Determination of Carbaryl in Vegetables Using an

Food and Agricultural Immunology ( 2000) 12, 101– 114

Determination of Carbaryl in Vegetables Using an Immunosensor Working in Organic Media J. PENALVA, J. A. GABALDON, A. MAQUIEIRA A N D R. PUCHADES Departamento de Qu´õ mica, Universidad Politécnica de Valencia, Camino de Vera s/n, 46071-Valencia, Spain (Original manuscript received 21 June 1999; revised manuscript accepted 25 October 1999)

Sensitive and rapid analysis of carbaryl using a flow immunosensor is described. An azlactone polymeric gel with covalently bound Protein A/G was used as immunofiltration support. The immunosensor was able to work at high concentrations of organic solvents ( e.g. up to 50% MeOH) using a competitive enzyme-immunoassay protocol, with an analysis rate of a complete assay cycle in 22 min. Good sensitivity ( I50 = 9.05 m g l– 1 ) was achieved in organic media and the reusability of the sensor was at least 300 cycles without loss of performance. The sensor was applied to the analysis of carbaryl extracted by both multiresidue method and methanol in fresh and processed vegetable samples. The analytical results were compared with those obtained by ELISA and high-performance liquid chromatography ( HPLC) methods. A good agreement was achieved by both immunological methods ( r = 0.98 ) and those obtained by HPLC technique. The results indicated the suitability of the immunosensor for carbaryl analysis in vegetable samples due to its reproducibility, rapidity and compatibility with organic solvents. Keywords: Immunosensor, ELISA, organic-media, carbaryl, vegetables

INTRODUCTION Carbaryl is an N-methylcarbamate pesticide extensively used as a broad-spectrum insecticide ( Worthing & Hence, 1991). This compound is an inhibitor of the acetylcholinesterase, but in spite of some adverse effects reported ( Farage-Elawar & Blaker, 1992; Casale et al., 1993 ), it is considered a safe insecticide due its low toxicity in mammals. Carbaryl shows high susceptibility to chemical hydrolysis and biodegradation, 1-naphthol being the main metabolite. Among the different methods used for carbaryl determination, high-performance liquid chromatography ( HPLC) with postcolumn fluorescence derivatization is the most Correspondence to: R. Puchades. [email protected] v.es

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accurate technique ( Barceló, 1993). However, laborious extraction, cleanup, and concentration steps are often necessary to obtain the desired sensitivity ( De Kock & Hiemstra, 1992). These procedures must be carried out in well-equipped laboratories with appropriate instrumentation and trained personnel. In order to solve the limitations showed by chromatographic methods, the development of new methodologies is demanded. In this sense, immunochemical methods are gaining relevance due to their proved rapidity, specificity, sensitivity and cost-effectiveness (Skerritt, 1995; Nunes et al., 1999). Nowadays, due to legal regulations and in order to guarantee the safety of fruits and vegetables (Fan & Bushway, 1997), the determination of pesticide residues in these types of samples should be carried out. ELISA is the most usual immunological technique for pesticide analysis and its application in monitoring of pesticide residues is attaining an enormous potential ( Van Emon, 1990). Currently, aqueous samples are analysed without any pre-treatment. However, the use of ELISA for analysis of pesticides in vegetable samples makes necessary the employment of extraction procedures and an effective sample preparation. Therefore, the immunological method should provide reliable data with minimum sample processing, or otherwise many of the potential advantages of immunoassay would be lost (Skerritt & Amita-Rani, 1996). Normally, multiresidue methodology ( MRMs) is applied for the extraction of pesticides from solid foods using a wide variety of organic solvents such as acetone, ether, petroleum ether, methanol, acetonitrile or hexane (Dankwart & Hock, 1997). These procedures are timeconsuming and labour-intensive, and they do not allow the generation of relevant data in time to prevent contaminated foods from entering the market place. Also, the amount of chemicals and toxic solvents used is usually a factor of 108 –1010 higher than that of the pesticide residues to be determined ( Torres et al., 1996). To solve these drawbacks a sample preparation scheme using a small volume of solvents should be optimised, not only for financial savings, safety, rapidity and environmental reasons, but also because high concentrations of organic solvents do affect negatively the performance of an immunoassay ( Meulenberg, 1997). For this reason, flow-through immunosensors are used since the contact time between the immunoreagents and organic media is sharply reduced. Generally, flow immunosensors are based on the heterogeneous immunological principles that combine good sensitivity and selectivity of immunological reactions with the speed, versatility, accuracy and applicability in control process as showed by flow-injection analysis methodology ( Puchades & Maquieira, 1996). However, due to the scarce literature available about the effects of the organic media on the Ab–Ag interactions, a more detailed study should be accomplished. In this sense, a study of the effects of this media and their mixtures on the Ab–Ag interactions for immunosensors development was previously done ( Penalva et al., 1999 ). The developed sensors were applied to the analysis of carbaryl residues in fresh and processed vegetables. Finally, the results were compared with those obtained by ELISA, using the same immunoreagents, and by HPLC as reference method. The viability for carbaryl analysis purposes in this type of matrices in a fast and simple way is discussed. MATERIALS AND METHODS Chemicals and Biochemicals Analytical standards of carbaryl, 1-naphthol, carbofuran, methiocarb and propoxur were purchased from Dr Ehrenstorfer ( Augsburg, Germany). Stock solutions of pesticides were prepared in N-N9 dimethylformamide ( DMF), except for carbaryl, which was dissolved in methanol (MeOH). From stock solutions kept at – 20°C, working standard solutions were prepared daily. 3-( p-Hydroxyphenyl )-propanoic acid ( HPPA), o-phenylenediamine ( OPD), Tween 20, and bovine serum albumin, fraction V ( BSA) were purchased from Sigma Chemical Co, ( St Louis, MO, USA). Horseradish peroxidase (HRP) was from Boehringer

