Abstract
Proquinazid is a new-generation fungicide authorized in the EU for combating powdery mildew infections in high-value crops. Due to the perishable nature of fruits, alternative analytical methods are necessary to protect consumer’s health from pesticide residues. Currently, immunoassays are a well-established approach for rapidly monitoring chemical contaminants. However, the production of high-quality immunoreagents, such as antibodies and bioconjugates, is essential. This study presents a newly designed hapten that maintains the characteristic moieties of proquinazid unmodified. The linear aliphatic substituents of this molecule were used to introduce the spacer arm. A three-step synthesis strategy was optimized to prepare a hapten that displays the entire 6-iodoquinazolin-4(3H)-one moiety with excellent yields. The N-hydroxysuccimidyl ester of the hapten was activated and purified to prepare a protein conjugate with high hapten density, which was used as an immunogen. Antibodies were raised and competitive enzyme-linked immunosorbent assays were developed. To enhance the assay’s sensitivity, two additional heterologous haptens were prepared by modifying the halogenated substituent at C-6. The optimized assays demonstrated low limits of detection in buffer, approximately 0.05 μg/L. When applied to the analysis of proquinazid in QuEChERS extracts of strawberry samples, the immunoassays produced precise and accurate results, particularly in the 10–1000 μg/kg range.
Graphical Abstract
This is a preview of subscription content, log in via an institution to check access.
References
Santos B, Blanco C, Porras M, Barrau C, Romero F. First confirmation of Sphaerothecamacularis on strawberry plants in southwestern Spain. Plant Dis. 2002;86(9):1049. https://doi.org/10.1094/PDIS.2002.86.9.1049C.
Gadoury DM, Asalf B, Heidenreich MC, Herrero ML, Welser MJ, Seem RC, Tronsmo AM, Stensvand A. Initiation, development, and survival of cleistothecia of Podosphaeraaphanis and their role in the epidemiology of strawberry powdery mildew. Phytopathology. 2010;100(3):246–51. https://doi.org/10.1094/PHYTO-100-3-0246.
Asalf B, Gadoury DM, Tronsmo AM, Seem RC, Cadle-Davidson L, Brewer MT, Stensvand A. Temperature regulates the initiation of chasmothecia in powdery mildew of strawberry. Phytopathology. 2013;103(7):717–24. https://doi.org/10.1094/PHYTO-09-12-0252-R.
Fungicide Resistance Action Committee. Fungal control agents sorted by cross-resistance pattern and mode of action. In FRAC Code List. 2022. https://www.frac.info/. Accessed 12 Mar 2024.
European Food Safety Authority. Conclusion on the peer review of the pesticide risk assessment of the active substance proquinazid. EFSA J. 2009;7(10):1350. https://doi.org/10.2903/j.efsa.2009.1350.
Esteve-Turrillas FA, Mercader JV, Parra J, Agulló C, Abad-Somovilla A, Abad-Fuentes A. Ready access to proquinazid haptens via cross-coupling chemistry for antibody generation and immunoassay development. PLoS ONE. 2015;10(7):e0134042. https://doi.org/10.1371/journal.pone.0134042.
Tully WR, Westwood R, Rowlands DA, Clements-Jewery S. Triazoloquinazolones and their salts, intermediates for preparing them, their use as medicines and compositions containing them. EP76199 A2. 1983;04–06.
Danilchenko SY, Drushlyak OG, Kovalenko SM. Synthesis of substituted 2-hydrazinoquinazolin-4-ones as intermediates for synthesis of heterocyclic compounds. Zhurnal Organichnoi ta Farmatsevtichnoi Khimii. 2014;12(3):66–73. https://doi.org/10.24959/ophcj.14.810.
Bereznak JF, Marshall EA, Sternberg CG, Sternberg JA, Sun K-M. Preparation of fungicidal quinazolinones and useful intermediates. WO9748684 A1. 1997;12–24.
Lehotay SJ, Son KA, Kwon H, Koesukwiwat U, Fu W, Mastovska K, Hoh E, Leepipatpiboon N. Comparison of QuEChERS sample preparation methods for the analysis of pesticide residues in fruits and vegetables. J Chromatogr A. 2010;1217(16):2548–60. https://doi.org/10.1016/j.chroma.2010.01.044.
