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Mycotoxins represent a major concern for human and animal health because of their harmful effects and high occurrence in food and feed. Rapid immunoanalytical methods greatly contribute to strengthening the safety of our food supply by efficiently monitoring chemical contaminants, so high-affinity and specific antibodies have been generated for almost all internationally regulated mycotoxins. The only exception is patulin, a mycotoxin mainly produced by Penicillium expansum for which such a target has not yet been achieved. Accordingly, no point-of-need tests commonly used in food immunodiagnostics are commercially available for patulin. In the present study, three functionalized derivatives conforming to generally accepted rules in hapten design were firstly tested to generate suitable antibodies for the sensitive immunodetection of patulin. However, these conventional bioconjugates were unable to elicit the desired immune response, so an alternative strategy that takes advantage of the high electrophilic reactivity of patulin was explored. Patulin was reacted with 4-bromothiophenol, and the obtained adduct was used to produce antibodies with nanomolar affinity values. These results demonstrated for the first time that targeting the adduct resulting from the reaction of patulin with a thiol-containing compound is a promising approach for developing user-friendly immunoanalytical techniques for this elusive mycotoxin. [ABSTRACT FROM AUTHOR]

Spirotetramat is employed worldwide to fight insect pests due to its high efficiency. This chemical is quickly metabolized by plants into spirotetramat-enol, so current regulations establish that both compounds must be determined in foodstuffs for monitoring purposes. Nowadays, immunochemical methods constitute rapid and cost-effective strategies for chemical contaminant analysis at trace levels. However, high-affinity binders and suitable bioconjugates are required. In this study, haptens with opposite functionalisation sites were synthesized in order to generate high-affinity monoclonal antibodies. A direct competitive enzyme-linked immunosorbent assay with an IC50 value for the sum of spirotetramat and spirotetramat-enol of 0.1 μg/L was developed using selected antibodies and a novel heterologous bioconjugate carrying a rationally-designed hapten. Studies with fortified grape, grape juice, and wine samples showed good precision and accuracy values, with limits of quantification well below the maximum residue limits. Excellent correlation of results was observed with a standard reference chromatographic method. As a step forward, a lateral flow immunoassay was developed for onsite screening analysis of spirotetramat in wine. This assay was successfully validated according to Regulation 519/2014/EU for semi-quantitative methods at concentrations in line with the legal levels of spirotetramat and spirotetramat-enol in grapes, with a satisfactory false suspect rate below 2%. [ABSTRACT FROM AUTHOR]

Mepanipyrim is a new-generation anilinopyrimidine pesticide that is employed nowadays to fight fungal diseases in a variety of crops. Rapid and user-friendly analytical methods that can complement the instrumental techniques are demanded. To analyse residues of this toxic compound in foodstuffs, monoclonal antibodies were generated for the first time, and an enzyme-linked immunosorbent assay was developed. In the present study, a series of haptens mimicking the mepanipyrim molecule, and with a linker at alternative sites of the molecular framework, were designed and prepared by total synthesis in order to study the influence of the linker tethering site over the binding properties of the generated antibodies. This strategy afforded high-affinity and specific monoclonal antibodies to mepanipyrim. Interestingly, the highest sensitivity was reached with antibodies that had been raised using haptens with the spacer arm at the most characteristic moiety of the analyte, the 1-propynyl group. Moreover, the influence of the triple bond was investigated both in immunizing bioconjugates and in assay antigens. Finally, a competitive enzyme-linked immunosorbent assay was optimized and characterized, showing an IC50 value of 0.3 μg/L for mepanipyrim. Good accuracy and precision values were found when grape juice and wine samples were analysed. [ABSTRACT FROM AUTHOR]

New-generation succinate dehydrogenase inhibitors with increased antifungal performance have been developed during the last years. Particularly, penthiopyrad has been recently introduced in the agrochemical market as a complementary fungicide for combined treatments with other active principles displaying a different mode of action. In the present study, rapid and high-throughput analytical methods for penthiopyrad residue determination in foodstuffs were developed. A novel functionalized hapten that completely mimics the molecule of penthiopyrad was designed for high-affinity and specific antibody generation. Moreover, heterologous haptens were synthesized, and the influence of their respective molecular modifications over antibody binding was evaluated by competitive enzyme-linked immunosorbent assay. Two immunoassays were optimized using alternative assay formats, affording limits of detection for penthiopyrad in buffer around 0.07 ng/mL in both cases. Performance, in terms of accuracy and precision, of the developed immunochemical methods was studied using apple juice and red and white grape juices. The obtained results showed that penthiopyrad could be determined in those food samples in compliance with European maximum residue limits. [ABSTRACT FROM AUTHOR]

Different highly selective sorbents have been evaluated for the treatment of food and biological samples to determine chloramphenicol residues by ion mobility spectrometry (IMS). Combination of a selective solid-phase extraction (SPE) and dispersive liquid-liquid microextraction allowed a highly sensitive determination of chloramphenicol in water, milk, honey, and urine samples. The performance of selective SPE supports such as immunoaffinity chromatography (IAC) and molecular imprinted polymers (MIP) have been compared in terms of selectivity, sensitivity, trueness, precision, and reusability. Quantitative recoveries were obtained for chloramphenicol residues, ranging from 91 to 123 % for water, from 99 to 120 % for skimmed milk, and from 95 to 124 % for urine using IAC-IMS and MIP-IMS methods. Quantitative recoveries (from 88 to 104 %) were also achieved for honey samples using IAC-IMS, but low recoveries were obtained using MIP-IMS. The limit of quantification was set at 0.1 μg L which is lower than the minimum required performance limit established by the EU. The proposed methodology is a simple and cost affordable alternative to chromatography methods for the highly sensitive and selective analysis of chloramphenicol residues in food and urine. [ABSTRACT FROM AUTHOR]

In recent years, a second generation of succinate-dehydrogenase-inhibitor (SDHI) fungicides has been introduced into the market for effective treatment of fruit and vegetable crops, with fluxapyroxad, boscalid, fluopyram, penflufen, bixafen, penthiopyrad, and isopyrazam being some of the members of this new class of agrochemical. We herein report the development of an analytical procedure for the determination of residues of these SDHI fungicides in food samples, based on a modification of the QuEChERS extraction method followed by ultra-performance liquid chromatography coupled to tandem-mass-spectrometry determination. The proposed method reached limits of detection from 0.8 to 2.0 μg L−1. Apple, strawberry, tomato, and spinach samples were used as model samples. Spiked samples, from 10 to 1000 μg kg−1, were analysed by the proposed method and quantitative recoveries were obtained (from 81 to 115 % for apples, from 84 to 136 % for strawberries, from 84 to 135 % for tomatoes, and from 80 to 136 % for spinach), with precision better than 20 % in all cases. Thus, the proposed method can be used for the analysis of SDHI fungicide residues to efficiently ensure that marketed fruits and vegetables comply with the maximum residue levels established by competent authorities. [ABSTRACT FROM AUTHOR]

Forchlorfenuron is a synthetic phytohormone with cytokinin-like activity used worldwide as a plant growth regulator to increase fruit size in a number of crops, mostly in kiwifruit and grape vines. A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for the determination of forchlorfenuron has been characterized and optimized. The selected immunoreagents afforded a highly selective assay with a limit of detection of 10 ng L in buffer. This direct competitive ELISA was validated in terms of trueness, precision, and robustness using both commercial juice and whole fruit samples. Recoveries from fortified kiwifruit juices and white and red musts were between 97 % and 131 %, with relative standard deviations below 16 %. When homogenized whole fruits were analysed after acetonitrile extraction, recoveries between 96 % and 113 % were found, with a limit of quantification of 5 μg kg. The proposed immunoassay was validated by comparison with a reference chromatographic method using fruits from in-field treated grape and kiwifruit vines. Linear regression analysis of ELISA and HPLC-UV determinations showed an excellent correlation ( r = 0.998), whereas analysis of the slope (0.99 ± 0.01) and of the intercept (−1 ± 3) clearly proved that the developed competitive immunoassay provided results that were statistically comparable to those obtained by the instrumental method for the analysis of forchlorfenuron in fruits at trace levels. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]