Middle East Journal of Applied Sciences ISSN 2077-4613 Volume : 06 | Issue :02 | April-June| 2016 Pages: 418-429 Corresponding Author: Emad R. Attallah, Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Foods, Agricultural Research Center, Ministry of Agriculture and Land Reclamation, P.O. Box 2311, 7, Nadi Elsaid st., Dokki, Giza, Egypt. E-mail: emadatala@ yahoo.com 418 Validation of Multiresidue Method for Determination of 340 Pesticide Residues in Fresh Pepper using LC–MS/MS Emad R. Attallah Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Foods, Agricultural Research Center, Ministry of Agriculture and Land Reclamation, P.O. Box 12311, 7, Nadi Elsaid st., Dokki, Giza, Egypt. Received: 16 May 2016 / Accepted: 14 June 2016 / Publication date: 30 June 2016 ABSTRACT Pepper is considered one of the promising exported crops in Egypt. Due to contamination risks of pepper with pesticide residue, the EU has included Egyptian exports of pepper to increased level of control (Regulation (EC) No 1021/2014). The current study covers as many pesticides registered in Egypt as possible in one multiresidue method to shorten the analysis turnaround time (TAT) and to fulfill the local and international requirements of pesticide maximum residue limits (MRLs) for conventional and organic products. Multiresidue method for determination of 340 pesticide residues in fresh pepper was validated using the quick, easy, cheap, effective, rugged and safe extraction method (QuEChERS) followed by high performance liquid chromatography with tandem mass spectrometry (LC–MSMS). Samples were extracted with acetonitrile, followed by a liquid-liquid partitioning step and phase separation by adding buffer–salt mixture consisting of magnesium sulfate, sodium chloride, disodium hydrogen citrate sesquihydrate and trisodium citrate dihydrate. Sample extract was centrifuged and an aliquot of the clear solution was filtered and injected directly into the LC-MSMS system. Quantitation and identity confirmation was attained by using atmospheric pressure electrospry positive ionization LC-MS/MS in multiple reactions monitoring (MRM) mode. Due to LC-MSMS signal suppression, determination of pesticide residues was based on matrix-matched standard calculations. Recoveries at three different concentration levels (0.01, 0.05 and 0.1 mg/kg) ranged from 70 to 120%. The reproducibility expressed as relative standard deviation (RSD %) was 4-24% (n=6). Key words: Pepper, pesticide residue, EU regulation, validation, LC-MSMS Introduction Pepper is considered one of the promising exported crops in Egypt, exports of fresh pepper reached14,000 tons during the season 2014/2015 which amounted to 16 million USD. The main imported countries were Saudia Arabia, United Arab Emirates, Hungary, USA, UK and Germany (General Organization for Export & Import Control database). Pesticides are used to protect agriculture products before and after harvest from infestation by pests and plant diseases. A possible consequence of their use may be the presence of pesticide residues in the treated products. The Egyptian Ministry of Agriculture through the Agriculture Pesticide Committee (APC) has approved the use of methomyl, penconazol, sulfur and thiamethoxam on pepper for pest control (Agriculture Pesticide Committee approved pesticide list, 2015). According to the data published in the official portal of the EU rapid alert system for food and feed (RASFF), there were 10 EU border rejections of Egyptians pepper consignments due to violations of the EU pesticide maximum residue limits (MRLs). The violated pesticides were carbendazim (MRL=0.1mg/kg), dodine (MRL=0.05 mg/kg), flusilazole (MRL=0.01mg/kg) and methomyl (MRL=0.02 mg/kg). Due to contamination risks of pepper with pesticide residue, the EU has included Egyptian exports of pepper to increased level of control with frequency of physical and identity checks of 10% (Commission Regulation (EC) No 1021/2014). It is very important to use accurate and rapid analytical methods for pesticide residue control. The use of multiclass, multiresidue methods is the most efficient approach to pesticide residue analysis in terms of analysis costs and turnaround time. Many multiresidue methods had been developed for determination of pesticide residue in fruits and vegetables. In 1996, Casanova used solid-phase extraction (SPE) to extract pesticides from diluted acetone extracts, thus completely avoiding liquid–liquid partitioning. Jansson et al. (2004) used extraction with ethyl acetate in presence of sodium sulfate followed LC-MS/MS determination.
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Middle East Journal of Applied Sciences ISSN 2077-4613
Corresponding Author: Emad R. Attallah, Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Foods, Agricultural Research Center, Ministry of Agriculture and Land Reclamation, P.O. Box 2311, 7, Nadi Elsaid st., Dokki, Giza, Egypt.
E-mail: emadatala@ yahoo.com 418
Validation of Multiresidue Method for Determination of 340 Pesticide Residues in Fresh Pepper using LC–MS/MS
Emad R. Attallah
Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Foods, Agricultural Research Center, Ministry of Agriculture and Land Reclamation, P.O. Box 12311, 7, Nadi Elsaid st., Dokki, Giza, Egypt.
