J. Sep. Sci. 2013, 36, 2593–2601 2593 Alma M. Hern ´ andez 1 Jos ´ e Bernal 1 Jos ´ e L. Bernal 1 Mar´ ıa T. Mart´ ın 1 Constantino Caminero 2 Mar´ ıa J. Nozal 1 1 I.U. CINQUIMA, Analytical Chemistry Group, University of Valladolid, Valladolid, Spain 2 Instituto Tecnol ´ ogico Agrario de Castilla y Le ´ on (ITACyL), Valladolid, Spain Received March 21, 2013 Revised May 20, 2013 Accepted May 24, 2013 Research Article Simultaneous determination of nine anticoagulant rodenticides in soil and water by LC–ESI-MS A new and sensitive analytical method is presented to determine nine anticoagulant ro- denticide (chlorophacinone, bromadiolone, pindone, diphacinone, warfarin, coumatetralyl, brodifacoum, floucomafen, and difenacoum) residues in water and soil samples by LC– ESI-MS. Rodenticides were extracted from soil using a methanol and ammonium formate 30 mM mixture, while ethyl acetate was employed in the water samples. A Gemini 5 mC 18 column was employed, and a mobile phase comprising a mixture of ammonium formate 30 mM and di-n-butylamine 30 mM in water (pH 3.5), ammonium formate 30 mM and di-n-butylamine 20 mM in water (pH 4.4), ammonium formate 30 mM in water (pH 6.5), and methanol in a gradient elution mode was selected. The method was fully validated and it was found to be selective and precise in terms of linearity and accuracy. Extraction recoveries ranged from 90 to 104% for the compounds studied, while the detection and quantification limits were between 0.09 and 2.2 g/kg in soil or 0.08 and 1.7 g/L in water. The method was applied to simultaneously measure these compounds in water and soil samples. Keywords: Anticoagulant rodenticides / Liquid chromatography / Mass spec- trometry / Soil / Water DOI 10.1002/jssc.201300310  Additional supporting information may be found in the online version of this article at the publisher’s web-site 1 Introduction The selection of a treatment based on anticoagulant roden- ticides against the country vole (Microtus arvalis) plague in Castilla y Le´ on came about as a result of the appearance in this region of cyclic plagues of this rodent. The last plague was so intense that it was necessary, as a last resort, to apply ex- tensive treatment with chlorophacinone (CP) and afterwards with bromadiolone (BD). Moreover, it was observed that cer- tain groups were able to resist several times the lethal dose. This implies the existence of resistance phenomena, making it appropriate to search for other compounds as alternative treatments. Correspondence: Dr. Jos ´ e Bernal, I.U. CINQUIMA, Analytical Chemistry Group, University of Valladolid, Paseo de Bel´ en 5, Val- ladolid 47011, Spain E-mail: jose.bernal@qa.uva.es Fax: +34-983-186347 Abbreviations: BD, bromadiolone; BF, brodifacoum; CP, chlorophacinone; CT, coumatetralyl; DAD, diode array de- tection; DBA, di-n-butylamine; DF, difenacoum; DP, diphaci- none; FC, flocoumafen; FLD, fluorescence detection; ITACyL, Instituto Tecnol ´ ogico Agrario de Castilla y Le ´ on; PD, pindone; QC, quality control; W, warfarin Several analytical methods involving flurorimetric, spec- trophotometric, and thin layer or ion chromatography [1–4] have been used to determine anticoagulant rodenticides, but due to their lack of specificity and sensitivity they are not suitable for determining the expected low-level residues that would be present in water and soil samples [5]. CE [6] and GC [7] were employed in some cases, but these procedures were usually lengthy and more complicated, and for these reasons were not frequently employed. However, the most common separation technique used to determine the com- pounds is LC in reverse mode with UV or diode array detec- tion (DAD) [5, 8–10], fluorescence detection (FLD) [11–16] or both [17–20], and more recently, LC coupled to MS [21–24]. Furthermore, it should be mentioned that chiral stationary phases have also been employed to perform the LC analy- sis of CP [25] and warfarin (W) [26], while in some cases monolithic stationary phases have been used to analyze W by liquid [27] and affinity [28] chromatography. To date, scant sci- entific literature can be found related to the determination of rodenticides in water and soil samples [4,5,8,10,13–15,19,20]; in such studies, LC has been used with UV or DAD [4,5,8,10], FLD [13–15] or FLD and DAD [19, 20]. Surprisingly, no LC–MS method has been encountered to determine antico- agulant rodenticides in water or soil. Different solvents were used to perform the extrac- tion of these compounds from soil and water. Acetone, C  2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com