Australian Journal of Basic and Applied Sciences. GC-FPD/NPD Method Development for Pesticide Multi-Residues Determination in Fresh Matrices

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1 AENSI Journals Australian Journal of Basic and Applied Sciences ISSN: Journal home page: GC-FPD/NPD Method Development for Pesticide Multi-Residues Determination in Fresh Matrices 1, 2 Sherif B. Abdel Ghani 1 Plant Protection Department, Faculty of Agriculture, Ain Shams University,P.O. Box 68 Hadayek Shoubra11241 Cairo, Egypt 2 Plant Production and Protection Department, Faculty of Agriculture and veterinary medicine, Qassim University, P.O. Box 6622 Buraydah, Al-Qassim, Saudi Arabia A R T I C L E I N F O Article history: Received 2 February 2014 Received in revised form 8 April 2014 Accepted 28 April 2014 Available online 25 May 2014 Keywords: GC-FPD, Method validation, Organophosphorus pesticides, Fresh vegetables A B S T R A C T Background: Many common insecticides used in fruit and vegetable farms belong to organophosphorus (OP) group of pesticides. They are mainly employed for insects, nematodes, and mites' control. Therefore, Eight organophosphorus pesticides were selected to be investigated, i.e., ethion, malathion, diazinon, chlorpyrifos, chlorpyrifosmethyl, cadusafos, profenofos and phenthoate. These pesticides are extensively used in fruits and vegetables. Grapes (high sugar content and acidic matrix) and green beans (high chlorophyll content matrix) were selected for the development and validation of the method. Objective: to develop and validate a rapid, specific and sensitive multiresidue-analytical method for the routine analysis of widely used organophosphorus pesticide residues in fresh grapes and green beans as representative materials for fresh products. Results: In green beans, accuracy data ranged from 87.9 % to 96.9 % at 0.01 mg/kg level with precision (RSD) of 9.9 as maximum while at the 0.05 mg/kg level recoveries ranged from to % and (RSD) of 5.7 % or less. In case of grapes, accuracy results ranged from 93.6 to 117.3% and from to 109.2% for 0.01 and 0.05 mg/kg levels, respectively while repeatability ranged from 5.5 to 11.9 % at 0.01 mg/kg level and from 4.2 to 5.0 % at 0.05 mg/kg level. Conclusion: A multi-residues analytical method for eight compounds in grapes and green beans was developed and validated using GC-FPD/NPD according to European Commission guidelines. Baseline separation (separation factor 1.5) was achieved for most of the tested pesticides. Matrix matched standards were prepared using pesticide free blank extract of both matrices as well as, interfering peaks. The presented method showed acceptable accuracy results that varied between 87.9 and % and precision (RSD) not higher than 11.9% in both matrices. The obtained results meet the EU standard requirements and legislations to consider a method accurate and reproducible AENSI Publisher All rights reserved. To Cite This Article: Sherif B. Abdel Ghani., GC-FPD/NPD Method Development for Pesticide Multi- residues Determination in Fresh Matrices. Aust. J. Basic & Appl. Sci., 8(7): , 2014 INTRODUCTION Pesticides have played a very important role in the development of agriculture since their invention. This role is still irreplaceable. Although pesticides found their way in wide applications and have played a significant part in boosting agricultural production, the hazards they have brought along with them to food safety and human health have increasingly become the focus of the world concern, (Pang et al., 2006). Several countries, the European Union and the Codex Alimentarius have established Maximum Residue Limits (MRLs), based on the assumption that good agricultural practice is applied at the use of pesticides in farming, for pesticide residues in foodstuff, European Commission Directive 90/642/EEC (1990) and its amendments. The QuEChERS method for pesticide residue analysis was first introduced by USDA (US Department of Agriculture) scientists in 2003, (Anastassiades and Lehotay, 2003). The method was modified to overcome some problematic pesticides analysis by including a buffer extraction system, resulting in the official methods, the AOAC , (Lehotay, 2007) and the European version EN 15662, (Payá et al., 2007). The method is a three steps process: extraction, dispersive SPE and analysis. In the first step, acetonitrile is added to the comminuted sample followed by salting out the water from the sample using anhydrous magnesium sulphate (MgSO 4 ), NaCl and buffering citrate salts to induce extraction/partitioning. After centrifugation, an aliquot of the extract is taken to the next step. The second step is a dispersive solid phase extraction SPE to minimize matrix effects using a combination of anhydrous MgSO 4 to eliminate the remaining water from the extract and Primary Secondary Amine modified silica (PSA) to remove polar impurities. Graphitized carbon black (GCB) Corresponding author: Sherif B. Abdel Ghani, Plant Protection Department, Faculty of Agriculture, Ain Shams University,P.O. Box 68 Hadayek Shoubra11241 Cairo, Egypt

