 and 650 nm by means of a microtiter plate reader (Wallac, model Victor 1420 multilabel counter, Turku, Finland). For GC analysis, a 6890 Hewlett-Packard devices automatic samplersequipped with a 5% phenyl-methyl siloxane capillary column (HP-5MS) model 19091S-433 (30 m length × 250 µm diameter × 0.25 µm film thickness) and a 5973 mass selective detector operating in selected ion monitoring mode were employed for peak identification. Haptens Synthesis. The haptens used throughout this work are depicted in Figure 1. Three types of haptens were synthesized depending on the site of the diazinon structure through which the spacer arm was attached: O-alkylation without the thiophosphate group (haptens 1-3), the aromatic ring (haptens 4-6), and the thiophosphate moiety (haptens 7 and 8). Haptens 1-3 were synthesized by reaction of sodium or potassium salt of 2-isopropyl-6-methyl-4-pyrimidinol with bromoesters of different lengths. Haptens 4-6 were synthesized attaching the spacer arm on the aromatic ring using 4-hydroxy-2-mercapto-6-methylpyrimidine as starting material. Hapten 7 was prepared following the procedure described by Beasely et al. (11), while hapten 8 was synthesized according to a previously reported procedure (10). [(2-Isopropyl-6-methylpyrimidin-4-yl)oxy]acetic Acid (1). Methyl bromoacetate (10 mmol, 0.95 mL) was added to a mixture of 2-isopropyl-6-methyl-4-pyrimidinol (10 mmol, 1520 mg) and anhydrous K2CO3 (10 mmol, 1.38 g) in anhydrous acetone (25 mL) under argon. The reaction mixture was stirred for 16 h at room temperature and for 2 h at 50 °C. Filtration of the crude, concentration, and purification by column chromatography (hexane/EtOAc, 80: 20) yield the methyl ester as a colorless oil (784 mg, 41%), TLC Rf 0.67 (hexane/EtOAc, 50:50); 1H NMR (CDCl3) δ (ppm): 1.23 (6H, d, J ) 6.9 Hz, CH(CH3)2), 2.42 (3H, s, CH3), 3.04 (1H, m, CH(CH3)2), 3.75 (3H, s, CH3O), 4.88 (2H, s, CH2O), 6.51 (1H, s, CH); 13C NMR (CDCl3) δ (ppm): 21.5 (CH(CH3)2), 24.0 (CH3), 37.3 (CH(CH3)2), 52.0 (CH3O), 62.3 (CH2O), 103.8 (CHar), 167.8, 168.4, 169.2, and 174.5 (Car and CO). 1116

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FIGURE 1. Chemical structure of diazinon and the diazinon-like haptens used in this work. The ester (2 mmol, 448 mg) was dissolved in tetrahydrofuran (THF) (1 mL), and 1 M NaOH (12 mL) was added. After reflux for 1 h, the mixture was extracted with diethyl ether (3 × 20 mL). The aqueous layer was acidified under ice cooling to pH 3 by careful addition of 14.5 M H3PO4 and then extracted with (3 × 20 mL) dichloromethane. The organic layer was dried over anhydrous Na2SO4 and concentrated, giving the acid as a white solid that did not need further purification (128 mg, 30%); 1H NMR (CDCl3) δ (ppm): 1.24 (6H, d, J ) 6.9 Hz, CH(CH3)2), 2.48 (3H, s, CH3), 3.12 (1H, m, CH(CH3)2), 4.95 (2H, t, J ) 6.2 Hz, CH2O), 6.56 (1H, s, CH); 13C NMR (CDCl ) δ (ppm): 21.4 (CH(CH ) ), 23.1 (CH ), 36.6 3 3 2 3 (CH(CH3)2), 62.4 (CH2O), 104.3 (CHar), 167.2, 168.8, 171.7, and 174.5 (Car and CO). 4-[(2-Isopropyl-6-methylpyrimidin-4-yl)oxy]butanoic Acid (2). 2-Isopropyl-6-methyl-4-pyrimidinol (9 mmol, 1370 mg) was added to a mixture of sodium hydride (NaH) (11.1 mmol, 486 mg) and washed twice with pentane in anhydrous acetonitrile (CH3CN) (32 mL) at 0 °C. The mixture was warmed to 30-40 °C, and ethyl 4-bromobutanoate (10.8 mmol, 2.1 g) in CH3CN (4 mL) was added dropwise. Then, it was refluxed for 7 h and left 15 h at room temperature. Filtration of the crude and concentration of the filtrate gave orange oil. Column chromatography (hexane/EtOAc, 80:20) yielded the methyl ester as a colorless oil (1030 mg, 42%), TLC Rf 0.69 (hexane/EtOAc, 50:50); 1H NMR (CDCl3) δ (ppm): 1.25 (3H, t, J ) 7.2 Hz, CH3CH2O), 1.27 (6H, d, J ) 6.8 Hz, CH(CH3)2), 2.09 (2H, m, CH2), 2.39 (3H, s, CH3), 2.46 (2H, t, J ) 7.4 Hz, CH2CO), 3.04 (1H, m, CH(CH3)2), 4.14 (2H, q, J ) 7.1 Hz, CH3CH2O), 4.40 (2H, t, J ) 6.3 Hz, CH2O), 6.33 (1H, s, CH); 13C NMR (CDCl ) δ (ppm): 14.2 (CH -CH ), 21.6 (CH(CH ) ), 3 3 2 3 2 23.9 (CH3), 24.3 (CH2), 30.9 (CH2CO), 37.4 (CH(CH3)2), 60.4 (OCH2CH3), 64.8 (CH2O), 103.6 (CHar), 167.1, 169.6, 173.1, and 174.9 (Car and CO). The ester (2 mmol, 532 mg) was dissolved in THF (1 mL), and 1 M NaOH (12 mL) was added. After reflux for 1 h, the mixture was extracted with diethyl ether (3 × 20 mL). The aqueous layer was acidified under ice cooling to pH 3 by careful addition of 14.5 M H3PO4 and then extracted with dichloromethane (3 × 20 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated, yielding the acid

