ORIGINAL PAPER Determination of benzimidazole fungicides in water samples by on-line MISPE–HPLC O. Zamora & E. E. Paniagua & C. Cacho & L. E. Vera-Avila & C. Perez-Conde Received: 2 December 2008 / Revised: 12 January 2009 / Accepted: 16 January 2009 / Published online: 1 February 2009 # Springer-Verlag 2009 Abstract An analytical methodology based on an on-line sample enrichment of water samples by means of an imprinted polymer, and the separation of benzimidazole compounds within a C 18 column by ion-pair reversed-phase liquid chromatography, has been developed. The molecularly imprinted polymer has been synthesized by precipitation polymerization using thiabendazole as template molecule, methacrylic acid as functional monomer, and divinylbenzene as cross-linker. Initial experiments carried out by solid-phase extraction on cartridges demonstrated a clear imprint effect for thiabendazole, as well as the ability of the imprinted polymer to selectively rebind several benzimidazole com- pounds. The developed methodology has been applied to the quantification of thiabendazole, carbendazim, and benomyl in river, tap, and well water samples within a single analytical run at concentration levels below the legislated maximum concentration levels. In this sense, detection limits of 2.3– 5.7 ng·L -1 have been obtained for the analysis of benzimid- azole fungicides in different water matrices. Recoveries obtained for the determination of benzimidazole fungicides in spiked samples ranged from 87% to 95%, with RSD below 5% in all cases. Keywords Benzimidazole fungicides . Molecularly imprinted polymers . On-line SPE . Ion-pair HPLC . Water samples Introduction Benzimidazole compounds are widely used both as anthel- mintic drugs in the treatment and prevention of parasitic infections and as fungicides to prevent crop spoilage. Although many public benefits have been realized by the use of benzimidazole compounds, their potential impact in both the environment and public health cannot be disregarded. Even if acute toxic effects of benzimidazole compounds are scarce due to their high lethal dose 50 values, several toxic effects have been associated to a chronic exposure to benzimidazole compounds, such as teratogenicity, congenic malformations, polyploidy, diarrhea, anemia, pulmonary edemas, or necrotic lymphoadenopathy [1]. In order to control the amount of benzimidazole deriva- tives that can be used under certain circumstances and the maximum amounts that can be tolerated in drinking water or food, legislation is under continuous revision by environ- mental protection agencies worldwide, so that exposure to benzimidazole compounds can be minimized as much as possible. In this sense, the European Water Framework Directive has established a maximum concentration level (MCL) of 0.1 μgL -1 for most benzimidazole compounds present in natural waters and a total concentration of all pesticides of 0.5 μgL -1 [2]. A large amount of effort has been invested in the past few decades to develop and validate analytical methodol- ogies able to quantify benzimidazole compounds and their metabolites in water samples at concentration levels below the legislated MCLs. An enrichment step of the water samples is usually required in order to diminish the detection limits of the analytical method and, thus, enable the quantification of benzimidazole compounds at concen- tration levels below the MCL. Additionally, as most detection systems are nonselective, a clean-up of the samples prior to Anal Bioanal Chem (2009) 393:1745–1753 DOI 10.1007/s00216-009-2631-1 O. Zamora : L. E. Vera-Avila Analytical Chemistry Department, Faculty of Chemistry, Universidad Nacional Autónoma de México, 04510 México DF, Mexico E. E. Paniagua : C. Cacho : C. Perez-Conde (*) Analytical Chemistry Department, Faculty of Chemistry, Universidad Complutense de Madrid, 28040 Madrid, Spain e-mail: cpconde@quim.ucm.es