2 242 Sherif B. Abdel Ghani, 2014 and C18 (octadecylsilane) can be added for pigment and fat removal, respectively. After mixing and centrifugation, the supernatant becomes ready for analysis. Analysis can be done using gas chromatography and/or liquid chromatography. Many common insecticides used in fruit and vegetable farms belong to organophosphorus (OP) group of pesticides. They are mainly employed for insects, nematodes, and mites control. Therefore, Eight organophosphorus pesticides were selected to be investigated, i.e., ethion, malathion, diazinon, chlorpyrifos, chlorpyrifos-methyl, cadusafos, profenofos and phenthoate. These pesticides are extensively used in fruits and vegetables. Grapes (Thomson) (high sugar content and acidic matrix) and green beans (Hama) (high chlorophyll content matrix) were selected as representative commodities for the development and validation of the method. In this study, the author developed a QuEChERS based method as a sample preparation process. Analysis was performed by selective GC FPD/NPD in multi-residue analysis fashion. Validation of the method was achieved according to the European guidelines. MATERIALS AND METHODS 1- Chemicals and Reagents: Pesticides standards (cadusafos, chlorpyrifos-methyl, chlorpyrifos, diazinon, ethion, malathion, phenthoate and profenofos) were purchased from Sigma Aldrich, Germany. Anhydrous magnesium sulfate was purchased from Merck, Germany. PSA was purchased from Supelco, UK. Glacial acetic acid was purchased from Merck. All organic solvents used in the study were pesticide grade or HPLC grade and purchased from Merck, Germany. 2- Standard Preparation: Individual stock solutions were prepared in toluene (Mastovska and Lehotay, 2004) at 1.0 mgml -1. A standard mixture solution, with all 8 pesticides was prepared at 1.0 µgml -1 in acetonitrile containing 0.1 % acetic acid to prevent degradation of the pesticides, (Hiemstra and Kok, 2007). All standard solutions were stored in the dark at -20 o C. Matrix-matched multi-standard solutions were prepared at 0.01 and 0.05 ppm using blank extracts of green beans or grapes. 3- Extraction Procedure: The green beans and grape samples were extracted using modified QuEChERS method (Anastassiades and Lehotay, 2003; CEN/TC 275, N 236, 2006). The method is summarized as follows (1) chop and homogenize 500 g of fresh sample; (2) weigh 10 g of the previously homogenized sample into 50 ml PTFE centrifuge tube; (3) add 10 ml of acetonitrile (1% acetic acid) and shake the sample vigorously for 1 min; (4) add 4 g magnesium sulfate anhydrous (MgSO 4 ) and 1 g sodium chloride (NaCl), and shake immediately for 1 min; (5) centrifuge the tube for 3 min at 5000 rpm; (6) transfer a 1 ml aliquot of the upper layer into a DSPE centrifuge tube (7) centrifuge the tube for 3 min at 5000 rpm; (8) filter through 0.45 micron filter; (9) transfer 0.7 ml of the extract into an PTFE capped autosampler vial. 4- Instrument: An Agilent 7890A gas chromatograph was used to conduct this study. The chromatograph is equipped with Flame Photometric Detector (FPD) for quantification and Nitrogen Phosphorus Detector (NPD) for confirmation. The system is provided with a Programmable Temperature Vaporization (PTV) injector. Two fused silica capillary columns HP-5, length 30 m, 0.25 mm i.d., film thickness 0.25 μm were used; each was attached to a different detector. Temperature programme consisted of 1 min. hold at 100 C, rise at 25 C/min. to 170 C, hold for 1 min., rise at 3 C/min. to 230 C, then hold for 1 min., rise at 8 o C/min. to 300 o C, then isothermal for 2min. Injector temperature program 70 C for 0.5 min. then rise to 380 C at 300 C /min. Carrier gas nitrogen, 2 ml/min. Detector FPD with phosphorus filters, temperature 200 C and NPD, temperature 320 C. FPD gases Nitrogen, 60 ml/min. hydrogen, 100 ml/min air, 100 ml/min. NPD gases, nitrogen, 8 ml/min hydrogen, 2.5 ml/min. air, 60 ml/min. Injection volume was 5 µl (splitless). 