as a white solid that did not need further purification (250 mg, 53%); 1H NMR (CDCl3) δ (ppm): 1.28 (6H, d, J ) 6.9 Hz, CH(CH3)2), 2.10 (2H, m, CH2), 2.41 (3H, s, CH3), 2.54 (2H, t, J ) 7.4 Hz, CH2CO), 3.06 (1H, m, CH(CH3)2), 4.42 (2H, t, J ) 6.2 Hz, CH2O), 6.35 (1H, s, CH); 13C NMR (CDCl3) δ (ppm): 21.6 (CH(CH3)2), 23.5 (CH3), 24.2 (CH2), 30.7 (CH2CO), 37.1 (CH(CH3)2), 64.8 (CH2O), 103.9 (CHar), 166.9, 169.7, 174.9 (Car), and 177.9 (CO). 6-[(2-Isopropyl-6-methylpyrimidin-4-yl)oxy]hexanoic Acid (3). 2-Isopropyl-6-methyl-4-pyrimidinol (6 mmol, 913 mg) was added to a mixture of NaH (7.4 mmol, 324 mg), washed twice with pentane in anhydrous acetonitrile (20 mL) at 0°C. The mixture was warmed to 30-40 °C, and ethyl 6-bromohexanoate (7.2 mmol, 1.6 g) in CH3CN (4 mL) was added dropwise. It was refluxed for 7 h and left 15 h at room temperature. Filtration of the crude and concentration of the filtrate gave orange oil. Column chromatography (hexane/ EtOAc, 80:20) yielded the methyl ester as a colorless oil (1027 mg, 58%), TLC Rf 0.77 (hexane/EtOAc, 50:50); 1H NMR (CDCl3) δ (ppm): 1.25 (3H, t, J ) 7.2 Hz, CH3CH2O), 1.28 (6H, d, J ) 6.7 Hz, CH(CH3)2), 1.45 (2H, m, CH2), 1.65-1.82 (4H, m, 2CH2), 2.32 (2H, t, J ) 7.5 Hz, CH2CO), 2.38 (3H, s, CH3), 3.05 (1H, m, CH(CH3)2), 4.12 (2H, q, J ) 7.2 Hz, CH3CH2O), 4.33 (2H, t, J ) 6.5 Hz, CH2O), 6.33 (1H, s, CH). 13C NMR (CDCl3) δ (ppm): 14.2 (CH3-CH2), 21.7 (CH(CH3)2), 23.9 (CH3), 24.7 (CH2), 25.6 (CH2), 28.5 (CH2), 34.2 (CH(CH3)2), 37.5 (CH2CO), 60.2 (O-CH2-CH3), 65.6 (CH2O), 103.6 (CHar), 167.0, 170.5, 178.0, and 183.4 (Car and CO). The ester (3.5 mmol, 1.03 g) was dissolved in THF (1.7 mL), and 1 M NaOH (17 mL) was added. After reflux for 1 h, the mixture was extracted with diethyl ether (3 × 20 mL). The aqueous layer was acidified with 14.5 M H3PO4 to pH 3 in an ice bath and extracted with dichloromethane (3 × 20 mL). The extract was dried over anhydrous Na2SO4 and concentrated, yielding the acid as a white solid that did not need further purification (490 mg, 46%); 1H NMR (CDCl3) δ (ppm): 1.28 (6H, d, J ) 6.8 Hz, CH(CH3)2), 1.51 (2H, m, CH2), 1.67-1.82 (4H, m, 2CH2), 2.38 (2H, t, J ) 7.0 Hz, CH2CO), 2.40 (3H, s, CH3), 3.05 (1H, m, CH(CH3)2), 4.35 (2H, t, J ) 6.5 Hz, CH2O), 6.33 (1H, s, CH); 13C NMR (CDCl3) δ (ppm): 21.6 (CH(CH3)2), 23.5 (CH3), 24.5 (CH2), 25.5 (CH2), 28.5 (CH2CO), 34.0 (CH2), 37.1 (CH(CH3)2), 65.8 (CH2O), 103.8 (CHar), 166.7, 169.8, 174.9 (Car), and 178.5 (CO). ({4-[(Diethoxyphosphorothioyl)oxy]-6-methylpyrimidin2-yl}thio)acetic Acid (4). 4-Hydroxy-2-mercapto-6-methylpyrimidine (50 mmol, 7.1 g) was added to a solution of sodium ethoxide (52.5 mmol) in ethanol (50 mL) at 0 °C under argon. The mixture was warmed to 50-60 °C, and ethyl bromoacetate (50 mmol, 5.6 mL) was added dropwise. It was warmed to 70-80 °C for 2.5 h. The crude was added over water (100 mL), and the product crystallized. It was isolated by filtration and washed with diethyl ether, giving a white solid (6.08 g, 53%). 1H NMR (CDCl3) δ (ppm): 1.28 (3H, t, J ) 7.1 Hz, CH2CH3), 2.22 (3H, s, CH3), 3.94 (2H, s, S-CH2), 4.21 (2H, q, J ) 7.1 Hz, CH2CH3), 6.06 (1H, s, CHar). 13C NMR (CDCl ) δ (ppm): 14.1 (CH CH ), 24.0 (CH ), 32.7 3 3 2 3 (S-CH2), 61.9 (OCH2), 108.6 (CHar), 158.9, 165.2, 165.7 (Car), and 168.2 (CO). Sodium (5 mmol, 115 mg) was added to a solution of the ethyl ester (5 mmol, 1.14 g) in anhydrous acetonitrile (25 mL) at 0 °C under argon. After 30 min. diethylchlorothiophosphate (6 mmol, 1.13 g) was added dropwise, and the mixture was refluxed for 2 h. Filtration, concentration at reduced pressure, and column chromatography (hexane/ EtOAc 1:1) gave the phosphate ester as a yellow oil (m ) 1.6 g, 81%). 1H NMR (CDCl3) δ (ppm): 1.27 (3H, t, J ) 7.1 Hz, CO2CH2CH3), 1.38 (6H, t, J ) 7.1 Hz, 2 OCH2CH3), 2.43 (3H, s, CH3), 3.95 (2H, s, S-CH2), 4.20 (2H, q, J ) 7.1 Hz, CO2CH2CH3), 4.31 (4H, m, 2 OCH2CH3), 6.52 (1H, s, CHar). 13C NMR (CDCl3) δ (ppm): 14.1 (CH3CH2), 15.8 and 15.9 (2CH3CH2),

