Optimization of the liquid–liquid extraction method and low temperature purification (LLE–LTP) for pesticide residue analysis in honey samples by gas chromatography Gevany Paulino de Pinho a, * , Antônio Augusto Neves b , Maria Eliana Lopes Ribeiro de Queiroz b , Flaviano Oliveira Silvério a a Institute of Agricultural Sciences, Federal University of Minas Gerais, 39404-006 Montes Claros, Brazil b Department of Chemistry, Federal University of Viçosa, 36570-000 Viçosa, Brazil article info Article history: Received 31 July 2009 Received in revised form 15 March 2010 Accepted 23 March 2010 Keywords: Extraction method Honey Pesticide Gas chromatography abstract This work optimized a simple and practical method for identification and quantification of the pesticides chlorpyrifos, k-cyhalothrin, cypermethrin and deltamethrin in honey samples. The method was based on liquid–liquid extraction and low temperature purification using acetonitrile: ethyl acetate (6.5 mL:1.5 mL) as the solvent for extraction. A final clean up step with 2 g florisil was performed before analysis by gas chromatography using electron-capture-detector. The technique was proven satisfactory with efficiency exceeding 85% and linear chromatographic response for the tested pesticides, ranging from 0.033 to 1.7 lgg À1 with correlation coefficients above 0.99. Detection and quantification limits were lower than 0.016 and 0.032 lgg À1 , respectively. The proposed method was applied to 11 honey samples. Chlorpyrifos and k-cyhalothrin residues were found in two samples at concentrations below maximum residue limit (MRL) established for food products. The presence of these compounds was con- firmed by mass spectrometry in SIM mode (GC–MS-SIM). Ó 2010 Published by Elsevier Ltd. 1. Introduction Honey is traditionally consumed by humans for being consid- ered a product of natural origin and healthy. Honey must therefore remain free of any chemical or biological contaminant to be safe for human consumption. However, some studies have reported the presence of pesticide residues in honey samples (Kadar & Faucon, 2006; Pirard et al., 2007; Rissato, Galhiane, Almeida, Gerenutti, & Apon, 2007). These residues may originate from the treatment of bee hives with acaricides in the control of Varroa jacobsonie and Ascosphera apis. The most commonly used pesti- cides are amitraz, cymiazole, bromopropylate, coumafos, flumeth- rin, fluvalinate, imidacloprid and fipronil (Korta, Bakkali, Berrueta, Gallo, & Vicente, 2001; Rial-Otero, Gaspar, Moura, & Capelo, 2007). Although the regulatory agencies of several countries have estab- lished the maximum residue limit (MRL) for some of these pesti- cides in honey samples, these limits are not included in the Codex Alimentarius (1998). Indirect honey contamination can occur during pesticide appli- cation in agriculture. Pesticide application in crops can contami- nate soil, air, water, and the flowers from which bees collect nectar for honey production (Kujawski & Namiesnik, 2008). Bees and honey may serve as indicators of environmental pollu- tion (Celli & Maccagnani, 2003; Kevan, 1999). High concentrations of pesticide residues lead to high mortality rate of bees, and the honey produced is unfit for human consumption. Rissato and col- laborators detected malathion residues in all honey samples ana- lyzed in the region of Bauru (São Paulo, Brazil). Presence of residues of these compounds in the samples was attributed to pes- ticide application for dengue vector control in the area. Analysis of pesticide residues in complex matrices consists of four steps: extraction, extract cleaning, identification and quantifi- cation of compounds. Among the extraction methods commonly used in honey analysis, are solid phase extraction (Albero, Sánchez-Brunete, & Tadeo, 2004; Blasco et al., 2003), supercritical fluid extraction (Rissato, Galhiane, Knoll, & Apon, 2004), conven- tional liquid–liquid extraction (Blasco et al., 2004, matrix solid phase dispersion (Fernández, Pico, & Manes, 2002) and solid phase microextraction (Campillo, Penalver, Aguinaga, & Hernandez- Cordoba, 2006). The clean up stage is based on techniques such as gel permeation chromatography and adsorption chromatogra- phy (Fernandez, Pico, & Manes, 2002; Rossi et al., 2001). The steps of identification and quantification of pesticide residues are based on gas chromatography (GC) and high performance liquid chroma- tography (HPLC) (Van der Hoff & Van Zoonen, 1999). More recently, liquid–liquid extraction and low temperature purification (LLE–LTP) has emerged as an alternative for pesticide 0956-7135/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.foodcont.2010.03.006 * Corresponding author. Tel.: +55 38 2101 7770. E-mail address: gevanypp@yahoo.com.br (G.P. de Pinho). Food Control 21 (2010) 1307–1311 Contents lists available at ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont