Determination of carbaryl, carbofuran and methiocarb in cucumbers and strawberries by monoclonal enzyme immunoassays and high-performance liquid chromatography with fluorescence detection: An analytical comparison

Laboratorio Integrado de Bioingenierı́a, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain
Journal of Chromatography A (Impact Factor: 4.17). 03/1999; 833(1):3-12. DOI: 10.1016/S0021-9673(98)00856-5


Carbaryl, carbofuran and methiocarb are three of the most important N-methylcarbamate pesticides. In the present work, the application of laboratory-developed monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) to the determination of these compounds in fruits and vegetables is described. Cucumbers and strawberries were spiked with the three carbamates at 10, 50 and 200 ppb. After extraction and clean-up, samples were analyzed by immunoassay and by HPLC with post-column derivatization and fluorescence detection (US Environmental Protection Agency Method 531.1). Results obtained by ELISA correlated well with those obtained by HPLC, both in terms of accuracy and precision. Recoveries were in the 60–90% range by ELISA and in the 50–90% range by HPLC, depending on the particular combination of commodity, pesticide, and fortification level under consideration. ELISAs were also applied to the analysis of non-purified sample extracts with excellent results: recoveries close to 100% were obtained, while maintaining similar precision values. This approach avoids the use of solid-phase extraction columns, saves time, and considerably increases the sample throughput. Results clearly indicate that the developed immunoassays may be suitable for the quantitative and reliable determination of carbaryl, carbofuran and methiocarb in fruits and vegetables even without including clean-up steps. These considerations make these ELISAs very useful analytical tools for monitoring and regulatory programs, without the need of complex and expensive instrumentation.

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    • "Many countries have developed programmes for pesticides monitoring, including the U.S. Environmental Protection Agency (EPA) that established the use of HPLC with a postcolumn derivatization and fluorescence for detection of carbaryl. Although this method is sensitive and well established, its regular use remains complex and expensive instrumentation is required [3] [4]. Electrochemical methods, including the use of amperometric biosensors, on the other hand, are simple, inexpensive and highly sensitive methods [5] [6]. "
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    ABSTRACT: We report a simple strategy to obtain an efficient enzymatic bioelectrochemical device, in which the enzyme acetylcholinesterase (AChE) was immobilized on gold electrodes functionalized with mixed self-assembled monolayer (SAMmix) of 11-mercaptoundecanoic acid (11-MUA) and 2-mercaptoethanol (C2OH). Development of the modified electrodes included the chemical adsorption of SAMmix on gold surface followed by immersion in AChE solution, resulting in the final Au/SAMmix/AChE configuration. For comparison, the electrochemical performance of Au/11-MUA/AChE and Au/C2OH/AChE electrodes were also investigated. The performance of the modified electrodes toward acetylthiocholine hydrolysis was investigated via cyclic voltammetry and chronoamperometric measurements, revealing a fast increase in anodic current with a well-defined peak upon addition of acetylthiocholine iodide to the electrolytic solution. The anodic currents for Au/SAMmix/AChE electrodes were significantly higher than those for Au/11-MUA/AChE and Au/C2OH/AChE electrodes. The Au/SAMmix/AChE architecture provided by the SAMmix surface promoted a high oxidation current of thiocholine at 0.31 V without the need of electron mediators. The chronoamperometric biosensor developed here provided a linear response to carbaryl in the concentration range of 0 to 1.75 μM. The detection limit and quantification values for carbaryl were found to be 3.45 × 10−10 M and 1.15 × 10−9 M, respectively. Michaelis–Menten kinetics, KMapp, of 0.46 mM was obtained, indicating that the electrode architecture employed is advantageous for fabrication of enzymatic devices via physical adsorption process with improvement of the biocatalytic properties.
    Electrochimica Acta 01/2013; 87:717–723. DOI:10.1016/j.electacta.2012.09.080 · 4.50 Impact Factor
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    • "Immunoassays, especially the enzyme-linked immunosorbent assay (ELISA), have been emerging as an attractive alternative to the traditional chromatographic methods. [1] [2] Due to their simplicity and cost-effectiveness, immunoassays can be used for the high sample throughput and on-site screening of pesticide residue. [3] [4] A successful immunoassay for detection of pesticide residue requires the production of antibodies against the targeted pesticide molecule. "
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    ABSTRACT: A new method for specific antibody production was developed using antibody (Ab)-pesticide complex as a unique immunogen. Parathion (PA) was the targeted pesticide, and rabbit polyclonal antibody (Pab) and mouse monoclonal antibody (Mab) were used as carrier proteins. The Ab-PA complexes were generated by conjugating the two antibodies with an excessive dosage of PA. It was shown that the sensitivity of homologous enzyme-linked immunosorbent assay (ELISA) using the new antibodies was similar to that using original antibodies. However, the new mouse Pab had not only similar positive recognizing spectrum as the original Mab, but also a significantly improved sensitivity in heterologous ELISA when some recognizable competitors were applied. IC(50) value of ELISA based on a combination of the new mouse Pab and hapten 9 was 0.24 ng/mL, which was 445.54 times as that of the homologous ELISA. An Ab-pesticide complex may be a suitable alternative immunogen for producing highly specific antibody to improve the immunoassay sensitivity.
    Journal of Environmental Science and Health Part B Pesticides Food Contaminants and Agricultural Wastes 08/2010; 45(6):540-6. DOI:10.1080/03601234.2010.493485 · 1.20 Impact Factor
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    ABSTRACT: There are many compounds that require analysis ranging from pesticide levels in corn to disease markers in human patients. There are copious challenges to be met when measuring analytes such as the matrix in which they are to be determined, the amounts present, the cost and the rapidity of the result required. Enzyme immunoassays, immunoaffinity chromatography, immunomagnetic polymerase chain reaction, flow cytometry and immunobiological biosensors have all characteristics that can enhance analytical techniques. Antibody-based methods have found applications in a large number of diverse areas such as food and water analysis, clinical diagnosis and therapeutics The structure and modes of production of antibodies and antibody-based derivatives is described and their applications in analysis critically examined.
    Analytical Letters 01/2000; 33(13):2563-2609. DOI:10.1080/00032710008543210 · 1.03 Impact Factor
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