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Abstract: Boscalid is a modern, broad-spectrum carboxamide pesticide highly efficient against most fungal diseases affecting valuable crops. In this study, a boscalid-mimicking derivative with a six-carbon spacer arm replacing the chlorine atom at the pyridine ring of the target molecule was synthesized and coupled to carrier proteins. Following rabbit immunization, antibodies against this agrochemical were obtained for the first time, and they were characterised in terms of affinity and specificity, tolerance to solvents, and robustness to changes in buffer pH and ionic strength, using two assay formats. Both of the optimised immunoassays showed limits of detection below 0.1μg/L. Moreover, matrix effects of grape, peach, apple, and tomato juices were evaluated. Finally, a simple and easy procedure was set up for boscalid determination with spiked samples, affording limits of quantification of 10μg/L, a value well below the sensitivity levels required for monitoring campaigns of pesticide residue analysis in food. [Copyright &y& Elsevier]

Abstract: Fenhexamid is a new-generation agrochemical extensively used nowadays to protect high valuable crops and fruits from fungus attacks. In this paper, the development of an indirect competitive immunoassay for the determination of fenhexamid residues in fruit samples is described. After optimisation of immunoreagent concentrations, the assay, based on monoclonal antibody FHo4#27, showed high sensitivity, and selectivity, with a limit of detection for fenhexamid standards in buffer of 0.04μg/L. The influence of several physico-chemical factors, including pH, ionic strength, solvent tolerance, and matrix interferences on the analytical parameters of the standard curve was studied. For spiked liquid food samples like grape must, just a direct 1:50 dilution in water was sufficient to attain recoveries between 100% and 110%. For kiwifruit and strawberry samples, a double extraction procedure with methanol is proposed in this study, which afforded recovery values that ranged between 103% and 116%. The limit of quantification of the developed assay was 5μg/kg for grape must, 10μg/kg for kiwifruits, and 25μg/kg for strawberries. The optimised assay was compared with a reference procedure based on liquid chromatography using incurred samples from the market, and an excellent correlation between both methods was found. [ABSTRACT FROM AUTHOR]

Abstract In 2013, quinoxyfen was included in the list of priority hazard pollutants of the European Water Framework Directive due to its toxicity to aquatic organisms. However, few analytical methods for the analysis of this fungicide have been reported and no rapid immunochemical methods have been published so far. In the present study, immunoreagents for quinoxyfen analysis were generated for the first time and an enzyme-linked immunosorbent assay was developed. Two carboxylated derivatives of quinoxyfen were designed on the basis of the minimum energy conformation of the target compound. Active esters of those novel compounds were prepared using N , N ′-disuccinimidyl carbonate, and purified for covalent coupling to proteins. Matrix-assisted laser desorption mass spectrometry of the prepared bioconjugates showed optimum hapten-to-protein molar ratios. Moreover, high-affinity antibodies specific of quinoxyfen were raised. As proof of concept, an immunoassay was evaluated using a heterologous conjugate, which afforded sensitivity values in the low nanomolar range. Moreover, excellent recoveries and coefficients of variation were obtained from the analysis of environmental water samples fortified with quinoxyfen. A limit of quantification of 60 μg/L was determined. The prepared bioconjugates and antibodies could be valuable immunoreagents for the development of a variety of rapid immunosensors for quinoxyfen determination in environmental samples. Graphical abstract Image 1 Highlights • Quinoxyfen is a priority hazard pollutant in the European Water Framework Directive. • Functionalized derivatives were synthesized for covalent conjugation to proteins. • Bioconjugates and high-affinity antibodies specific of quinoxyfen were prepared. • A sensitive immunoassay (LOQ = 60 μg/L) was optimized and developed. • Precision and accuracy of the method were verified with environmental water samples. [ABSTRACT FROM AUTHOR]

