Development and application of kinetic-spectrophotometric method for analysis of diflubenzuron in soil samples using SPE followed by HPLC method

Chemia Naissensis Volume 4, No.1 (2021) (стр. 40-61) 

АУТОР(И) / AUTHOR(S):  Emilija Pecev-Marinković, Ana Miletić, Aleksandra Pavlović and Vidoslav Dekić


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DOI: 10.46793/ChemN4.1.40PM


The purpose of this paper is to present a new sensitive and simple kinetic-spectrophotometric method for the determination of the insecticide diflubenzuron (DFB). The kinetic method is based on the inhibition effect of DFB on the oxidation of sulfanilic acid (SA) by potassium periodate in acetate buffer in the presence of Fe(III) ion as a catalyst and 1,10-phenantroline. The reaction was monitored spectrophotometrically by measuring the increase in absorbance with time of the reaction product at 368 nm. Diflubenzuron was determined with linear calibration graph in the interval from 0.0374 to 0.374 μg/cm3 and from 0.374 to 26.18 μg/cm3. The detection limit and quantification limit of the method with 3σ criteria were 0.0039 μg/cm3 and 0.0131 μg/cm3, respectively. The relative standard deviations for five replicate determinations of 0.0374, 0.188 and 0.374 μg/cm3 DFB were 2.24, 2.11 and 1.10%, respectively. The method was successfully applied to determine DFB residues in soil samples. Solid-phase extraction (SPE) was used for extraction of DFB from soil and samples with Chromabond® (Macherey-Nagel) C18 cartridges. The HPLC method was used as a comparative method to verify the results. The results obtained by two different methods showed good agreement.


Diflubenzuron, Kinetic method, HPLC method, Solid-phase extraction, Soil samples


  • Amelin, V. G., Lavrukhin, D. K., & Tret’yakov, A. V. (2013). Dispersive liquid-liquid microextraction for the determination of herbicides of urea derivatives family in natural waters by HPLC. Journal of Analytical Chemistry, 68, 822-830.
  • Ambike, V., & Argekar, A. (2017). Stability indicating HPLC method for the determination of diflubenzuron insecticide. Science Journal of Energy Engineering, 5(6), 152-157.
  • Bendito, D. P., & Silva, M. (1988.). Kinetic Methods in Analytical Chemistry. Ellis Horwood: Chichester.
  • Carneiro, R. P., Oliveira, F. A. S., Madureira, F. D., Silva, G., de Souza W. R., & Pereira Lopes, R. (2013). Development and method validation for determination of 128 pesticides in bananas by modified QuEChERS and UHPLC-MS/MS analysis. Food Control, 33, 413-423.
  • Chen, B., Jin, B., Jiang, R., Xie, L., Lin, Y., Feng, W., & Ouyang, G. (2014). Screening and quantification of 304 pesticides and related organic pollutants in surface water using dispersive Liquid–Liquid Microextraction Coupled with Gas Chromatography–Mass Spectrometry. Analytical Methods, 6, 1743–1752.
  • Chen, X., Zhao, K., Ge, B., & Chen, Q. (2013). Simultaneous determination of 44 pesticides in tobacco by UPLC/MS/MS and a modified QuEChERS procedure. Journal of AOAC International, 96, 422-431.
  • Choi, S., Kim, S., Shin, J.Y., Kim, M, & Kim, J.-H. (2015). Development and verification for analysis of pesticides in eggs and egg products using QuEChERS and LC-MS/MS. Food Chemistry, 173, 1236-1242.
  • Diaw, P. A., Maroto, A., Mbaye, O. M. A., Gaye-Seye, M. D., Stephan, L., Coly, A., Deschamps, L., Tine, A., Aaron, J. J., & Giamarchi, P. (2013). Determination of phenylurea pesticides by direct laser photo-induced fluorescence. Talanta, 116, 569–574.
  • De Clercq, P., De Cock, A., Tirry, L., Viñuela, E., & Degheele, D. (1995). Toxicity of diflubenzuron and pyriproxyfen to the predatory bug Podisus maculiventris. Entomologia Experimentalis et Applicata, 74, 17–22.