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( Mannheim, Germany). Ultralink Immobilised Protein A/G support (Protein A/G) was purchased from Pierce Chemical Company (Rockford, IL, USA). C1 8 cartridges ( Sep-Pak from Waters-Millipore, Milford, MA, USA) containing 360 mg of 40 m m C1 8 -bonded silica were used. All other reagents used were analytical or biochemical grade.

Immunoreagents Mouse monoclonal anti-carbaryl antibodies CNA 36, CNH 32, CNH 36, CNH 37, CNH 45, CNH 89, CNH 101 and CNH 103 and the haptens 6-[(( 1-naphthyloxy ) carbonil)-amino] hexanoic acid ( CNH), 6-[(( 1-naphthyloxy ) carbonyl )-amino] propanoic acid ( CNA), and 1-(5-carboxypentyl )-3-( 1-naphthyl ) urea ( CPNU) were a gift from Laboratorio Integrado de Bioingenier´õ a ( Abad et al., 1997). Rabbit anti-carbaryl polyclonal serum R2114 and the hapten N-( 1-naphthoyl )-6-aminohexanoic acid (H4), were kindly provided and characterised by Marco et al. ( 1993 ). The enzymatic tracers CNH-HRP, CNA-HRP, CPNU-HRP and H4-HRP were prepared using a modification of the anhydride mixed method ( Rajskowsky et al., 1997 )

Immunosorbent to Sensor Development The UltraLink Immobilised Protein A/G support ( Protein A/G) is able to bind specifically and reversibly all IgG subclasses of mouse and rabbit immunoglobulins through their Fc domains. To ensure high binding capacities, this sorbent has to work in binding buffer ( 20 mM -sodium phosphate, pH 8) ( PB), as suggested by the manufacturer ( Pierce Chemical Company, 1993). The support was placed into a small polymethylmethacrylate tube ( 7 mm length, 4 mm inner diameter, approximate volume 80 m l) and all the available volume of the reactor was filled with the immunosorbent. The Protein A/G binds the immunocomplexes formed in solution and after the regeneration step, the sorbent is able to run a new assay. The regeneration is easily achieved using flow techniques by passing a 0.1 M -glycine/HCl solution, pH 2, according to manufacturer’s suggestions.

System Design The system consists of a syringe pump connected to two eight-way distribution valves ( Kloehn Ltd; Las Vegas, NV, USA). The reactor is connected between the first valve port and the fluorimeter ( Turner Designs, model 450, CA, USA) equipped with a Hellma flow cell (type No. 176.052-QS). Liquid handling and fluorimetric signals (l E x = 320 nm, l E m = 404 nm) were managed by means of Winpump (Kloehn Ltd) and Chrom-Card Manager software packages (Fisons Instrument, Rodano, Italy), respectively. The scheme of the used manifold is shown in Figure 1. Sigmaplot software package ( Jandel Scientific, Erkrath, Germany) was used for data treatment.

Assay Protocols Immunosensor. Briefly, a mixture of 300 m l of Ab solution, 300 m l of diluted enzymatic tracer and 500 m l of organic media with or without analyte was injected through the reactor. Therefore, the competition between the immunoreagents occurred in solution and the immunocomplexes were trapped by the support. After this, a washing step was carried out in order to remove the unbound immunoreagents. The scheme of the assay protocol is shown in Table 1. The total assay time was approximately 22 min.

FIG. 1. Scheme of the manifold used.