Highlights • Fluopyram regioisomeric haptens with opposite linker tethering sites were used. • High-affinity monoclonal antibodies to fluopyram were generated for the first time. • Two ELISAs were optimized for fluopyram analysis with LOD values below 0.05 µg L−1. • Immunoassay performance was verified in plum, grape, must, and wine samples. • Immunochemical results were validated with LC-MS using contaminated samples. Abstract Monoclonal antibody-based techniques have become a useful analytical technology in the agro-food sector. Nowadays, residues of the recently registered fungicide fluopyram are increasingly being found in quality control programs. In the present study, novel chemical derivatives of this pesticide were prepared and specific and high-affinity monoclonal antibodies to fluopyram were raised for the first time. Moreover, immunoassays to fluopyram were developed in two alternative enzyme-linked immunosorbent assay formats, using homologous and heterologous assay conjugates, with limits of detection below 0.05 µg L−1. The optimized immunoassays were applied to the analysis of fluopyram in fortified plums and grapes of four different varieties as well as in in-house prepared musts and wines. Recoveries were between 76.3% and 109.6% and coefficients of variation were below 20%. Quantification limits were well below the maximum residue limits. Immunoassay performance was statistically validated with a reference chromatographic technique using samples from fluopyram-treated plum and grape cultivars. [ABSTRACT FROM AUTHOR]

Boscalid is an agrochemical recently developed for crop protection and the most significant member of the succinate dehydrogenase inhibitor group of fungicides. In this study, a collection of high-affinity monoclonal antibodies was generated to boscalid. By using a series of haptens with a linker at alternative tethering sites of the boscalid framework, specific antibodies were isolated as well as antibodies that also recognized the main boscalid metabolite. Two immunoassays were developed using different ELISA formats. Optimized assays displayed very high sensitivities (limits of detection were near 0.01 µg/L). Trueness and precision for the determination of the target analyte in strawberry samples was evaluated. Moreover, immunoassay performance was validated with a reference chromatographic method using QuEChERS extracts of fruits from fungicide-treated crops. A monitoring study with strawberry samples from local markets was carried out by immunoassay, showing an occurrence of boscalid of 15% with a maximum residue concentration of 43 µg/kg. [ABSTRACT FROM AUTHOR]

Abstract Anatoxin-a and homoanatoxin-a are highly neurotoxic compounds produced by cyanobacteria, principally during surface water-blooms (SWBs). Owing to their powerful biological activity and unique structural characteristics, these natural alkaloids have been the subject of extensive research work in both pharmacological and synthetic studies. In this contribution we report a simple and efficient synthetic approach for the preparation of both the natural and unnatural enantiomers of these cyanotoxins, [(+)- 1 and (+)- 2 ] and [(−)- 1 and (−)- 2 ] respectively. Key features of this approach include: i) construction of the azabicyclic homotropane framework in both enantiomeric forms from cis -5,6-epoxycyclooctene, based on a microwave mediated epoxide ring opening reaction by a chiral benzyl amine followed by a transannular amine-alkene cyclization; ii) elaboration of the characteristic methyl or ethyl enone by means of a Sonogashira cross-coupling reaction of an enol-triflate with a C2 or C3 terminal alkyne, followed by a chemo- and regio-selective hydration of the resulting conjugated enyne group. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

Azoxystrobin, boscalid, cyprodinil, fenhexamid, and pyrimethanil are new generation fungicides extensively employed in order to combat diseases affecting vineyards worldwide. Owing to their physico-chemical characteristics, residues of these compounds on grapes are transferred to must and wine. In this study, a survey of the occurrence of these fungicides in international wines was carried out by using rapid antibody-based assays. Results are discussed as a function of wine type and sample geographical origin. 44.4% of the samples contained at least one of the targets (>10 μg L −1 ). Fungicide residue occurrences were 22.4%, 19.2%, 18.8%, 6.8%, and 1.2% for pyrimethanil, boscalid, fenhexamid, cyprodinil, and azoxystrobin, respectively, while residue contents higher than 100 μg L −1 were found in 8.4% of the samples. This study shows that contamination of commercial wines with pesticides is an issue of worldwide relevance with potential implications for consumer health and international trade. [ABSTRACT FROM AUTHOR]