24.0 (CH3), 33.5 (S-CH2), 61.6 (CH2-CH3), 65.5 (2CH2-O), 105.4 (CHar), 163.9, 169.0, 170.1 (Car), and 170.5 (CO). The ester (3.9 mmol, 1.55 g) was dissolved in 1:1 THF/ methanol (20 mL). Aqueous sodium carbonate (744 mg in 7 mL of water) was added with stirring for 30 min at 0 °C. Water (7 mL) was added, and the mixture was stirred for a further 4 h at room temperature, followed by the addition of another 40 mL of water. The mixture was extracted with diethyl ether (3 × 10 mL), and the ethereal layer was backwashed with 30 mL of water. The combined aqueous layers were acidified with 12 M HCl at 0 °C and then extracted with ethyl acetate (3 × 25 mL). The extract was dried and evaporated, giving a yellow oil (743 mg, 52%). 1H NMR (CDCl3) δ (ppm): 1.39 (6H, t, J ) 7.1 Hz, 2 OCH2CH3), 2.50 (3H, s, CH3), 3.86 (2H, s, S-CH2), 4.32 (4H, q, J ) 7.1 Hz, 2 OCH2CH3), 6.63 (1H, s, CHar). 13C NMR (CDCl3) δ (ppm): 15.8 and 15.9 (2CH3CH2), 23.7 (CH3), 33.7 (S-CH2), 65.8 and 65.9 (2CH2-O), 106.2 (CHar), 164.2, 170.5, 170.6, and 171.3 (Car and CO). 4-({4-[(Diethoxyphosphorothioyl)oxy]-6-methylpyrimidin-2-yl}thio)butanoic Acid (5). 4-Methyl-2-thiouracyl (50 mmol, 7.1 g) was added to a solution of sodium ethoxide (52.5 mmol) in ethanol (50 mL) at 0 °C under argon. The mixture was warmed to 50-60 °C, and ethyl 4-bromobutanoate (50 mmol, 7.2 mL) was added dropwise. It was warmed to 70-80 °C for 2.5 h. The crude was added over water (130 mL), and the product crystallized. It was isolated by filtration, giving a white solid (2.95 g, 76%). 1H NMR (DMSO) δ (ppm): 1.17 (3H, t, J ) 7.2 Hz, CH2CH3), 1.89 (2H, m, CH2), 2.15 (3H, s, CH3), 2.40 (2H, t, J ) 7.4 Hz, CH2CO), 3.12 (2H, t, J ) 6.7 Hz, S-CH2), 4.05 (2H, q, J ) 7.1 Hz, CH2CH3), 5.98 (1H, s, CHar). 13C NMR (DMSO) δ (ppm): 14.1 (CH3CH2), 23.2, 24.3, 28.9, 32.3 (3 CH2 and CH3), 59.9 (OCH2), 103.7 (CHar), 153.1, 161.0, 163.9, and 175.9 (Car and CO). Sodium (5 mmol, 115 mg) was added to a solution of the ethyl ester (5 mmol, 1.28 g) in anhydrous acetonitrile (25 mL) at 0 °C under argon. After 30 min. diethylchlorothiophosphate (6 mmol, 1.13 g) was added dropwise. The mixture was refluxed for 3 h. Filtration, concentration at reduced pressure, and column chromatography (hexane/EtOAc 9:1) gave the phosphate ester as a yellow oil (0.58 g, 28%). The ester (1.37 mmol, 0.56 g) was dissolved in THF/ methanol 1/1 (7 mL). Aqueous sodium carbonate (265 mg in 2.5 mL of water) was added and stirred for 30 min at 0 °C. Water (3.7 mL) was added, and the was mixture stirred for 5 days at room temperature, followed by the addition of another 26 mL of water. The mixture was extracted with diethyl ether (3 × 10 mL), and the ethereal layer was backwashed with 11 mL of water. The combined aqueous layers were acidified with 14.5 M H3PO4 at 0 °C and then extracted with dichloromethane (3 × 25 mL). The extract was dried and evaporated, leading to a yellow oil (217 mg, 42%) that did not require further purification. 1H NMR (CDCl3) δ (ppm): 1.40 (6H, t, J ) 7.1 Hz, 2 OCH2CH3), 2.10 (2H, m, CH2), 2.46 (3H, s, CH3), 2.57 (2H, t, J ) 7.4 Hz, CH2CO), 3.23 (2H, t, J ) 7.3 Hz, S-CH2), 4.25-4.38 (4H, q, J ) 7.1 Hz, 2 OCH2CH3), 6.51 (1H, s, CHar). 13C NMR (CDCl3) δ (ppm): 15.8 and 15.9 (2 CH3CH2), 24.0, 24.4, 30.0, 32.7 (3 CH2 and CH3), 65.4 and 65.5 (2 OCH2), 104.9 (CHar), 164.0, 170.5, 171.5 (Car), and 178.9 (CO). 6-({4-[(Diethoxyphosphorothioyl)oxy]-6-methylpyrimidin-2-yl}thio)hexanoic Acid (6). 4-Methyl-2-thiouracyl (50 mmol, 7.1 g) was added to a solution of sodium ethoxide (52.5 mmol) in ethanol (50 mL) at 0 °C under argon. The mixture was warmed to 50-60 °C, and ethyl 6-bromohexanoate (50 mmol, 8.9 mL) was added dropwise. It was warmed to 70-80 °C for 2.5 h. The crude product was added over water (100 mL) and extracted with dichloromethane (3 × 25 mL) giving an orange oil. Column chromatography (hexane/ AcEt 1:1) gave a white solid (9.96 g, 70%). TLC Rf 0.25 (hexane/ VOL. 38, NO. 4, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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EtOAc, 50:50); 1H NMR (CDCl3) δ (ppm): 1.23 (3H, t, J ) 7.1 Hz, CH2CH3), 1.45 (2H, m, CH2), 1.77-1.60 (4H, m, 2CH2), 2.23 (3H, s, CH3), 2.29 (2H, t, J ) 7.4 Hz, CH2CO), 3.17 (2H, t, J ) 7.4 Hz, S-CH2), 4.10 (2H, q, J ) 7.1 Hz, CH2CH3), 6.04 (1H, s, CHar). 13C NMR (CDCl3) δ (ppm): 14.2 (CH3CH2), 24.1, 24.3, 28.0, 28.6, 30.2, 34.0 (5 CH2 and CH3), 60.2 (OCH2), 108.1 (CHar), 160.6, 165.4, 165.8 (Car), and 173.5 (CO). Sodium (5 mmol, 115 mg) was added to a solution of the ethyl ester (5 mmol, 1.42 g) in anhydrous acetonitrile (25 mL) at 0 °C under argon. After 30 min, diethylchlorothiophosphate (6 mmol, 1.13 g) was added dropwise. The mixture was refluxed for 2 h and stirred for 14 h at room temperature. Filtration, concentration at reduced pressure, and isolation by column chromatography (hexane/EtOAc 9:1) gave the phosphate ester as a colorless oil (1.5 g, 68%); Rf (hexane/ EtOAc 7:3) ) 0.57. 1H NMR (CDCl3) δ (ppm): 1.24 (3H, t, J ) 7.1 Hz, CO2CH2CH3), 1.38 (6H, t, J ) 7.1 Hz, 2 OCH2CH3), 1.40-1.52 (2H, m, CH2), 1.58-1.80 (4H, m, 2CH2), 2.30 (2H, t, J ) 7.4 Hz, CH2CO), 2.42 (3H, s, CH3), 3.13 (2H, t, J ) 7.3 Hz, S-CH2), 4.11 (2H, q, J ) 7.1 Hz, CO2CH2CH3), 4.25-4.36 (4H, m, 2 OCH2CH3), 6.47 (1H, s, CHar). 13C NMR (CDCl3) δ (ppm): 14.2 (CH3CH2), 15.8 and 15.9 (2CH3CH2), 24.1, 24.5, 28.3, 28.9, 30.7, and 34.2 (5 CH2 and CH3), 60.2 (OCH2), 65.3 and 65.4 (2 OCH2), 104.8 (CHar), 163.9, 170.4, 172.0, and 173.6 (CO and Car). The ester (2.55 mmol, 1.1 g) was dissolved in 1:1 THF/ methanol (13 mL). Aqueous sodium carbonate (486 mg in 4.6 mL of water) was added with stirring for 30 min at 0 °C. Then, the solution was stirred with 7 mL water for 4 h at room temperature, followed by the addition of another 26 mL of water. The mixture was extracted with diethyl ether (3 × 10 mL), and the ethereal layer was back-washed with 30 mL of water. The combined aqueous fractions were acidified with 14.5 M H3PO4 at 0 °C and then extracted with dichloromethane (3 × 25 mL). The extract was dried, evaporated, and purified by column chromatography (hexane/AcEt 7:3) leading to a colorless oil (310 mg, 30%). 1H NMR (CDCl3) δ (ppm): 1.38 (6H, t, J ) 7.1 Hz, 2 OCH2CH3), 1.45-1.55 (2H, m, CH2), 1.60-1.85 (4H, m, 2CH2), 2.37 (2H, t, J ) 7.4 Hz, CH2CO), 2.43 (3H, s, CH3), 3.13 (2H, t, J ) 7.3 Hz, S-CH2), 4.20-4.40 (4H, q, J ) 7.1 Hz, 2 OCH2CH3), 6.49 (1H, s, CHar). 13C NMR (CDCl3) δ (ppm): 15.8 and 15.9 (2 CH3CH2), 24.0, 24.2, 28.2, 28.9, 30.6, 33.8 (5 CH2 and CH3), 65.3 and 65.4 (2 OCH2), 104.7 (CHar), 163.9, 170.3, 171.9 (Car), and 179.5 (CO). Immunogens, Enzyme Tracers, and Coating Conjugates. For immunizing purposes, haptens were covalently attached through their carboxylic acid moieties to the lysine groups of BSA by use of a modification of the active ester method (19). Additionally, for enzyme tracers preparation, the battery of haptens was covalently attached to HRP (horseradish peroxidase). Similarly, the set of haptens was reacted to OVA for preparing coating conjugates using the mixed anhydride method (20). Hapten 7 was conjugated through its alcohol group to BSA, OVA, and HRP following the method previously described (11). Finally, immunogens, tracers, and coating conjugates were purified by gel-exclusion chromatography on Sephadex G-25 using PBS for elution. The conjugates were stored at -20 °C till use. Immunization Schedule and Antiserum Preparation. Each immunizing conjugate (0.20 mg in 0.5 mL of PBS) was suspended in 0.5 mL of Freund’s complete adjuvant and injected intramuscularly into two (numbered I and II) female New Zealand white rabbits. Animals were boosted at 21-day intervals with the same immunogen suspended in 0.5 mL of Freund’s incomplete adjuvant. Ten days after each boost, blood was obtained by bleeding the ear vein of the rabbit. When no titers enhance were observed, whole blood was collected and allowed to coagulate overnight at 4 °C. Then serum was separated by centrifugation. An overall of 16 sera 1118

