Optimization of the liquid–liquid extraction method and low temperature purification (LLE–LTP) for pesticide residue analysis in honey samples by gas chromatography

Abstract

This work optimized a simple and practical method for identification and quantification of the pesticides chlorpyrifos, λ-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 μg g⁻¹ with correlation coefficients above 0.99. Detection and quantification limits were lower than 0.016 and 0.032 μg g⁻¹, respectively. The proposed method was applied to 11 honey samples. Chlorpyrifos and λ-cyhalothrin residues were found in two samples at concentrations below maximum residue limit (MRL) established for food products. The presence of these compounds was confirmed by mass spectrometry in SIM mode (GC–MS-SIM).

Introduction

Honey is traditionally consumed by humans for being considered 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 and Faucon, 2006, Pirard et al., 2007, Rissato et al., 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 pesticides are amitraz, cymiazole, bromopropylate, coumafos, flumethrin, fluvalinate, imidacloprid and fipronil (Korta et al., 2001, Rial-Otero et al., 2007). Although the regulatory agencies of several countries have established the maximum residue limit (MRL) for some of these pesticides in honey samples, these limits are not included in the Codex Alimentarius (1998).

Indirect honey contamination can occur during pesticide application in agriculture. Pesticide application in crops can contaminate 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 pollution (Celli and 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 collaborators detected malathion residues in all honey samples analyzed in the region of Bauru (São Paulo, Brazil). Presence of residues of these compounds in the samples was attributed to pesticide application for dengue vector control in the area.

Analysis of pesticide residues in complex matrices consists of four steps: extraction, extract cleaning, identification and quantification of compounds. Among the extraction methods commonly used in honey analysis, are solid phase extraction (Albero et al., 2004, Blasco et al., 2003), supercritical fluid extraction (Rissato, Galhiane, Knoll, & Apon, 2004), conventional 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 chromatography (Fernandez et al., 2002, Rossi et al., 2001). The steps of identification and quantification of pesticide residues are based on gas chromatography (GC) and high performance liquid chromatography (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 extraction in water (Vieira, Neves, & Queiroz, 2007) and milk (Goulart, Queiroz, & Neves, 2008). The method is based on the partition of analytes between the aqueous and organic phase resulted from temperature lowering (−20 °C). The advantage of this method is that the sample components are frozen with the aqueous phase, whereas pesticides are extracted by the organic phase.

This work aimed to optimize and validate a method using liquid–liquid extraction with low temperature purification for pesticide residue analysis in honey by gas chromatography. The method was applied for the determination of chlorpyrifos, λ-cyhalothrin, cypermethrin and deltamethrin which are often detected in monitoring studies of food samples. The insecticide chlorpyrifos is one of the most widely used crop protection products in the world, while the pyrethroids are insecticides included in over 3500 registered products, many of which are used in agriculture (Environmental Protection Agency, 2009).