Cyprodinil is among the most common agrochemical residues found in highly perishable fruits, such as strawberries. In the present study, high-affinity monoclonal antibodies to this anilinopyrimidine fungicide were raised for the first time with the aim to produce valuable immunochemical analytical assays. Cyprodinil bioconjugates and the generated novel monoclonal antibodies were employed for sensitive competitive immunoassay development in two different formats. The limits of detection of the optimized assays were 20 and 30 ng L −1 for the indirect and direct assay, respectively. Influence over assay parameters of different physicochemical factors was studied. Strawberry samples were extracted following the recommended QuEChERS procedure for pesticide residues in food, and analyzed by the optimized immunoassays. Recoveries and coefficients of variation from fortified samples were within standard values. In addition, the obtained immunochemical results with naturally contaminated samples were statistically comparable, according to Deming regression analysis, to those of a reference chromatographic method. [ABSTRACT FROM AUTHOR]

Cyprodinil is a new-generation anilinopyrimidine fungicide widely used in crop protection and frequently found in fruits. In this study, novel derivatives of cyprodinil with linker site heterologies were synthesized and employed in order to produce antibodies with enhanced affinity. Moreover, moiety-heterologous haptens were designed and prepared for assay sensitivity improvement. Two competitive enzyme-linked immunosorbent assays for the analysis of this active substance were developed using direct and indirect formats, achieving IC 50 values around 0.15 μg/L. Analytical figures of merit and usability of the optimized assays were evaluated with wine and cider as model food processed matrices. The obtained recoveries were from 90% to 120%, and the limit of quantification was in the 1–5 μg/L range. Finally, a monitoring study ( n = 150) was performed to estimate the occurrence and the concentration of cyprodinil in commercial wine and cider products from different origins. We found that 28% of the analysed wine samples contained cyprodinil residues at levels higher than 5 μg/L. [ABSTRACT FROM AUTHOR]

Highlights: [•] Two new monoclonal antibody-based immunoassays to trifloxystrobin were characterised. [•] An indirect assay for trifloxystrobin and its metabolite was developed. [•] Competitive ELISAs were validated with blind-spiked and in-field treated samples. [Copyright &y& Elsevier]

Highlights: [•] Seven functionalized regioisomeric derivatives of trifloxystrobin were synthesized. [•] Immunizing and assay conjugates were produced by a single coupling procedure. [•] A collection of monoclonal antibodies was generated. [•] Immunoreagents were evaluated by direct and indirect ELISA. [•] Fine specificity of antibodies was investigated using analogous compounds. [Copyright &y& Elsevier]

Abstract: Strobilurins are a family of synthetic natural-derived pesticides widely used for fungi control in many crops. As part of a larger research project, a collection of monoclonal antibodies to one of the most relevant strobilurins, i.e. pyraclostrobin, has been generated using functionalized haptens with the spacer arm located at different sites. The toxophore aliphatic moiety, as observed with other strobilurins, and the planar aromatic two-ring system characteristic of this molecule were revealed as the best tethering sites for high-affinity monoclonal antibody production. A central derivatization position did not generate a good immune response, but such derivatives were useful as heterologous competitors in order to improve assay sensitivity. Direct and indirect assays were characterized in terms of solvent influence, pH, and ionic strength. Immunoassays with limits of detection below 0.1 μg/L were developed. A QuEChERS-based procedure was applied to the extraction of blueberry, strawberry, raspberry, peach, apricot, and plum jams. Clean acetonitrile extracts were properly diluted in water and analyzed by both optimized immunoassays with good accuracy and precision at levels covering the European maximum residue limits for those fruits. [Copyright &y& Elsevier]

