CORE Metadata, citation and similar papers at core.ac.uk Provided by UGD Academic Repository American Journal of Applie d Chemistry 2014; 2(4): 46-54 Published online August 10, 2014 (http://www.sciencepublishinggroup.com/j/ajac) doi: 10.11648/j.ajac.20140204.11 ISSN: 2330-8753 (Print); ISSN: 2330-8745 (Online) Development and validation of a method for the simultaneous determination of 20 organophosphorus pesticide residues in corn by accelerated solvent extraction and gas chromatography with nitrogen phosphorus detection Vesna Kostik *, Biljana Gjorgeska, Bistra Angelovska Medical Faculty, Department of Pharmacy, University “Goce Delchev”, Shtip, Republic of Macedonia Email address: [email protected] (V. Kostik), [email protected] (B. Gjorgeska), [email protected] (B. Angelovska) To cite this article: Vesna Kostik, Biljana Gjorgeska, Bistra Angelovska. Development and Validation of a Method for the Simultaneous Determination of 20 Organophosphorus Pesticide Residues in Corn by Accelerated Solvent Extraction and Gas Chromatography with Nitrogen Phosphorus Detection. American Journal of Applied Chemistry. Vol. 2, No. 4, 2014, pp. 46-54. doi: 10.11648/j.ajac.20140204.11 Abstract: The method for simultaneous determination of 20 organophosphorus pesticide residues in corn samples has been developed and validated. For the extraction of organophosporus pesticide residues from the samples, the accelerated solvent technique with the mixture of dichloromethane: acetone (1:1, V/V ) was used. Clean up was done using liquid – liquid extraction with n – hexane, followed by solid phase extraction on primary secondary amine adsorbent, and elution with the mixture of acetone: toluene (65:35). The determination of the pesticides was carried out by gas chromatography with nitrogen phosphorus detection. Separation and quantitative determination of the analytes were performed on a fused silica capillary ZB-35 column (30 m x 0.25 mm i.d. x 0.25 µm, Phenomenex). The recovery was investigated in blank corn samples fortified with mevinphos, diazinon, dimethoate, bromofos-methyl, chlorfenvinphos, fenamiphos, ethion and phosalone at 5 ng/g, 10 ng/g, 15 ng/g , 20 ng/g and 25 ng/g, respectively and with methacrifos, phorate, etrimfos, parathion-methyl, pirimiphos - methyl, fenitrothion, chlorpyrifos, malathion, parathion, bromofos-ethyl, phosmet and azinphos-methyl at 10 ng/g, 20 ng/g, 30 ng/g, 40 ng/g and 50 ng/g, respectively. The recovery ranged from 76.0% to 112.0%. Repeatability expressed as relative standard deviation (RSD) was less than 8.2%. Linearity expressed as correlation coefficient (R 2) ranged from 0.9935 to 0.9996. Measurement uncertainty (Ux) was lower than 14.2% for all tested pesticides. The limits of quantification (LOQ) were bellow 5 ng/g for all tested pesticides. The satisfactory Z-score results of international proficiency tests confirm good analytical performances of the developed method. Keywords: Organophosporus Pesticide Residues, Gas Chromatography, Accelerated Solvent Extraction, Solid Phase Extraction 1. Introduction Most crops are treated with pesticides on several control pests. A particular insecticide targets specific insects occasions during the growing season. Pesticides enable [2]. Fungicides are applied to control a considerable number farmers to produce some crops in areas that otherwise would of diseases caused by Venturia inaequalis , Un ci nul a neca tor, not be suitable, increase their yields, preserve product Sclerotinia blight , Botrytis cinerea etc [3]. At the same time, quality, and extend shelf life [1]. Without pesticides, pesticides can pose risks if they are not applied according to commercial crop’s production would not be economically Good Agricultural Practice (GAP). Pesticide levels tend to viable in many regions of the world. Insecticides and decline over time as the residues in crops fungicides are likely to remain the major class of pesticides degrade/metabolize during the growing period and used for crop’s protection [2, 3]. Insecticides are used to following harvest if they are washed and processed before 47 Vesna Kostik et al. : Development and Validation of a Method for the Simultaneous Determination of 20 Organophosphorus Pesticide Residues in Corn by Accelerated Solvent Extraction and Gas Chromatography with Nitrogen Phosphorus Detection reaching the markets. M any auth ors hav e al so s hown t ha t if ethion; fenamiphos; fenitrothion; malathion; parathion; pest ici des are applied in accordance with GAP then the parathion-methyl; pirimiphos-methyl; phorate; phosalone; Maximum Residue Levels (MRLs) will not be exceeded. [4, phosmet in corn including etrimfos, mevinphos and 5] So, levels are usually well below legal limits by the time methacrifos, which have not appeared