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TABLE 1. Scheme of immunosensor assay protocola

Step

Event

1 2 3 4 5 6 7 8

Mixing c sample ( 0.5 ml) with tracer solution ( 0.3 ml) and antibody solution ( 0.3 ml) Injection of 1 ml of mixture at 0.25 ml min– 1 Reactor conditioning with 0.02 M -PBT Mixing HPPA ( 0.3 ml, 0.8 g l– 1 ) with H2 O2 (0.2 ml, 0.012%). Injection of 0.2 ml Incubation for 3 min Eluting reaction product with 1.5 ml of PBT at 2 ml min– 1 . Peak registration Desorption with 2.5 ml 0.1 M -glycine/HCl pH 2.0 solution at 0.5 ml min– 1 Washing of the reactor with PBT. End of cycle Total assay time

Time ( min)b 2 4 3 1 3 1 5 3 22

a

Previous to each step, the manifold ran a washing cycle with the next solution to be used. Values included all the washing cycles performed in each step. c Syringe dead volume ( 0.1 ml) filled with sample. PBT: 20 mM -phosphate buffer, pH 8, and 0.05% Tween 20. b

ELISA procedure. The protocol was based on a competitive direct ELISA format. First, microtitre plates ( Costar Corporation, Cambridge, MA, USA) were coated with 1 mg 1– 1 ( 100 m l/well) CNH 45 antibody in coating buffer (50 mM -carbonate buffer, pH 9.6). The following day, the plates were washed ( 12-channel microplate washer, Nunc-Immuno, Denmark) four times with 10 mM -phosphate buffer saline, pH 7.4, containing 0.05% ( v/v) of Tween 20 ( PBS-T). Carbaryl standards ( 0.02 to 2000 m g l– 1 ) or samples were added to the coated plates (50 m l/well) by quadruplicate, followed by 50 m l/well of a 5 m g ml– 1 solution of the tracer CPNU-HRP, and the mixture was incubated for 1 h. Then, the plates were washed as described before and 100 m l of the substrate solution ( 2 mg ml– 1 OPD substrate solution in 25 mM -sodium citrate, 62 mM -sodium phosphate buffer, pH 5.4, containing 0.012% H2 O2 ) was added. After 5 min, the enzymatic reaction was stopped by adding 100 m l of 2.5 M -sulphuric acid and the absorbance at 490 nm was read in a dual-wavelength mode ( 490– 650 ) using a Victor Multilabel Counter 1420 microplate reader from Wallac ( Turku, Finland). HPLC/Fluorescence detection analysis. The HPLC analyses were performed using a Hewlett-Packard Model 1100, quaternary pump system. The HPLC was equipped with a 4.0 ´ 250 mm reverse phase column, packed with 5 m m C8 Silica (Pickering 0840250). The flow rate of the mobile phase was 0.8 ml min– 1 with a sample injection volume of 10 m l. Prior the analysis of carbaryl, a post-column derivatization device was coupled to the system. Briefly, the separated carbaryl was first hydrolysed by NaOH at 100°C releasing an alcohol, carbonate, and methylamine. In the second post-column reaction, methylamine reacted with o-phthalaldehyde (OPA) and the nucleophilic Thiofluor or 2-mercaptoethanol (Du Pont, France ), formed a highly fluorescent 1-methyl-2-alkylthioisoindole derivative. The derivatisation post column system ( Pickering, Model PCX 5100) was equipped with a programmable fluorescence detector from Hewlett-Packard Model 1046 A ( l E x = 330 nm, l E m = 465 nm). The temperature of the reactor and column was 100 and 42°C, respectively. Organic Solvents The organic solvents were selected after a review of the most usual solvents utilized in pesticide extraction ( Moye, 1990; Sawyer et al., 1990 ). These organic solvents are: ( a) water-

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immiscible: hexane, ethyl acetate and dichloromethane; ( b) water-miscible: methanol, acetonitrile and acetone. Due to its general miscibility properties, other water-miscible solvents such as tetrahydrofurane, 2-propanol and N-N9 dimethylformamide were also used as transference solvents to aqueous media ( 20 mM -sodium phosphate buffer, pH 8, containing 0.05% ( v/v) of Tween 20, PBT).

Study of the Organic Solvents Generally, the use of pure organic solvents produced the precipitation of immunoreagents. Moreover, incompatibility effects with the working material were observed. Therefore, in basic previous work ( Penalva et al., 1999 ), an evaluation of miscibility and compatibility of the organic solvents with the working conditions was carried out. This study was performed adding to different solvent– PBT mixtures, small amounts of enzyme-tracer, and immobilisation support, as well as plastic and poly ( tetrafluoroethylene ) bits, and checking visually whether protein precipitation or material degradation occurs. Thus, the selected binary mixtures were: 50% MeOH–50% PBT and 25% acetonitrile–75% PBT. The best ternary mixtures assayed were: 20% MeOH–20% isopropanol –60% PBT; 10% ethyl acetate–25% isopropanol –65% PBT; 10% ethyl acetate–25% MeOH– 65% PBT and 5% ethyl acetate– 25% acetonitrile– 70% PBT.