5- Method Validation: The validation of the analytical method was carried out according to the European guidelines, (European Commission Document SANCO/10684/2009). The method was performed to assess precision and accuracy, limits of quantification, and repeatability. Validation studies were carried out by spiking blank samples of green beans and grape with multi standards mixture at levels of 0.01and 0.05 mg/kg. The spiked samples were analyzed in six replicates. The accuracy of the method was estimated by means of recovery experiments at two concentration levels while repeatability of the method was evaluated through the Relative Standard Deviation (RSD). The Limits Of Quantification (LOQs) were calculated according to Fajgelj and Ambrus (2000). The LOQ was established as the lowest concentration assayed, which gave satisfactory recovery (80 120%) and

3 243 Sherif B. Abdel Ghani, 2014 precision ( 20% RSD). For this purpose, six independent replicates of green beans and grape blank samples were spiked with pesticides at a level of 0.01 mg/kg and analyzed against matrix matched reference Linearity and LOQ: The evaluation of the calibration curve s linearity was done based on injections of the matrix matched standard solutions of each matrix at the concentrations 0.005, 0.01, 0.05 and 0.1 µgml -1. Calculations were performed on the average peak areas. Relative standard deviations (RSDs) and calibration curve equations, also the determination coefficients (r 2 ) were determined for each pesticide Accuracy and precision: Accuracy and precision of the method were determined via performing recovery studies at two levels i.e and 0.05 mgkg -1. Pesticide free samples of green beans and grape samples were used as blank control matrices in recovery experiments and for the preparation of matrix-matched multi-level calibration solutions. The blank samples were previously analyzed to assure they are free of the tested pesticides (Fig. 1). 10 g sample (six replicates) was fortified with the multi-standard solution in acetonitrile to give the required concentration and left to stand for 30 min before extraction to allow pesticides absorption onto the matrix. Samples were extracted using the mentioned procedure. Matrix matched external standard method was employed for the residues quantification Matrix matched standard: 10 g blank sample of green beans or grapes was extracted and clean-up was performed according to the method. Multi-standard solution was added prior the GC determination step to give the required final concentration. RESULTS AND DISCUSSION 1- Pesticides Selection: Eight organophosphorus pesticides were selected to be investigated in the present study. The list of the analyzed pesticides comprises quite distinct physico-chemical characteristics (Table 1). The molecular weights ranged from (cadusafos) to (ethion). While the polarity (expressed as K ow ) varied from relative polar (malathion) to moderate polar (e.g. diazinon and phenthoate) and non polar compounds (e.g. chlorpyrifosmethyl and chlorpyrifos. Concerning the WHO acute toxicity classification, the tested pesticides varied from highly hazardous (cadusafos); moderately hazardous (e.g. Diazinon, chlorpyrifos and profenofos); slightly hazardous (malathion) and unlikely to present acute hazard in normal use (chlorpyrifos-methyl), (Tomlin, ). However, the selected pesticides have different bioactivity against certain pest including one nematicide (cadusafos), one acaricide (ethion) and 6 insecticides. Table 1: Analyzed pesticides: polarity, molecular weight, action and toxicity class. Pesticide K ow M.W Action Toxicity class WHO Cadusafos Nematicide Ib Diazinon Insecticide II Chlorpyrifos-methyl Insecticide U Malathion Insecticide III Chlorpyrifos Insecticide II Phenthoate Insecticide II Profenofos Insecticide II Ethion Acaricide II 2- Chromatographic separation, detector response range and confirmation: Baseline separation (separation factor 1.5) was achieved for most of the tested pesticides under the above mentioned chromatographic conditions. In the case of malathion and chlorpyrifos the separation factor reached 1.34 and area under peak was accurately measured using the Agilent Chemstation software in the same way for both matrix-matched standard and samples (Table 2). FPD dynamic linear range was up to 1 ppm for all the tested pesticides. Nitrogen Phosphorus Detector (NPD) was used for confirmation of the results. European directives, mentioned that selective detectors employed with GC such as, FPD and NPD, when used together, offer acceptable level of confirmation and should be acknowledged when reporting the results, (European Commission Document SANCO/10684/2009).

4 244 Sherif B. Abdel Ghani, 2014 Table 2: Analyzed pesticides: retention times, width at half peak and separation factor. Pesticide Rt min Width (1/2) min separation factor Cadusafos Diazinon Chlorpyrifos-methyl Malathion Chlorpyrifos Phenthoate Profenofos Ethion Fig. 1: Blank extract of green beans (a), and fortified extract at 0.05 mg/kg (b). 3- Method Validation: Two common types of food commodities were selected for the development of a multiresidue analysis method i.e. green bean (high chlorophyll content) and grape (high sugar content). The method is still valid for variety of vegetable and fruit matrices Linearity range and determination coefficient (r 2 ): The evaluation of the analytical curves linearity was achieved based on injections of the standard solutions prepared in blank extract at the concentrations 0.005, 0.01, 0.05 and 0.1 µgml 1. Determination coefficient (r 2 ) ranged from 0.98 to Matrix matched calibration standard: Matrix-matched standards were prepared in two levels i.e and 0.05 mg/kg (LOQ of the method and 5 times LOQ) using blank extract of green beans or grapes. The specificity of the method was tested by the analysis of blank samples. Chromatographic analysis of the blank samples showed no peaks corresponding to the tested pesticides. Thus, indicating that the used blank samples were free of the tested pesticides, and of interfering matrix compounds that would give false positive results (Fig. 1 and Fig. 2) Accuracy, precision and method LOQ: Following the European Commission guidelines, this method was validated at two different concentrations. At each concentration, six measurements were performed (n=6). Results are reported in tables 3 and 4. The method accuracy varied between 87.9 and % and precision (RSD) not higher than 11.9% in both matrices; hence the obtained results of the method meet the European Commission requirements, indicating that a method

5 245 Sherif B. Abdel Ghani, 2014 can be considered accurate and precise when accuracy data are comprised between 80 and 120% with relative standard deviations not higher than 20% Green beans: Table 3 presents recovery percent and repeatability (RSD) data for the pesticides analyzed at two different spiking levels in green beans. The recoveries ranged from 87.9 % to 96.9 % at 0.01 mg/kg level with RSD of 9.9 as maximum. At the 0.05 mg/kg level recoveries ranged from to % with repeatability RSD of 5.7 % or less Grapes: The recovery percents and repeatability of the spiking levels 0.01 and 0.05 mg/kg in grapes are shown in table 4. The obtained results showed excellent rate of recoveries of the tested pesticides in both levels. It ranged from 93.6% (profenofos) to 117.3% (diazinon) at 0.01 mg/kg level while in 0.05 mg/kg it ranged from 102.7% to 109.2% (malathion and ethion). However, the tested pesticides showed good repeatability at 0.01 mg/kg level, ranged from 5.5% (profenofos) to 11.9% (malathion) while at 0.05 mg/kg level the obtained RSD ranged from 4.2 to 5.0 %. Fig. 2: Blank extract of grape (c), and fortified extract at 0.05 mg/kg (d). Table 3: Accuracy and precision data of the analyzed pesticides in green beans (n=6). Pesticide 0.01 mg/kg 0.05 mg/kg Recovery (%) RSD Recovery (%) RSD LOQ (mg/kg) MRL (mg/kg) Cadusafos Diazinon Chlorpyrifos-methyl Malathion Chlorpyrifos Phenthoate Profenofos Ethion

6 246 Sherif B. Abdel Ghani, 2014 Table 4: Accuracy and precision data of the analyzed pesticides in grapes (n=6). Pesticide 0.01 mg/kg 0.05 mg/kg Recovery (%) RSD Recovery (%) RSD LOQ (mg/kg) MRL (mg/kg) Cadusafos Diazinon Chlorpyrifos-methyl Malathion Chlorpyrifos Phenthoate Profenofos Ethion Worth noting that, in both matrices, recoveries were more accurate at the 0.05 mg/kg than at 0.01 mg/kg level. In addition, at the 0.05 mg/kg level, RSDs were lower than at 0.01 mg/kg. However, results obtained by Oliva et al. (2000); Mol et al. (2003); Banerjee et al. (2007) and Pérez et al. (2008) are supporting the same trend obtained in the present