Vashist SK. The importance of bioanalytical parameters and statistical analysis in immunoassays for food analytes. In: O’Kennedy R, editor. Rapid antibody-based technologies in food analysis. Food chemistry, function and analysis. London: Royal Soc Chem. 2019;15:32–45.
Esteve-Turrillas FA, Mercader JV, Agulló C, Abad-Somovilla A, Abad-Fuentes A. A class-selective immunoassay for simultaneous analysis of anilinopyrimidine fungicides using a rationally designed hapten. Analyst. 2017;142:3975–85. https://doi.org/10.1039/C7AN01138E.
Ceballos-Alcantarilla E, Agulló C, Abad-Fuentes A, Escamilla-Aguilar M, Abad-Somovilla A, Mercader JV. High-affinity antibodies from a full penthiopyrad-mimicking hapten and heterologous immunoassay development for fruit juice analysis. Food Anal Methods. 2017;10:4013–23. https://doi.org/10.1007/s12161-017-0974-3.
Cho YA, Kim YJ, Hammock BD, Lee YT, Lee H-S. Development of a microtiter plate ELISA and a dipstick ELISA for the determination of the organophosphorus insecticide fenthion. J Agric Food Chem. 2003;51:7854–60. https://doi.org/10.1021/jf0346915.
Shim JY, Kim YA, Lee YT, Hammock BD, Lee H-S. Monoclonal antibody-based enzyme-linked immunosorbent assays for the organophosphorus insecticide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN). J Agric Food Chem. 2010;58:5241–7. https://doi.org/10.1021/jf904528y.
Luo L, Shen Y-D, Wang H, **ao Z-L, Sun Y-M, Lei H-T, Yang J-Y, Xu Z-L. Production of polyclonal antibody and development of a competitive enzyme-linked immunosorbent assay for benzoic acid in foods. Food Anal Methods. 2015;8:1101–11. https://doi.org/10.1007/s12161-014-9975-7.
Acknowledgements
Proteomic analysis was performed at the Proteomics Unit of the Instituto de Parasitología y Biomedicina “López – Neyra”.
Funding
This study was funded by MCIU/AEI/https://doi.org/10.13039/501100011033 (grant numbers PID2021-125721OB-C21/C22 and PDC2022-133559-C21/C22), and cofinanced by “ERDF A way of making Europe” and by the “European Union NextGenerationEU/PRTR”. The Accreditation as Center of Excellence Severo Ochoa CEX2021-001189-S also funded by MCIU/AEI/https://doi.org/10.13039/501100011033 is fully acknowledged.
Author information
Authors and Affiliations
Contributions
Conceptualization: F.A. Esteve-Turrillas, C. Agulló, I. Navarro-Fuertes. Methodology: F.A. Esteve-Turrillas, J.V. Gimeno-Alcañiz, I. Navarro-Fuertes. Formal analysis and investigation: F.A. Esteve-Turrillas, J. Gimeno-Alcañiz, C. Agulló. Writing — original draft preparation: J.V. Mercader, A. Abad-Somovilla. Writing — review and editing: J.V. Mercader, A. Abad-Somovilla, A. Abad-Fuentes. Funding acquisition: J.V. Mercader, A. Abad-Somovilla, A. Abad-Fuentes. Supervision: J.V. Mercader, A. Abad-Somovilla, A. Abad-Fuentes.
Corresponding author
Ethics declarations
Ethics approval
The study has been performed according to the ethical standards approved by the University of Valencia bioethic committee.
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Published in the topical collection Optical Biosensors and Biomimetic Sensors for Chemical Analysis with guest editors Elena Benito-Peña and Guillermo Orellana.
In honor of Professor María Cruz Moreno Bondi.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Gimeno-Alcañiz, J.V., Esteve-Turrillas, F.A., Abad-Fuentes, A. et al. Rapid immunochemical methods for the analysis of proquinazid in strawberry QuEChERS extracts. Anal Bioanal Chem (2024). https://doi.org/10.1007/s00216-024-05428-0
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00216-024-05428-0