Simultaneous Determination of Terbuthylazine and Its Major Hydroxy and Dealkylated Metabolites in Wetland Water Samples Using Solid-Phase Extraction and High-Performance Liquid Chromatography with Diode-Array Detection NIKOLAOS PAPADOPOULOS, †,‡ EVAGELOS GIKAS, † GEORGIOS ZALIDIS, ‡ AND ANTHONY TSARBOPOULOS* ,†,§ Bioanalytical Laboratory, GAIA Research Center, The Goulandris Natural History Museum, Othonos 100 Street, 145 62 Kifissia, Greece, Laboratory of Applied Soil Science, School of Agriculture, Aristotle University of Thessaloniki, 540 06 Thessaloniki, Greece, and Laboratory of Instrumental Pharmaceutical Analysis, Department of Pharmacy, University of Patras, 265 04 Patras, Greece A method based on high-performance liquid chromatography with diode-array detection was developed and validated aiming at the simultaneous determination of terbuthylazine (TER) and its five major metabolites, desisopropyl-hydroxy-atrazine, desethyl-hydroxy-terbuthylazine, desisopropyl-atrazine, hydroxy-terbuthylazine, and desethyl-terbuthylazine. Although s-triazines are used worldwide as herbicides for agricultural and nonagricultural purposes, there is limited information on the environmental impact of TER degradation products. The proposed method includes a solid-phase extraction procedure (using MCX cartridges) with adequate recovery efficiency (70-80%). The statistical evaluation of the method reveals good linearity, accuracy, and precision for the compounds determined, with RSD values less than 14.6%, while the detection limit was found to be 0.05 μgL -1 for DIHA and 0.01 μgL -1 for the other substances. This method can be employed in biodegradation studies of TER and its metabolites in water samples from constructed wetlands, thus assisting the evaluation of their environmental impact. 1. INTRODUCTION s-Triazines are used worldwide as selective pre- and post- emergence herbicides for the control of both grasses and broadleaf weeds in many agricultural crops like corn, wheat, maize, barley, sorghum, grape, peaches, apple, and asparagus as well as for nonagricultural purposes such as soil sterilization and road maintenance (1). During and after the herbicide application to the farming land, triazines may be transported to both ground and surface water and also into the atmosphere (2-4). Atrazine (AT) is the most commonly used and the main representative of s-triazines (5). Due to environmental pollution, the commercial use of atrazine has been forbidden in the European Union (6) and has been gradually replaced by terbuthylazine (TER) (7, 8). TER has lower water solubility (8.5 vs 33.0 mg L -1 (pH 7, 20 °C)) than atrazine and stronger soil sorption (9). However no methodology exists for routine analysis of TER together with its degradation products. As a result there has been limited information on the environmental impacts of TER degradation products. In water and soil the parent TER molecule is subjected to various biotic and abiotic degradation processes such as pho- tolysis, oxidation, hydrolysis, and biodegradation, leading to dealkylation of the amine groups, dechlorination, and subsequent hydroxylation (10-15). The main degradation products in ground and surface waters via biotic mechanism are the dealkylated chloro metabolites, such as deethyl-terbuthylazine (DET) and deisopropyl-atrazine (DIA) (16, 17). Hydroxy- terbuthylazine (HT) is the major abiotic degradation product in water and soil. Other major metabolites of TER are deethyl- hydroxy-terbuthylazine (DEHT) and deisopropyl-hydroxyatra- zine (DIHA) (10) as shown in Figure 1. To date, many analytical methods have been developed aiming at the determination of TER. As AT shares common metabolites with TER, several analytical methods have also been proposed for their determination. A method for the simultaneous determination of AT and its dealkylated products in agricultural soil and in associated surface and groundwater has been reported employing a high-performance liquid chromatography-ultraviolet (HPLC-UV) system and a solid-phase extraction (SPE) pre- treatment step using cyclohexyl Bond elut cartridges (18). In another method, the determination of the dechlorinated products has also been realized using an LC-MS system (19). Nonpolar SPE sorbents are generally selected for extracting triazines from water samples such as the parent molecule of * Corresponding author phone: +30 210 6233255; fax: +30 210 8087084; e-mail: atsarbop@gnhm.gr, atsarbop@upatras.gr. Corresponding author address: GAIA Research Center, The Goulandris Natural History Museum, 13 Levidou Steet, Kifissia 145 62, Greece. † The Goulandris Natural History Museum. ‡ Aristotle University of Thessaloniki. § University of Patras. 7270 J. Agric. Food Chem. 2007, 55, 7270-7277 10.1021/jf0706777 CCC: $37.00 © 2007 American Chemical Society Published on Web 08/03/2007