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were obtained: S1-I and S1-II for BSA-1, S2-I and S2-II for BSA-2, S3-I and S3-II for BSA-3, S4-I and S4-II for BSA-4, S5-I and S5-II for BSA-5, S6-I and S6-II for BSA-6, S7-I and S7-II for BSA-7, and S8-I and S8-II for BSA-8. Aliquots of sera were stored at 4 °C in 50% ammonium sulfate. Screening of Sera, Enzyme Tracer, and Coating Conjugate. Optimum concentrations of coating conjugates, sera dilution, and enzyme tracers were chosen to produce absorbance values of approximately 0.5-1.2 units in the absence of analyte. For this purpose, two assay formats were carried out using a checkerboard titration assay. First, the avidity of sera against different coating antigens was determined on a noncompetitive indirect ELISA format by measuring the binding of serial dilutions (from 1/1000 to 1/64 000) of each antiserum to microtiter plates coated with different concentrations (from 0.001 to 1.0 mg/L) of each conjugate. Similarly, their avidities against enzyme tracers was tested on a direct ELISA by measuring the binding of serial dilutions of the enzyme tracers (from 0.001 to 1.0 mg/ L) to the microtiter plates coated with different dilutions of each serum (from 1/500 to 1/512 000). Conjugate-Coated Format. Flat-bottomed polystyrene ELISA plates were coated with 100 µL/well of the appropriate concentration of the OVA-hapten conjugate solution in coating buffer (50 mM carbonate-bicarbonate buffer, pH 9.6 (CB)). The plates were then sealed and incubated overnight at 4 °C. The following day, plates were washed six times with PBS-T (10 mM phosphate buffer, 137 mM NaCl, and 2.7 mM KCl, pH 7.5, containing 0.05% Tween 20). For competitive assays, a volume of 50 µL of the appropriate sera dilution (in 2-fold concentrated PBST) and 50 µL of standards in deionized water were added to the coated plates and incubated for 1 h at room temperature. After being washed as earlier, plates were incubated for 1 h with peroxidaselabeled goat anti-rabbit immunoglobulins (GAR-HRP) diluted 1:4000 in PBST (100 µL/well). Once washed, peroxidase activity was determined by adding 100 µL/well of substrate solution (2 mg/mL OPD and 0.012% H2O2 in 25 mM sodium citrate and 62 mM sodium phosphate, pH 5.5). After 10 min, the enzymatic reaction was stopped by adding 2.5 M H2SO4 (50 µL/well), and the absorbance was read in dual-wavelength mode (490 nm as test wavelength and 650 nm as reference wavelength). Antibody-Coated Format. Polystyrene ELISA plates were coated overnight at 4 °C with 100 µL/well of the appropriate dilution of antibody in CB. The following day, plates were washed six times with PBST. Then 100 µL/well of a mixture of 50 µL of HRP-hapten in 2-fold concentrated PBST with 50 µL of standards in deionized distilled water was added to coated plates and incubated for 1 h at room temperature. After being washed, the HRP tracer activity was measured using the same procedure described for the conjugate-coated format. ELISA Optimization. Assay optimization was performed using diazinon as the competitor analyte. A set of experimental parameters (pH, ionic strength, and surfactant concentration) was studied sequentially to improve the sensitivity of the immunoassay. Criteria used to evaluate immunoassay performance were sensitivity (IC50), maximum absorbance (A0), dynamic range (RD), and limit of detection (LD, estimated at 10% inhibitory concentration). The optimization was performed only to the two most sensitive assays (S6-I/OVA-7; S7-I/OVA-7). The effect of pH was evaluated by use of different PBST solutions ranging from pH 6.5 to pH 8.0. PBS at 0.5, 1.0, 2.0, and 4.0X (PBS), always containing 0.05% (v/v) Tween 20, were then used to estimate the ionic strength effect. Finally, the influence of surfactant Tween 20 concentration (0, 0.01, 0.05, 0.1, and 0.5% v/v) on immunoassay performances was also studied.