  • Du, P., Liu, X., Gu, X., Dong, F., Xu, J., Kong, Z., Wu, Y., Zhu, Y., Li, Y., & Zheng, Y. (2013).
  • Rapid residue analysis of pyriproxyfen, avermectins and diflubenzuron in mushrooms by ultra-performance liquid chromatography coupled with tandem mass spectrometry. Analytical Methods, 5, 6741-6747.
  • Erler, F., Polat, E., Demir, H., Catal, M., & Tuna, G. (2011). Control of mushroom sciarid fly Lycoriella ingenua populations with insect growth regulators applied by soil drench. Journal of Economic Entomology, 104(3), 839-844.
  • Graça Melo, M., Carqueijo, A., Freitas, A., Barbosa, J., & Sanches Silva, A. (2019). Modified QuEChERS extraction and HPLC-MS/MS for simultaneous determination of 155 pesticide residues in rice (Oryza sativa L.). Foods 9, 18. doi:10.3390/foods9010018.
  • Grahovac, Z. M., Mitić, S. S., Pecev, E. T., & Pavlović, A. N. (2010). Determination of the insecticide diflubenzuron in mushrooms by kinetic method and high-performance liquid chromatographic method. Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 45, 783-789.
  • Guimarães de Oliveira, L., Ramkumar, A., Moloney, M., Scherer Kurz, M. H., Ferreira Gonçalves, F., Prestes, O. D., & Danaher, M. (2019). Vibrational extraction QuEChERS for analysis of antiparasitic agents in fish by liquid chromatography coupled with tandem mass spectrometry. Analytical and Bioanalytical Chemistry, 411, 6913-6929.
  • Huang, X., Qiao, K., Li, L., Liu, G., Xu, X., Lu, R., Gao, H., & Xu, D. (2019a). Preparation of a magnetic graphene/polydopamine nanocomposite for magnetic dispersive solid-phase extraction of benzoylurea insecticides in environmental water samples. Scientific Reports, 9:8919. DOI: 10.1038/s41598-019-45186-z
  • Huang, Y., Zhou, Q., Xie, G., Liu, H., & Lin, H. (2011). Titanium dioxide nanotubes for solid phase extraction of benzoylurea insecticides in environmental water samples, and determination by high performance liquid chromatography with UV detection. Microchimica Acta, 172, 109– 115.
  • Hartmann, C., Smeyers-Verbeke, J., Penninckx, W., Vander Heyden, Y., Vankeerberghen, P., & Massart, D. L. (1995). Reappraisal of hypothesis testing the method validation: detection of systematic error by comparing the means of two methods or of two laboratories. Analytical Chemistry, 67, 4491-4499.
  • Huang, Y., Shi, T., Luo, X., Xiong, H., Min, F., Chen, Y., Nie, S., & Xie, M. (2019b). Determination of multi-pesticide residues in green tea with a modified QuEChERS protocol coupled to HPLC-MS/MS. Food Chemistry, 275, 255-264.
  • Irungu, J., Raina, S., & Torto, B. (2016). Determination of pesticide residues in honey: a preliminary study from two of Africa’s largest honey producers. International Journal of Food Contamination, 3, 1-14.
  • Choi, S., Kim, S., Shin, J. Y., Kim, M., & Kim, J.-H. (2015). Development and verification for analysis of pesticides in eggs and egg products using QuEChERS and LC–MS/MS. Food Chemistry, 173, 1236–1242.
  • Kim, J.-H., Seo, J.-S., Moon, J.-K., & Kim, J.-H. (2013). Multi-residue method development of 8 benzoylurea insecticides in mandarin and apple using high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. Journal of the Korean Society for Applied Biological Chemistry, 56, 47-54.
  • Klima of Forensnings Direktoratet (Klif). (2011). Environmental screening of veterinary medicines used in aquaculture – diflubenzuron and teflubenzuron. TA 2773. Utført av NIVA (Norwegian Institute for Water Research).
  • Łozowicka, B., Rutkowska, E., & Jankowska, M. (2017). Influence of QuEChERS modifications on recovery and matrix effect during the multi-residue pesticide analysis in soil by GC/MS/MS and GC/ECD/NPD. Environmental Science and Pollution Research, 24, 7124–7138.
  • Lee, S. W., Choi, J.-H., Cho, S.-K., Yu, H.-A., Abd El-Aty, A.M., & Shim, J.-H. (2011).
  • Development of a new QuEChERS method based on dry ice for the determination of 168 pesticides in paprika using tandem mass spectrometry. Journal of Chromatography A, 1218, 4366–4377.
  • Matsumura, F. (2010). Studies on the action mechanism of benzoylurea insecticides to inhibit the process of chitin synthesis in insects: A review on the status of research activities in the past, the present and the future prospects. Pesticide Biochemistry and Physiology, 97, 133–139.
  • Macken, A., Lillicrap, A., & Langford, K. (2015). Benzoylurea pesticides used as veterinary medicines in aquaculture: risks and developmental effects on non-target crustaceans. Environmental Toxicology and Chemistry, 34(7), 1533-1542.
  • Martínez-Domínguez, G., Nieto-García, A. J., Romero-González, R., & Frenich, A. G. (2015). Application of QuEChERS based method for the determination of pesticides in nutraceutical products (Camellia sinensis) by liquid chromatography coupled to triple quadrupole tandem mass spectrometry. Food Chemistry, 177, 182–190.
  • Martín-Pozo, L., de Alarcón-Gómez, B., Rodríguez-Gómez, R., García-Córcoles, M. T., Çipa, M., & Zafra-Gómez, A. (2019). Analytical methods for the determination of emerging contaminants in sewage sludge samples. A review. Talanta, 192, 508-533.
  • Mei, M., Huang X., Liao, K., & Yuan, D. (2015). Sensitive monitoring of benzoylurea insecticides in water and juice samples treated with multiple monolithic fiber solid-phase microextraction and liquid chromatographic analysis. Analytica Chimica Acta, 860, 29–36.
  • Mei, M., Huang, X., & Yuan, D. (2014). Multiple monolithic fiber solid-phase microextraction: A new extraction approach for aqueous samples. Journal of Chromatography A, 1345, 29-36.
  • Miller, J. N. (1991). Basic statistical methods for analytical chemistry. Part 2. Calibration and regression methods. A review. Analyst, 116, 3-14.
  • Mottola, H. A. (1988). Kinetic Aspects of Analytical Chemistry. Wiley: New York.
  • Ouyang, H., He, Y., & Lin, L. (2015). Determination of four kinds of pesticide residues in edible mushrooms by HPLC. Journal of Southern Agriculture, 46, 437-440.
  • Olsvik, P. A., Samuelsen, O. B., Erdal, A., Holmelid, B., & Lunestad, B. T. (2013). Toxicological assessment of the anti-salmon lice drug difubenzuron on Atlantic cod Gadus morhua. Diseases of Aquatic Organisms, 105, 27–43.
  • Pecev-Marinković, E. T., Grahovac, Z. M., Pavlović, A. N., Tošić, S. B., Rašić Mišić, I. D., Mitić, M. N., Miletić, A. S., & Sejmanović, D. M. (2018). Development of a kinetic spectrophotometric method for insecticide diflubenzuron determination in water and baby food samples. Hemijska Industrija, 72(5), 305–314.
  • Prichard, E., & Barwick, V. (2007). Quality Assurance in Analytical Chemistry. John Wiley & Sons: Teddington.
  • Shi, X., Tang, Z., Sun, A., Zhou, L., Zhao, J., Li, D., Chen, J., & Pan, D. (2014). Simultaneous analysis of polychlorinated biphenyls and organochlorine pesticides in seawater samples by membrane–assisted solvent extraction combined with Gas Chromatography–Electron Capture Detector and Gas Chromatography–Tandem Mass Spectrometry. Journal of Chromatography B, 972, 58–64.
  • Sun, R., Liu, C., Zhang, H., & Wang, Q. (2015). Benzoylurea chitin synthesis inhibitors. Journal of Agricultural and Food Chemistry, 63, 6847–6865.
  • Skoog, D. A., West, D. M., & Holler, F. J. (1996). Fundamentals of Analytical Chemistry. Saunders College Publishing: Philadelphia.