study. Conclusion: A multi-residues analytical method for eight compounds in grapes (high sugar content) and green beans (high chlorophyll content) was developed and validated using GC-FPD/NPD according to European Commission 2009 guidelines. Baseline separation (separation factor 1.5) was achieved for most of the tested pesticides. Matrix matched standards were prepared using pesticide free blank extract of both matrices as well as, interfering peaks. The presented method showed acceptable accuracy results that varied between 87.9 and % and precision (RSD) not higher than 11.9% in both matrices. The obtained results meet the EU standard requirements and legislations to consider a method accurate and reproducible. REFERENCES Anastassiades, M. and S.J. Lehotay, Fast and Easy Multiresidue Method Employing Acetonitrile Extraction/Partitioning and Dispersive Solid-Phase Extraction for the Determination of Pesticide Residues in Produce. J. AOAC Int., 86: Banerjee, K., D.P. Oulkar, S. Dasgupta, S.B. Patil, S.H. Patil, R. Savant and P.G. Adsule, Validation and uncertainty analysis of a multi-residue method for pesticides in grapes using ethyl acetate extraction and liquid chromatography tandem mass spectrometry. J. Chromatography A, 1173: CEN/TC 275, N 236, Food of plant origin Determination of pesticide residues using GC MS and/or LC-MS/(MS) following acetonitrile extraction/partitioning and cleanup by dispersive SPE QuECheRS-method. European Committee for Standardisation. European Commission Directive 90/642/EEC., fixing of maximum levels for pesticide residues in and on certain products of plant origin, including fruit and vegetables. Off. J. Eur. Commun. No. L350/71. Fajgelj A. and A. Ambrus, Principles and practices of method validation. The Royal Society of Chemistry, UK. Hiemstra, M. and A. Kok, Comprehensive multi-residue method for the target analysis of pesticides in crops using liquid chromatography tandem mass spectrometry. J. Chromatography A, 1154: Lehotay, S.J., Determination of pesticide residues in foods by Acetonitrile extraction and partitioning with magnesium sulphate: collaborative study. J. AOAC int., 90: Mastovska, K. and S.J. Lehotay, Evaluation of common organic solvents for gas chromatographic analysis and stability of multiclass pesticide residues. J. Chromatography A, 1040: European Commission Document SANCO/10684/2009. Method Validation and Quality Control Procedures for Pesticide Residues Analysis in Food and Feed. pp: Mol, H.G., R.C. Van Dam and O.M. Steijger, Determination of polar organophosphorus pesticides in vegetables and fruits using liquid chromatography with tandem mass spectrometry: selection of extraction solvent. J. Chromatography A, 1015: Oliva, J., A. Barba, N. Vela, F. Melendreras and S. Navarro, Multiresidue method for the rapid determination of organophosphorus insecticides in grapes must and wine. J. Chromatography A, 882: Pang, G., Y.Z. Cao, J.J. Zhang, C.L. Fan, Y.M. Liu, X.M. Li, G.Q. Jia, Z.Y. Li, Y.Q. Shi, Y.P. Wu and T. Guo, Validation study on 660 pesticide residues in animal tissues by gel permeation chromatography cleanup/gas chromatography mass spectrometry and liquid chromatography tandem mass spectrometry. J. Chromatography A, 1125: 1-30.

7 247 Sherif B. Abdel Ghani, 2014 Payá, P., M. Anastassiades, D. Mack, I. Sigalova, B. Tasdelen, J. Oliva and A. Barba, Analysis of pesticide residues using the quick easy cheap effective rugged and safe (QuEChERS) pesticide multiresidue method in combination with gas and liquid chromatography and tandem mass spectrometric detection. Anal. Bioanal. Chem, 389: Pérez, L., J. Borges and M. Delgado, Multi-walled carbon nanotubes as efficient solid-phase extraction materials of organophosphorus pesticides from apple, grape, orange and pineapple fruit juices. J. Chromatography A, 1211: Tomlin, C.D.S., Ed., ( ). The British Crop Protection Council. UK. The e-pesticide Manual, Version 3.1, 13th ed.

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