Evaluation of the Immunoassays. (i) Cross-Reactivity Determinations. The specificity of the ELISA assays was determined against several organophosphorus insecticides, two commonly used s-triazines, and structurally related compounds with diazinon. (ii) Effect of Organic Solvents. The use of organic solvents has to be adequately tested since antibodies generally are not very tolerant to them. Using the optimized conditions, competitive curves were carried out for diazinon standards containing percentages from 0 to 10% of acetonitrile, 2-propanol, methylsulfoxide, and methanol. (iii) Analysis of Drinking Water by ELISA. Eight bottled drinking water samples with a low mineralization (electrical conductivity between 25 and 874 µS/cm at 20 °C) were spiked with diazinon within the analytical working range and analyzed through the optimized ELISA without previous extraction or cleanup. Furthermore, several surface water samples (BDO5 < 3 mg of O2/L) from the Turia River Basin (Spain) were also analyzed through the optimized diazinon immunoassay. Competitive curves were mathematically analyzed by fitting experimental points to a four-parameter logistic equation by the Sigmaplot software package (Jandel Scientific, Erkrath, Germany). Standards and samples were run in three replicates recording the absorbance values. GC-MS Determination. Prior to GC-MS analysis, water samples (30 mL) were homogenized for 60 s with 20 mL of acetonitrile and 20 mL of dichloromethane using an Ultraturrax T-25 apparatus mixer (Hanke and Junkle, Germany). After filtration, the organic phase was transferred to a conicalbottom flask; the solvent was evaporated to dryness and reconstituted with 2 mL of hexane. Finally, 2 µL of each sample was injected, by pulsed split-less, on the GC column for diazinon quantification. Helium was the carrier gas at a flow rate of 1.2 mL min-1. Samples were injected in the splitless mode. The column temperature was held at 60 °C for 1 min, then increased 30 °C/min to 120 °C, 5 °C/min to 195 °C, 30 °C/min to 280 °C, and held at this temperature for 4 min. Injector temperature was 250 °C. Diazinon was detected by selected ion monitoring of three characteristic fragment ions (m/z 248, 276, and 304).