Sample Treatment Fresh ( green peppers) and processed ( tomatoes and red peppers) vegetables were collected from a local market in Valencia ( Spain). Previously to the extraction procedure, samples were chopped and homogenised in a blender Osterizer ( Milwaukee, WI, USA). After this, samples were fortified at different levels with carbaryl, mixed, homogenised during 1 h and extracted according to the following procedures: ( a) Multiresidue extraction. This procedure is a modification of the multiresidue method for the extraction of N-metilcarbamate pesticides in fruit and vegetables ( Ting et al., 1984 ). Basically, 20 ml of acetonitrile and 5 ml of distilled water were added to vegetable samples ( 10 g) and the mixture blended for 10 min. The supernatant was filtered by suction through a porous plate filter ( G-3 ). Then, 30 ml of distilled water, 10 ml of petroleum ether and 1 g of NaCl were added to the filtrate and shaken during 1 min in a decantation funnel. The organic phase was separated from the aqueous one, which was extracted again with 5 ml of fresh petroleum ether. This procedure was repeated until no coloration was observed in the organic phase ( five extractions were necessary). The gathered ether phases were washed with 10 ml of distilled water carrying out a last decantation, and anhydrous Na2 SO4 was added in order to remove aqueous traces. The organic phase was taken to dryness in a water bath at 40°C using an N2 stream and the residue was redissolved with 5 ml of MeOH. A clean-up with C18 cartridges was then performed. Finally, 2 ml of the cleaned extract was conditioned with 2 ml of PBT for immunosensor analysis. ( b) Methanolic extraction. Vegetable samples ( 5 g) and 10 ml of MeOH were blended for 15 min and the supernatant vacuum filtered using a porous plate filter ( G-3 ). The extract was then cleaned and conditioned for immunosensor analysis as mentioned above. For ELISA determinations no clean up was necessary and only a dilution of the extract 1/10 ( v/v) with PBS-T was required. For HPLC analysis, the extracts were taken to dryness, redissolved on 2 ml of MeOH and cleaned using C1 8 cartridges. Finally, carbaryl was eluted with 2 ml of acetonitrile/water ( v/v).

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Recovery Studies The samples were fortified with carbaryl at 50, 100 and 150 m g kg– 1 , homogenised (for 1 h) and extracted in triplicate using both multiresidue and methanolic procedures. Finally, the sample extracts were analysed by organic-sensor, ELISA and HPLC techniques. RESULTS AND DISCUSSION Performance of the Immunosensor As mentioned above, the injection of 2.5 ml 0.1 M -Gly/HCl pH 2 solution completed the regeneration of the sensor. This process assured the breakthrough of all Ab– protein A/G complexes allowing the immunosorbent to run new assays. The selection of the most suitable antiserum-enzyme tracer combination was based on the sensitivity criteria in flow immunosensor experiences. From a set of one polyclonal ( pAb), eight monoclonal (mAb) antibodies and four haptens conjugated to HRP, the best results concerning to sensitivity and reusability were achieved with the mAb CNA 36, CNH 36 and CNH 45 in combination with CNH-HRP and CPNU-HRP tracers. These combinations were also selected as optimal in other studies ( Gonz a´ lez-Mart´õ nez et al., 1997 ). The conjugate CNA–HRP had not enough activity in any case. The optimisation of the immunoreagent concentrations ( combination tracer/Ab) for reaching an acceptable signal (200–300 fluorescence arbitrary units) was carried out for each mixture selected. The antibody concentrations studied ranged from 1 to 3 mg 1– 1 , CNA 36 being the antibody that needed the lowest concentration. Regarding concentration of the tracers, the range varied from 0.5 to 3 mg 1– 1 , CPNU– HRP being that which showed highest activity. Immunorreagent concentrations were lower when methanol mixtures were used. The optimal immunosensor assay protocol ( e.g. solutions, volumes and flow rates) is shown in Table 1. Table 2 shows the sensitivity ( I50 values) obtained with the selected immunoreagents and organic media. As can be observed, 50% MeOH mixture was the most sensitive with I5 0 values of 9.05 m g l– 1 for [CNH 45/CPNU– HRP], 11.86 m g 1– 1 for [CNA 36/CPNU–HRP] and 43.44 m g l– 1 for [CNH 36/CNH–HRP]. On the other hand, only up 25% of acetonitrile could be used since in 50% acetonitrile mixture not enough signal was presented to make competitive assays. When mixtures of non-miscible water solvents were used, only the antibody CNA 36 showed good competition. In these media, higher tracer concentrations were needed to obtain an appreciable signal, which indicates that the diffusion of the antigen towards the recognition sites of the antibody was hindered and a major antibody denaturation

TABLE 2. I50 ( m g l– 1 ) values obtained with the immunosensor using the best immunoreagents and organic mixtures CNA 36