  • Tian, F., Qiao, C., Luo, J., Guo, L., Pang, T., Pang, R., Li, J., Wang, C., Wang, R., & Xie, H. (2020). Method development and validation of ten pyrethroid insecticides in edible mushrooms by modifed QuEChERS and gas chromatography-tandem mass spectrometry. Scientific Reports. 10:7042.
  • Tfouni, S.A.V., Furlani, R.P.Z., Carreiro, L.B, Loredo, I.S.D., Gomes, A.G, Alves, L.A, Mata, R.S.S., Fonseca, A.M.D., & Rocha, R.M.S. (2013). Determination of diflubenzuron residues in milk and cattle tissues. Arquivo Brasileiro de Medicina Veterinaria e Zootecnia, 65, 301-307.
  • Tran, K., Eide, D., Nickols, S. M., Cromer, M. R., Sabaa-Srur, A., & Smith, R. E. (2012). Finding of pesticides in fashionable fruit juices by LC–MS/MS and GC–MS/MS. Food Chemistry, 134, 2398–2405.
  • Wang, J., Jiao, C., Li, M., Wang, X., Wang, C., Wu, Q., & Wang, Z. (2018). Porphyrin based porous organic polymer modified with Fe3O4 nanoparticles as an efficient adsorbent for the enrichment of benzoylurea insecticides. Microchimica Acta, 185, 36.
  • Wang, X., Yang, C., Huang, M., Wang, M., Zha, Y., Lin, L., & Zeng, S. (2014). Determination of diflubenzuron residues in vegetables by UPLC-MS/MS. Advanced Materials Research, 852, 266-269.
  • Willrich, M. M., & Boethel, D. J. (2001). Effects of diflubenzuron on Pseudoplusia includens (Lepidoptera: Noctuidae) and its parasitoid Copidosoma floridanum (Hymenoptera: Encyrtidae). Environmental Entomology, 30, 794-797.
  • WHO/FAO (2020). Fao specifications and evaluations for agricultural pesticides, Food and Agriculture Organisation of the United Nations.
  • Wang, H., Hu, L., Li, W., Lu, R., Zhang, S., Zhou, W., & Gao, H. (2016). A rapid and simple pretreatment method for benzoylurea insecticides in honey samples using in-syringe dispersive liquid–liquid microextraction based on the direct solidification of ionic liquids. Journal of Chromatography A, 1471, 60–67.
  • Wang, H., Hu, L., Li, W., Yang, X., Lu, R., Zhang, S., Zhou, W., Gao, H., & Li, J. (2017). In-
  • syringe dispersive liquid-liquid microextraction based on the solidification of ionic liquids for the determination of benzoylurea insecticides in water and tea beverage samples. Talanta, 162, 625–633.
  • Wang, S., Zhang, S., Dong, C., Wang, G., Guo, J., & Sun, W. (2013). Simultaneous determination of four herbicide and pesticide residues in Chinese green tea using QuEChERS purification procedure and UPLC-MS/MS analysis. Advanced Materials Research, 634-638, 1586-1590.
  • Yang, X., Qiao, K., Ye, Y., Yang, M., Li, J., Gao, H., Zhang, S., Zhou, W., & Lu, R. (2016). Facile synthesis of multifunctional attapulgite/Fe3O4/polyaniline nanocomposites for magnetic dispersive solid phase extraction of benzoylurea insecticides in environmental water samples. Analytica Chimica Acta, 934, 114–121.
  • Yang, M., Xi, X., Yang, X., Bai, L., Lu, R., Zhou, W., Zhang, S. & Gao, H. (2015). Determination of benzoylurea insecticides in environmental water and honey samples using ionic-liquid-mingled air-assisted liquid–liquid microextraction based on solidification of floating organic droplets. RCS Advances, 32(5), 25572-25580.
  • Zhang, Z., Lefebvre, T., Kerr, C., & Osprey, M. (2014). Simultaneous extraction and determination of various pesticides in environmental waters. Journal of Separation Science, 37, 3699–3705.
  • Zainudin, B. H., Salleh, S., Mohamed, R., Choy Yap, K., & Muhamad, H. (2015). Development, validation, and determination of multiclass pesticide residues in cocoa beans using gas chromatography and liquid chromatography tandem mass spectrometry. Food Chemistry, 172, 585–595.