Results and Discussion Hapten Design and Synthesis. The initial and decisive step in the development of immunoassays for diazinon lies on the selection of appropriate haptens. The diazinon molecule has apparently three significant antigenic determinants that can participate in establishing noncovalent bonds with the antibody. These are the oxygen atom, the thiophosphate group, and the aromatic moiety. In this work, a set of different types of diazinon haptens (haptens 1-8, Figure 1) has been synthesized. The haptens differ on the site of the diazinon structure through which the spacer arm was attached and on the length of the spacer arm as well. Haptens 1-3 were synthesized by reaction of sodium or potassium salt of 2-isopropyl-6-methyl-4-pyrimidinol with bromoesters of different length followed by ester hydrolysis. Direct alkylation of this compound with bromoacids would lead to the carboxylic acids in just one step. However, it is well-known that 4-pyrimidinols react with alkyl halides giving O- and N-alkylation products. Since esters are easier to separate than acids, we decided to carry out the reaction with bromoesters as alkylating agents. These reactions led to the expected O- and N-alkylation products that could be easily isolated by column chromatography, with the first one being the less polar compound. This double alkylation gives the desired O-alkylated product in poor yield. The use of potassium carbonate instead of sodium hydride in the alkylation with methyl bromoacetate increased appreciably the O-alkylation product yield. Hydrolysis of the obtained esters was carried out with sodium