CNH 36

CNH 45

Organic Media

CNH– HRP

CPNU– HRP

CNH– HRP

CPNU– HRP

CNH– HRP

CPNU– HRP

50%M–50%PBT 20%M–20%I–60%PBT 25%A– 75%PBT 10%E–25%I– 65%PBT 10%E–25%M– 65%PBT 5%E–25%A–70%PBT

17.51 14.48 78.43 49.87 59.73 96.33

11.86 12.27 35.39 83.46 58.72 110.61

35.58 57.31 47.29 n·a· n·a· n·a·

43.44 100.95 71.59 n·a· n·a· n·a·

11.06 21.32 16.69 n·a· n·a· n·a·

9.05 24.74 34.18 n·a· n·a· n·a·

M: methanol, I: isopropanol, A: acetonitrile, E: ethyl acetate, PBT: 20 mM -phosphate buffer, pH 8, and 0.05% Tween 20. n.a: no activity.

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FIG. 2. Normalised competition curves obtained by immunological methods: ( m ) sensor using 50% MeOH50% PBT as organic media; ( d ) ELISA using PBS-T containing 5% MeOH ( v/v).

occurred. In this case, the best results were obtained when CNH–HRP tracer was used, being up to 1.67 times more sensitive than CPNU–HRP tracer when worked on 10% ethyl acetate– 25% isopropanol –65% PBT as organic media. The normalised competition curve obtained with the immunosensor under the most sensitive conditions (mAb CNH 45 = 1 mg l– 1 , tracer CPNU–HRP = 0.5 mg l– 1 and 50% MeOH–50% PBT as organic media) is shown in Figure 2. Under these conditions, the working range (inhibition of signal between 20 and 80%) was from 2.11 to 44.80 m g l– 1 with an I50 value ( concentration value at the inflexion point) of 9.05 m g l– 1 . The reproducibility of the results along several days using a 10 m g l– 1 carbaryl standard dilution was 11.20% ( CV, n = 8). The Protein A/G support was kept at 4°C in PB containing 0.02% sodium azide as preservative when not in use. This reactor could be used for more than 300 assay cycles with no detectable loss of activity. Selection of Extraction Procedure To evaluate a rapid and reliable extraction method for vegetable samples, direct extraction with methanol was performed using multiresidue method as confirmatory. For this study, four portions of each sample fortified at 0 ( control), 10, 20 and 30 m g kg– 1 were used. Each portion was extracted using methanol and multiresidue procedures, and the extracts were analysed by means of the immunosensor. Carbaryl concentrations obtained in fortified tomato and pepper ( red and green) samples are shown in Table 3. Control samples ( after the elimination of the colour by the cleaning treatment) did not present matrix interferences. The best carbaryl recoveries were obtained from red pepper with mean values of 99.1 and 93.0% for multiresidue and methanol extracts, respectively. For tomato samples, mean recoveries of 119.0% for multiresidue extracts and 113.7% for methanol extracts indicated an overestimation of carbaryl level. Anyway, good

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TABLE 3. Immunosensor determination of carbaryl concentrations in vegetable samples using two extraction procedure s Found level ( m g kg– 1 ) Fortified Level ( m g kg– 1 )

Tomato

Green pepper

Multiresidu e

Methanolic

Multiresidu e Methanolic

Multiresidu e

Methanolic

10 20 30

13.0 ± 2.0 25.0 ± 1.4 30.6 ± 3.4

10.8 ± 0.6 23.6 ± 2.6 34.5 ± 1.7

9.8 ± 0.4 21.9 ± 3.6 34.1 ± 6.6

10.1 ± 0.9 19.6 ± 2.3 29.5 ± 3.5

8.2 ± 1.0 21.7 ± 0.9 26.5 ± 2.9

7.8 ± 2.6 14.8 ± 2.7 26.2 ± 3.1

Red pepper

Values expressed as mean ± SD (three determination s). In all cases, 50% MeOH-50% PBT ( 20 mM phosphate buffer, pH 8, 0.05% Tween 20) was used as organic media.

recoveries were found in all cases ( around 100%) with RSD values £ 6.6. Therefore, the methanol extraction is very suitable for these matrices and has the advantage of its speed and low organic solvent consumption. Cross-reactivity To determine the selectivity of the immunosensor a study of cross-reactivity was carried out under the optimized conditions ( see above). For this study, 1-naphthol and other N-methylcarbamates such as carbofuran, propoxur, aldicarb, methomyl and methiocarb were analysed. As can be seen in Table 4, the percent CR values, estimated as the ratio between I50 values of carbaryl and related compounds, were very low and only 1-naphtol and methiocarb showed a very little interference ( 0.7 and 0.1%, respectively). To assess these results, tomato samples were fortified with a pesticide mixture ( 20 m g kg– 1 of carbaryl and 200 m g kg– 1 of 1-naphthol), extracted using methanol and analysed by the immunosensor. Mean carbaryl recovery ( 104% ) demonstrated that 1-naphthol at 10-fold concentrated did not interfere in the analysis of carbaryl using this methodology. ELISA Procedure First, tolerance level of the ELISA to MeOH solvent was studied. The optimal percentage of MeOH was estimated taking into account two approaches: no practical effects on the ELISA performances and minimal dilution of the extracts, in order to provide accurate and sensitive TABLE 4. Cross-reactivity values obtained for N-methilcarbamates using the organic-senso ra Compound