Abstract: Picoxystrobin is a new generation fungicide primarily developed to be used in cereal crops. In the present study a novel collection of specific monoclonal antibodies has been produced using different immunizing haptens based on a carboxy-functionalized identical spacer arm attached to alternative positions of the target pesticide molecule. Two competitive enzyme-linked immunosorbent assays have been developed employing hapten heterology, one using the antibody-coated direct format and the other in the conjugate-coated indirect format. Both immunoassays have been characterized in terms of selectivity, solvent tolerance, and buffer conditions, affording similar limits of detection at or below 0.1 μg/L. Finally, the optimized assays were applied to the analysis of picoxystrobin in wheat, corn, oat, barley, and soybean flours. Average recovery values from spiked samples were between 84 and 115%. [Copyright &y& Elsevier]

Abstract: The use of agrochemicals for crop protection may result in the presence of toxic residues in soils and aquatic environments, besides in foodstuffs. Most often just the parent compound is included in the definition of pesticide residue, even though chemicals resulting from biotransformation and degradation routes might also be of toxicological relevance. Azoxystrobin is a broad-spectrum systemic fungicide widely used worldwide to combat pathogenic fungi affecting plants. We herein report the synthesis and detailed chemical characterization of several of the most relevant metabolites and degradates of azoxystrobin. These compounds were further employed as ligands for screening a collection of monoclonal antibodies to azoxystrobin, which had been previously generated from haptens functionalized at different positions of the target chemical. As a result, an antibody was identified capable of binding, with subnanomolar affinity, not only azoxystrobin but also its main transformation products, such as the so-called acid and enol derivatives, as well as the azoxystrobin (Z)-isomer. The selected binder was demonstrated as a useful immunoreagent for the development of immunochemical assays as novel analytical tools for the qualitative determination of azoxystrobin and its metabolites and degradates. [Copyright &y& Elsevier]

Abstract: The immune response to regioisomeric haptens of azoxystrobin with varied derivatization sites was studied. Based on the Sonogashira and Suzuki–Miyaura couplings and following a straightforward modular design, we have synthesized four haptens with the same linker anchored through C–C bonds and located at different sites of the molecule. The most stereoselective antibodies were produced from immunogens with the spacer arm at a distal position from the β-methoxyacrylate moiety characteristic of strobilurins. Moreover, we observed that assay cross-reactivity was reliant on the functionalization site of the competitor derivative. Finally, the antibody binding site was explored using synthetic chemical analogues. [Copyright &y& Elsevier]

• ELISA and LFIA based on novel immunoreagents were developed for OTA detection in wine and must. • The ELISA allows the analysis of OTA at concentrations below the limit set by the EU (2 ng/mL). • The LFIA showed 80% and complete inhibition at 0.5 and 2 ng/mL, respectively. • LFIA was validated following EU Regulation 519/2014 for semi-quantitative screening methods. • The immunoassays are suitable for on-site monitoring of samples contaminated with OTA. Immunochemical methods are highly deployed in analytical laboratories worldwide for monitoring the incidence of mycotoxins in the food chain. Nevertheless, most conventional immunoassays for ochratoxin A (OTA), including commercial kits, show limitations to robustly determine this mycotoxin in grape-derived products below regulated levels (2 ng/mL). Herein, two rapid tests for sensitive OTA determination in wine and must were developed capitalizing on a collection of bioconjugates from innovative synthetic haptens and monoclonal antibodies with subnanomolar affinity. The ELISA (LOD = 8 pg/mL) showed excellent performance in recovery studies, and it was applied to survey commercial wines and musts for OTA contamination. Concerning LFIA, validation according to the Commission Regulation 519/2014 showed that samples exceeding 2 ng/mL were properly scored as uncompliant. More importantly, illegal samples provided a complete inhibition of the test signal, making this test an easy-to-use, rapid, and convenient screening method for in-house control of OTA in wineries. [ABSTRACT FROM AUTHOR]