hydroxide in tetrahydrofuran/methanol, followed by acidification with 12 M HCl. Haptens were obtained with moderate yields, recovering 2-isopropyl-6-methyl-4-pyrimidinol used as the starting material in some cases. This fact can be explained by a nucleophilic attack of hydroxide ion to position 4 of the ring. However, hydrolysis with sodium carbonate, which does not permit such an attack, did not yield better results. Despite this, the approach provides a fast and easy way to obtain haptens for diazinon. Haptens 4-6 were synthesized using 4-hydroxy-2-mercapto-6-methyl pyrimidine as a suitable commercial starting product. Given that the sulfur atom in this kind of ring suffers alkylation with high quimioselectivity, the compound provides a way to attach the spacer arm through alkylation of thiol group, keeping the hydroxyl group necessary to synthesize the corresponding phosphate ester. Therefore, reaction of the selected ring with an equivalent of sodium methoxide and the corresponding bromoester led, as we expected, to the sulfide. It must be noted that in all cases sulfides were easily isolated from the reaction mixture by precipitation after the addition of water. Generation of the sodium salt of the subsequent pyrimidinols followed by reaction with diethylchlorothiophosphate led to the introduction of the phosphate group. Finally, it was necessary to make a selective hydrolysis of the spacer arm ester. The use of sodium carbonate provided mild hydrolysis conditions. Although a long reaction time was necessary to improve hydrolysis in the synthesis of hapten 6, this approach led to the desired carboxylic acids in good yields. For the synthesis of haptens 7 and 8, we followed previously reported procedures (10, 11). Using haptens 1-8, we expected to raise high-quality antisera and therefore improve on the quality of previously reported diazinon immunoassays (4, 17). Protein Conjugates. The conjugation of each hapten to proteins (BSA, OVA, and HRP) was estimated by measuring the hapten/protein molar ratio according to UV absorbance. By this method, a mean diazinon/protein ratio ranging from 14 to 39 was obtained for coating and immunizing conjugates, respectively. Meanwhile, for HRP conjugates, a hapten/HRP molar ratio ranging from 1 to 2 was calculated. Sera Screening. The serum of each rabbit was tested against homologous and heterologous coating conjugates on an indirect ELISA format after every bleeding. Because none of HRP-hapten conjugates were recognized by any sera tested (minimum sera dilution factor of 1/500 and 1 mg/mL HRP-conjugates) in the antibody-coated ELISA format, we decided to evaluate a battery of eight different coating antigens in the conjugate-coated format. The result of the screening of the 16 sera against each of the 8 coating antigens is presented in Table 1. L (low), M (medium), and H (high) are arbitrary units corresponding to the dilution factor applied to the sera for coating conjugate concentrations ranging from 0.001 to 1.0 mg/L to obtain absorbance signal between 0.5 and 1.2. L < 1:10 000, M ) 1/10 000-1/30 000, and H > 1/30 000. A notable feature in Table 1 is that, for each coating antigen, the titer difference among the antisera is significant. It is difficult to draw solid conclusions from these experiments; inter-individual variation among animals and the nature of the target compounds may affect the final behavior of the sera. However, the results of the experiment showed that there are certain trends in the behavior of the antibodies. Those antibodies obtained through the same group of haptens that is used for coating purposes show high or medium titer. In contrast, some low titer values (S6-II) appear to have resulted from divergence in the host animal rather than the difference in spacer arm length because the other sera against the same hapten (S6-I) showed much higher titers. Another feature to note in Table 1 is that titer difference VOL. 38, NO. 4, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Sera Titration Experiments with the Different Coating Antigens coating conjugatesb seruma OVA-1 OVA-2 OVA-3 OVA-4 OVA-5 OVA-6 OVA-7 OVA-8 S1-I S1-II S2-I S2-II S3-I S3-II S4-I S4-II S5-I S5-II S6-I S6-II S7-I S7-II S8-I S8-II

H H L M N N N N N N H L L N N N

M H H H N L L L M H H L L L N N

L L M M L L N L L L H L L N N N

L L N N M N H L M L M L N N N N

L N N N N N M N H H L L N N N N

N N N N L N L N L L L L N N N N

L L L L L L L L L L M M M M N N

N N N N N N N N N N N N N N N N

a Arabic numbers corresponds to the hapten, and the Roman numeral corresponds to each immunized rabbit. b L, M, and H are arbitrary units corresponding to the diltuion factor applied to the sera (L < 1:10 000, M ) 1/10 000-1/30 000, and H > 1/30 000) giving a signal between 0.5 and 1.2 units of absorbance. N, no recognition.

TABLE 2. Serum-Coating Antigen Combination Given the Lowest IC50 serum

coating antigen

IC50 (ng/mL)

S1-I S2-II S3-I S4-I S5-I S6-I S7-I

OVA-4 OVA-1 OVA-7 OVA-4 OVA-2 OVA-7 OVA-7

2010 69.1 29.5 27.3 627 7.5 7.5

between the homologous and heterologous assays is small, suggesting that heterology in spacer arm length is not important for the antigen recognition by antibodies. The examination of Table 1 also reveals that the avidity of some sera continues to decrease with increasing spacer arm length of plate-coating hapten. Of the 16 antisera, 13 show the lowest titer or no recognition against the hapten with longest spacer arm (hapten 3, 6, and 8). Theoretically, this may result from masking the functional groups of coating hapten due to folding of the hapten molecule. Coating the plates with OVA-8 conjugate, the avidity of all sera was negligible. Nevertheless, the rest of the coating conjugates probed under both homologous and heterologous conditions was fairly recognized. All the combinations of serum/coating conjugates that showed specific recognition were used to carry out competitive assays in order to determine the most sensitive assay for diazinon. Table 2 shows IC50 values. From these results, it was further noticeable that haptens containing the thiophosphate moiety produced polyclonal sera that were inhibited by lower concentration of diazinon than sera obtained from haptens lacking that moiety or containing smaller aliphatic chains. Before optimization, the optimum sera/coating conjugate concentrations giving the lowest IC50 were S6-I/OVA-7 (1:1000/1.0 mg/L; assay A) and S7-I/OVA-7 (1:2000/1.0 mg/L; assay B) for diazinon determination. The sensitivity (IC50) achieved for diazinon with both assay A and assay B was 7.5 ng/mL. ELISA Optimization. The effect of pH, ionic strength, and surfactant concentration on assay performance (signal and 1120