I50 ( m g l– 1 )

Carbaryl 1-Naphthol Methiocarb Methomyl Aldicarb Carbofuran Propoxur

9.05 1.3 103 6.5 103 2.6 104 9.0 104 9.3 104 1.9 105

CR ( %) 100 0.7 0.1 < 3.4 < 1.0 < 0.9 < 4.7

10– 2 10– 2 10– 2 10– 3

a mAb CNH 45: 1 mg l– 1 ; CPNU-HRP tracer: 0.5 mg l– 1 ; organic madia: 50% MeOH—50% PBT

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FIG. 3. Influence of MeOH concentration on the sensitivity of the ELISA method for carbaryl determination.

determinations. As shown in Figure 3, the sensitivity ( I5 0 = 4.14 m g l– 1 ) remained constant practically in proportion as MeOH increased to 5% whereas the signal in absence of analyte ( B0 ) decreased up to 85% ( 1.06– 0.90 ). MeOH percentages higher than 5% caused a rapid decrease of the sensitivity ( I5 0 = 11.21 m g l– 1 for 20% MeOH). Therefore, MeOH extracts were diluted 1/10 ( v/v) in PBS-T before the analysis ( 5% MeOH into the well). In these conditions, the mean dose–reponse curve for carbaryl (n = 10 runs ) was obtained ( see Figure 2) and the error bars illustrate the good reproducibility of the standard curve ( RSD < 0.934 ). The different parameters of this curve are gathered in Table 5. The ELISA sensitivity was 4.35 m g l– 1 with a dynamic range from 0.61 to 22.75 m g l– 1 . TABLE 5. Variability of the standard curve parameters for carbaryl determination analysis using ELISA method Parameter a

Mean value SDb Minimum Maximum

A

B

C(I50 )

D

0.933 0.079 0.829 1.054

1.961 0.351 1.497 2.404

4.352 0.934 3.443 6.074

0.014 0.006 0.007 0.021

Asymptotic maximum absorbance ( A ), slope at the inflexion point (B ), concentration value in m g l– 1 at the inflexion point ( C ), and asymptotic minimum absorbance (D ) are the parameters which fit the sigmoidal equation y = (A – D )/ [1 + (C/x )B/ln 10] + D b Standard deviation, n = 10 a

Tomato

b

a

150

100

50

150

100

50

150

46.3 31.9 40.4 84.4 63.4 73.3 148.4 120.8 128.2

55.4 44.2 49.1 128.3 101.7 115.1 180.9 163.5 172.1

36.0 45.6 41.3 112.9 104.3 107.1 150.5 121.7 138.3

Recovered ( m g kg– 1 )

132.4 ± 14.3

73.7 ± 10.5

39.5 ± 7.2

172.1 ± 8.7

115.0 ± 13.3

49.5 ± 5.6

136.8 ± 14.4

108.1 ± 4.3

40.9 ± 4.8

Mean ± SD ( m g kg– 1 )

Organic-sensor

88.3

73.7

79.0

114.7

115.0

99.1

91.2

108.1

81.9

Recovery ( %)

44.0 54.9 51.7 70.0 80.0 98.5 146.6 157.7 131.0

42.0 45.0 46.0 96.0 94.0 77.5 140.2 118.0 112.0

58.5 46.7 58.8 105.0 78.0 98.0 143.0 129.0 133.0


145 ± 13.4

82.8 ± 14.4

50.2 ± 5.6

123.4 ± 14.8

89.0 ± 10.0

44.3 ± 2.0

135 ± 7.2

93.6 ± 14.0

54.6 ± 6.9

Mean ± SD ( m g kg– 1 )

ELISAa

To ensure accurate results, ELISA plates included a calibration curve (PBS-T with a 5% of MeOH) in each assay. Due to cost and time reasons only a sample of each fortified level was analysed using HPLC.

Red pepper

Green pepper

50

Sample

100

Fortification level ( m g kg– 1 )

96.7

82.8

100.4

82.3

89.0

88.6

90.0

93.6

109.2

Recovery ( %)

140.1

90.3

44.8

132.9

94.7

42.7

140.2

87.4

41.3


93.4

90.3

89.6

88.6

94.7

85.4

93.5

87.4

82.6

Recovery ( %)

HPLCb

TABLE 6. Determination of carbaryl in fortified vegetable samples using the immunosensor and organic media. Comparison to ELISA and HPLC techniques


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FIG. 4. Correlation between the results for carbaryl obtained using immunosensor, ELISA (a) and HPLC ( b) techniques in vegetable samples: ( m ) red pepper; ( d ) tomato; ( j ) green pepper.