in recent reports. food reaches the retail outlets. Extraction of the samples was performed with a low A large number of pesticides are used on corn, which in volume of organic solvent using ASE technique. N – the Republic of Macedonia is grown throughout the country hexane was used for purification of the extracts, followed both for human consumption and animal feed [6]. by SPE on the primary secondary amine adsorbent Conventionally grown corn requires many applications of (PSA). The OPPs in corn samples were determined by GC insect-killers, weed-killers, plant growth regulators and – NPD. other types of pesticides [7]. Corn is treated with organophosphorus pesticides (OPPs) in pre harvest interval, while corn grains are treated in post harvest period for 2. Experimental prevention the infestation with insects of stored 2.1. Instrumentation commodities. The use of OPPs as post harvest pesticides is mainly due to the lower rate of degradation under the storage The sample extraction was performed in a DIONEX conditions that exist in storehouses.The post-harvest Accelerated Solvent extractor, ASE-100 (USA). Stainless pesticides have been attracting much attention because their steel extraction cells (10 mL) were used for the extraction. residues in stored cereal grains may be hazardous to human Helium (purity 99.999 %) was used as a purge gas. health [8]. The presence of OPPs residues in corn is still The chromatographic analysis was performed on a reported [9, 10]. The monitoring of pesticide residues in Shimadzu 2010 Gas Chromatography system (GC) system corn and corn products is of a significant importance in equipped with nitrogen-phosphorus detector (NPD), and terms of consumer safety. auto injector (AOC- 20i), and the ChromSolution software. To protect the health of consumers and to eliminate unsafe Chromatographic separation was achieved on a fused silica food, as well as to comply with the requirements of ZB-35 capillary column (30 m x 0.25 mm i.d. x 0.25 µm film international food trade, MRLs of pesticide in food were laid thickness), supplied by Phenomenex (Torrance, USA). down in Regulation (EC) 396/2005 [11]. For the majority of Operating conditions were as follows: injector port the studied pesticides, MRLs in corn have been established temperature, 250 0C; injection volume, 2 µL in splitless at the analytical limit of determination (between 10 and 50 mode (constant pressure 110.8 kPa; total flow 10.2 mL/min); µg/kg). detector temperature 280 0C (make up gas - helium flow Due to the complex nature of cereals and the presence of 27.5 mL/min; hydrogen flow 1.5 mL/min; air flow 145 lipids, fiber-carbohydrates (hemicellulose, cellulose and mL/min; current 3 pA); helium as carrier gas at a flow rate of lignin), non fiber carbohydrates (starch, sucrose and reducing 1.2 mL/min; oven temperature programme, 90 0C (1 min), sugars) and proteins, the selection of the appropriate increased with the rate of 25 0C/min to 200 0C, held for 15 technique of extraction, concentration and purification of the min, then increased to 220 0C with the rate of 10 0C min, and pesticides, and its optimization is the most laborious, but very held for 25 min. The total run time of the chromatographic important aspect of the analysis [12]. A lot of different analysis was 47.40 min. The column equilibration time was extraction and cleanup methods including multiple solvent 3 min. extractions, supercritical fluid extraction (SFE), accelerated solvent extraction (ASE), gel permeation chromatography 2.2. Reagents and Chemicals (GPC), solid phase extraction (SPE) were used in the All chemicals and solvents were a special grade for pesticide determination of pesticides in cereals but there is no universal residue analysis. Acetone, dichloromethane (DCM), n-hexane, technique, which would be entirely better than the others in ethyl acetate, acetonitrile (ACN) and toluene were obtained terms of all analytical scopes [13-20]. Many of the traditional from Merck (Darmstadt, Germany). Anhydrous sodium procedures used to perform the extractions for these analyses sulphate, (prepared 3 hours at 650 0C), sodium chloride and are time consuming and solvent intensive [13, 17]. PSA /SPE cartridges (PSA, SPE 500 mg/ 6 mL tubes) were Organophosphorus pesticides are usually determined by purchased from Sigma-Aldrich/Fluka/Riedel-de-Haen gas chromatography (GC) with flame photometric detection (Zwijndrecht, The Netherlands). Water was deionized then (FPD) [13, 18], nitrogen-phosphorus detection (NPD) [14, distilled from glass apparatus. 15, 21], mass spectrometry detection (MS) [16, 17, 21-25], The analytical standards of azinphos-methyl (97.3 % ± 2), and liquid chromatography coupled with tandem mass bromofos-methyl (98.5 % ± 2), bromofos-ethyl