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sensitivity) was studied via a 1-h step reaction using the conjugate-coated ELISA format at room temperature. These conditions were selected for ELISA optimization. The effect of the pH of the media for both assays A and B is shown in Figure 2. As diazinon is hydrolyzed at both low and high pH, we decided to evaluate the influence of a narrow pH range. It was observed that immunoassays for diazinon are more sensitive (lower IC50) between pH 7.5 and pH 8 than at other tested pH values. Furthermore, above pH 7.5 the signal diminished, particularly for assay B, whose signal was less than 0.5 absorbance units. This signal loss had a distinct influence on assay performance, yielding standard curves having large slopes and narrow dynamic range. Similar pH effects of polar compounds have been reported (21). Therefore, a pH value of 7.5 was chosen as optimum to keep an acceptable signal-to-sensitivity ratio. On the other hand, the effect of the ionic strength on the assays performance is also shown in Figure 2. Different concentrations of PBS, ranging from 0.5 to 4.0-fold of the original PBS buffer concentration and always 0.05% (v/v) Tween 20, were tested. Essentially, for both immunoassays IC50 value decreased gradually as the buffer salt concentration increased. It seems that the effect of the salt concentration on the assay is due to biochemical interactions, mainly hydrophobic, between analyte and antibody. These interactions are generally favored by increasing the ionic strength of the reaction medium, while the increase exerts a detrimental effect on interactions where ionic driving forces prevail. However, some studies suggested that antibody affinity rises for polar analytes by increasing the buffer concentration (22). As diazinon is relatively polar analyte (log Kow ) 3.81), the interactions with the antibody are weak type such as dipole-dipole, etc. In this study, the optimum concentration of the buffer, which provided the highest A0 and lowest IC50, was 10 mM phosphate buffer, 137 mM NaCl, 2.7 mM KCl, pH 7.5 containing 0.05% Tween 20 for both assays for diazinon. Using the established pH and buffer concentration conditions, the effect of Tween 20 as surfactant on signal and sensitivity was studied. The usual concentration of Tween 20 for most pesticide immunoassay is 0.05%. However, for several pesticides there is a marked effect of detergent concentration on ELISA performance (23). In this study (Figure 2), the negative influence (lower signal and lower sensitivity) observed as surfactant concentration increased may be related with nonspecific hydrophobic interactions between the surfactant and the interferences with the specific immunoreaction. Therefore, 0.05% Tween 20 was selected as a trade between sensitivity and absence of nonspecific interactions. Figure 3 shows the competitive curves of the optimized assays for diazinon detection. The limit of detection (LD), calculated as the concentration corresponding to 10% inhibition of the maximum signal, was 0.40 and 0.85 ng/mL for the assay A and B, respectively. The dynamic range, established between the concentrations producing 20% and 80% inhibition was between 1.3 and 71.3 ng/mL and between 1.2 and 32.0 ng/mL for assay A and assay B, respectively. Although, for diazinon determination, the optimization procedure did not enhance assay sensitivity; the assay can be considered sensitive enough to determine diazinon at nanograms per milliliter level. In this sense, it is worth mentioning the better sensitivity and specificity obtained in comparison with the immunoassay reported by ten Hoeve et al. (10). On the other hand, though we followed the route proposed by Beasely et al. (11) for the synthesis of a diazinon hapten, our immunoassays are slightly less sensitive. In this case, the most likely explanation is the individual variation among animals. In comparison with the performances of commercial immunoassay kits for diazinon, it can be

FIGURE 2. Influence of pH, buffer concentration (PBS), and Tween 20 on the maximum signal (A0) and assay sensitivity (IC50) in the diazinon ELISA for antiserum S6-I (assay A) and S7-I (assay B). designed novel synthetic strategies to generate diazinon haptens yielding sensitive antibodies. Consequently, following herein described hapten synthesis in combination with sera production (polyclonal, monoclonal, etc.) and purification and setting up other assay formats (coating with protein A, magnetic particles, etc.) further improvement on sensitivity and selectivity for diazinon immunoassays could be achieved.

FIGURE 3. Calibration curves for diazinon using antiserum S6-I (assay A, b) and S7-I (assay B, 0). Each point represents the mean ( SD of six plates with three replicates per plate. noted that our immunoassay offers poorer sensitivity even though it works under heterology conditions. Given the limited information about how the kit immunoreagents were obtained and how the haptens were synthesized, we

Cross-Reactivity Studies. The antibodies were further characterized by determining the cross-reactivity with a variety of pesticides. As is shown in Table 3, a weak crossreactivity was observed for the set of organophosphorus insecticides, and the diazinon hydroxy-metabolite checked. These values can be considered virtually negligible taking into account the sensitivity level for the diazinon assay. Thus, the developed immunoassays for diazinon are highly specific against the main organophosphorus insecticides and its main metabolite. However, diazoxon, which several studies have suggested may be a relatively significant cross-reactant, was not tested. In addition, the cross-reactivity of several nonchemically related herbicides was also determined because of their widespread agricultural and domestic use. As it can be seen, none of them was detected at concentrations above 10 mg/L. When comparing the results obtained in this VOL. 38, NO. 4, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Organic solvent tolerance of the diazinon immunoassay. Data were obtained from calibration curves carried out in the buffer containing different concentrations of the solvents (from 0 to 10%). Maximum absorbance is expressed relative to the control inhibition curve performed without organic solvent.

TABLE 3. Cross-Reactivity of Extensively Used Pesticides and Their Metabolitesa assay A (S6-I/OVA-7) compound diazinon diazinon hydroxy metabolite pirimiphos-methyl pirimiphos-ethyl fenitrothion azinphos-methyl fenthion dichlorvos parathion-methyl tetrachlorvinphos chlorpyriphos gliphosate AMPA atrazine simazine

IC50 (ng/mL)

CR (%)

7.5 100 157 4.8 268 662 >10 000 >10 000 >10 000 >10 000 6 166 >10 000 >10 000 >10 000 >10 000 >10 000 >10 000

2.8 1.1 10 000 3 084

0.96 0.39