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Application to Real Samples To assess the analytical performance of the developed organic-sensor, ELISA as a reference method and a HPLC as confirmatory technique were performed. For this purpose, a set of fresh and processed vegetable samples spiked with carbaryl at 50, 100 and 150 m g kg– 1 ( three fortifications of each level) were extracted with methanol and analyzed using the three procedures (immunosensor, ELISA and HPLC). The effectiveness of the extraction procedure was evidenced by the good recoveries obtained in all cases. Using organic-sensor and ELISA methods recoveries of 94.5 and 92.5%, respectively, were achieved with RSD values £ 14.8% in both cases. For HPLC technique, the recovery was about 89.5%. The results obtained by both immunochemical protocols and by the chromatographic method, for all samples analysed, are showed in Table 6. As it can be seen, the best results were obtained from tomato samples with mean recoveries of 93.7%, 97.6% and 87.8% for the sensor, ELISA and HPLC techniques, respectively. For pepper samples, mean recoveries obtained with the sensor ( 109.6% for green and 80.3% for red) although good, were worse than those obtained by ELISA ( 86.6% for green and 93.3% for red) and HPLC ( 89.5% for green and 91.1% for red) that were the same in practice. Figure 4 shows the comparison of the results obtained for carbaryl using both immunological methods. As it can be seen, a good agreement was found with correlation coefficient values ( r ) of 0.956 for red pepper, 0.988 for tomato and 0.996 for green pepper. Also, slopes lower than 1.02 indicated that no bias between both techniques was found in any case. Finally, carbaryl concentrations measured by organic-sensor in tomato, red and green pepper. ( see Figure 4) were highly correlated with those obtained by HPLC ( r = 0.992, r = 0.998 and r = 0.964, respectively). CONCLUSIONS A selective and sensitive flow immunosensor able to work at high concentration of organic solvents has been developed for the determination of carbaryl in vegetable samples. This system allows the analysis of non-polar analytes by immunological techniques in a very sensitive and rapid way. Even reusability, the most troublesome aspect of the development of a reversible immunoanalysis system especially when working with non-aqueous solutions, has been successfully solved by means of the use of Protein A/G support. The fact of carrying out immunological reactions in high concentrations of organic solvents will ease the analysis of carbaryl residues recovered from extraction procedures. The methanolic extraction, here proposed, has shown to be very appropriate for the analysis of carbaryl in food samples such as fruits and vegetables and it can be considered as an attractive approach versus the multiresidue extraction. The rapidity and the moderate use of organic solvent are the main advantages of the methanolic extraction. The good correlation obtained between organic-sensor, ELISA and HPLC techniques for all samples have demonstrated the suitability of the developed immunoassay method. Furthermore, the proposed extraction procedure allows the analysis of a great number of samples. In addition, the sensor shows a high throughput and autonomy, and its applicability for on-line analysis is demonstrated. This system can also be used as screening method when the analysis of a great number of samples is attempted. ACKNOWLEDGEMENTS This work was supported by the projects CICYT ( AMB96–1079 ) and DGES ( PB95– 0740 ). J. Penalva acknowledges a grant from Ministerio de Educación y Cultura, Spain, to carry out Ph.D. studies. J. A. Gabaldo´ n also acknowledges a grant from Instituto de Fomento de la Región de Murcia, Spain, by means of the Seneca Program. Also, we thank M. D. Ibañez Talavera from CTC, Murcia, Spain, for valuable HPLC determinations. The authors wish to

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thank LIB from UPV and Dr B. D. Hammock’s laboratory for providing and supplying the immunoreagents. REFERENCES AB A D , A., PRIM O , J. & MO N T O YA , A. ( 1997 ) Development of an enzyme-linked immunosorbent assay to carbaryl. 1. Antibody production from several haptens and characterization in different immunoassay formats, Journal of Agricultural and Food Chemistry, 45, 1486–1494. BA RC E L O´ , D. ( 1993) Environmental-protection-agency and other methods for the determination of priority pesticides and their transformation products in water, Journal of Chromatography, 643, 117–143. CA SA L E , G. P., VE N N E R S T RO M , J. L., BAVA RI , S. & WA N G , T. L. ( 1993) Inhibition of interleukin-2 driven proliferation of mouse ctll2 cells by selected carbamate and organophosphate insecticides and congener s of carbaryl, Immunopharmacology and Immunotoxicolog y, 15, 199– 215. DA N K WA RT, A. & HO C K , B. ( 1997) Enzyme immunoassays for analysis of pesticides in water and food, Food Technology and Biotechnolog y, 35, 165–174. DE KO C K , A. & HIE M S T RA , M. ( 1992) Optimization, automation, and validation of the solid-phase extraction cleanup and online liquid chromatographic determination of N-methylcarbamate pesticides in fruits and vegetables, Journal of the Association of Official Analytical Chemists Internationa l, 75, 1063–1072. FA N , Z. & BU S H WAY, R. J. (1997 ) Determination of diazinon in fruits and vegetables by ELISA, Food Technology and Biotechnolog y, 35, 205– 208. FA RA G E -EL AWA R , M. & BL A K E R , W. D. ( 1992) Chick embryo exposure to carbamates alters neurochemica l parameters and behaviour, Journal of Applications and Toxicology, 12, 421– 426. GO N Z A L E Z -MA RT IN E Z , M. A., MO R A IS , S., PU C H A D E S , R., MA Q U IE IR A , A., AB A D , A. & MO N T O YA , A. ( 1997) Monoclonal antibody-based flow-through immunosensor for analysis of carbaryl, Analytical Chemistry, 69, 2812–2818. MA R C O , M. P., GE E , S. J., CH E N G , H. M., LIA N G , Z. Y. & HA M M O CK , B. D. (1993 ) Development of an enzymelinked-immunosorbent assay for carbaryl, Journal of Agricultural and Food Chemistry, 41, 423– 430. ME U L E N B E RG , E. P. ( 1997) Immunochemical detection of environmental and food contaminants: development, validation and application, Food Technology and Biotechnology, 35, 153–163. MO Y E , H. ( 1990) in Analysis of Pesticide Residues ( MO Y E , H., Ed.), Malabar, Florida, USA, 247 pp. NU N E S , G. S., MA R CO , M. P., FA R RE , M. & BA R CE L O , D. ( 1999 ) Direct application of an enzyme-linke d immunosorbent assay method for carbaryl determination in fruits and vegetables. Comparison with a liquid chromatography-postcolumn reaction fluorescence detection method, Analytica Chimica Acta, 387, 245– 253. PE N A LVA , J., PU C H A D E S , R. & MA Q U IE IR A , A. ( 1999 ) Study of the analytical properties of immunosensor s working in organic media, Analytical Chemistry, 71, 3862–3872. PIE RC E CH E M ICA L CO M PA N Y (1993 ) Instructions 0497, Rockford, IL, USA. PU CH A D E S , R. & MA Q U IE IR A , A. (1996 ) Recent developments in flow injection immunoanalysis, Critical Reviews in Analytical Chemistry, 26, 195– 218. RA JS K O W SK Y, K. M., CIT TA N O VA , N., DE S FO S S E S , B. & JAY L E , M. F. ( 1977 ) The conjugation of testosteron e with horseradish peroxidase and a sensitive enzyme assay for the conjugate, Steroids, 29, 701–713. SAW Y E R , L. D., MC MA H O N , B. M., NE W SO M E , W. H. & PA R K E R , G. A. ( 1990) Pesticide and industrial chemical residues, Journal of the Association of Official Analytical Chemists, 73, 127– 132. SK E RR IT T, J. H. (1995 ) Analytical Aspects of Immunoassays of Agrochemicals, in New Frontiers in Agrochemical Immunoassay ( KU RT Z , D. A., SK E RR IT, J. H. & STA N K E R , L., Eds), Journal of the Association of Official Analytical Chemists Internationa l, Arlington, VA, USA, pp. 1–16. SK E RR IT T, J. H. & AM ITA -RA N I , B. E. ( 1996) Detection and removal of sample matrix effects in agrochemical immunoassays in Immunoassay of Insecticides, Fungicides and Pesticides ( BE IE V, R. C. & STA N K E R , L. H., Eds), ACS Symposium Series 621, American Chemical Society, Washington, DC, USA, pp. 29– 43. TIN G , K. C., KH O , P. K., MU S SE L M A N , A. S., RO O T, G. A. & TIC H E L A A R , G. A. ( 1984) High performance liquid chromatographic method for determination of six N-methylcarbamates in vegetables and fruits, Bulletin of Environmental Contamination and Toxicology, 33, 538– 547. TO R RE S , C. M., PIC O´ , Y. & MA N˜ E S , J. ( 1996) Determination of pesticide residues in fruit and vegetables , Journal of Chromatography A, 754, 301– 331. VA N EM O N , J. M. (1990 ) Immunoassay Methods: EPA Evaluations, in Immunochemical Methods for Environmental Analysis ( VA N EM O N , J. M. & MU M M A , R., Eds), ACS Symposium Series 442, American Chemical Society, Washington, DC, USA, pp. 58– 64. WO RT H IN G , C. R. & HE N CE , R. J. ( 1991) in The Pesticide Manual: A World Compendium, 9th edn, BCPC Publications, Berks, UK.

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