Received: 16 May 2016 / Accepted: 14 June 2016 / Publication date: 30 June 2016 ABSTRACT
Pepper is considered one of the promising exported crops in Egypt. Due to contamination risks of pepper with pesticide residue, the EU has included Egyptian exports of pepper to increased level of control (Regulation (EC) No 1021/2014). The current study covers as many pesticides registered in Egypt as possible in one multiresidue method to shorten the analysis turnaround time (TAT) and to fulfill the local and international requirements of pesticide maximum residue limits (MRLs) for conventional and organic products. Multiresidue method for determination of 340 pesticide residues in fresh pepper was validated using the quick, easy, cheap, effective, rugged and safe extraction method (QuEChERS) followed by high performance liquid chromatography with tandem mass spectrometry (LC–MSMS). Samples were extracted with acetonitrile, followed by a liquid-liquid partitioning step and phase separation by adding buffer–salt mixture consisting of magnesium sulfate, sodium chloride, disodium hydrogen citrate sesquihydrate and trisodium citrate dihydrate. Sample extract was centrifuged and an aliquot of the clear solution was filtered and injected directly into the LC-MSMS system. Quantitation and identity confirmation was attained by using atmospheric pressure electrospry positive ionization LC-MS/MS in multiple reactions monitoring (MRM) mode. Due to LC-MSMS signal suppression, determination of pesticide residues was based on matrix-matched standard calculations. Recoveries at three different concentration levels (0.01, 0.05 and 0.1 mg/kg) ranged from 70 to 120%. The reproducibility expressed as relative standard deviation (RSD %) was 4-24% (n=6). Key words: Pepper, pesticide residue, EU regulation, validation, LC-MSMS
Introduction
Pepper is considered one of the promising exported crops in Egypt, exports of fresh pepper reached14,000 tons during the season 2014/2015 which amounted to 16 million USD. The main imported countries were Saudia Arabia, United Arab Emirates, Hungary, USA, UK and Germany (General Organization for Export & Import Control database).
Pesticides are used to protect agriculture products before and after harvest from infestation by pests and plant diseases. A possible consequence of their use may be the presence of pesticide residues in the treated products. The Egyptian Ministry of Agriculture through the Agriculture Pesticide Committee (APC) has approved the use of methomyl, penconazol, sulfur and thiamethoxam on pepper for pest control (Agriculture Pesticide Committee approved pesticide list, 2015).
According to the data published in the official portal of the EU rapid alert system for food and feed (RASFF), there were 10 EU border rejections of Egyptians pepper consignments due to violations of the EU pesticide maximum residue limits (MRLs). The violated pesticides were carbendazim (MRL=0.1mg/kg), dodine (MRL=0.05 mg/kg), flusilazole (MRL=0.01mg/kg) and methomyl (MRL=0.02 mg/kg). Due to contamination risks of pepper with pesticide residue, the EU has included Egyptian exports of pepper to increased level of control with frequency of physical and identity checks of 10% (Commission Regulation (EC) No 1021/2014).
It is very important to use accurate and rapid analytical methods for pesticide residue control. The use of multiclass, multiresidue methods is the most efficient approach to pesticide residue analysis in terms of analysis costs and turnaround time. Many multiresidue methods had been developed for determination of pesticide residue in fruits and vegetables.
In 1996, Casanova used solid-phase extraction (SPE) to extract pesticides from diluted acetone extracts, thus completely avoiding liquid–liquid partitioning.
Jansson et al. (2004) used extraction with ethyl acetate in presence of sodium sulfate followed LC-MS/MS determination.
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Anastassiades et al. (2003) recently introduced the so-called quick, easy, cheap, effective, rugged, and safe (QuEChERS) method of pesticide residue analysis.
In a follow-up study, Lehotay et al. (2005) demonstrated its effectiveness for >200 pesticides in lettuce, orange, and several other matrixes using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) for analysis.
Banerjee et al. (2007) validated a method for the multi-residue analysis of 82 pesticides in grapes at ≤25 ng/g level. Berry samples (10 g) mixed with sodium sulphate (10 g) were extracted with ethyl acetate (10 ml); cleaned by dispersive solid phase extraction and the results were obtained by liquid chromatography–tandem mass spectrometry.
Attallah et al. (2012) has validated QuEChERS analytical method for determination of 93 pesticide residues in apples and potatoes using LC–MS/MS.
Lesueur et al. (2008) analysed 140 pesticides with GC/SQ-MS and 46 pesticides with HPLC/IT-MS after extraction with the QuECheRS method in four matrices (grape, lemon, onion and tomatoes).
A rapid and sensitive liquid chromatography–tandem mass spectrometry method, in electrospray ionization positive mode, has been developed by Kmellar et al. (2008) for the determination of 160 selected multi-class pesticides over a 33-min run time.
Materials and Methods Apparatus:
PFTE or polyethylene 15 ml and 50 ml with screw cap tubes. Centrifuge (Heraues up to 4000 rpm). LC–
MS/MS was performed with an Agilent 1200 Series HPLC instrument coupled to an API 4000 Qtrap MS/MS from AB Sciex with an electrospray ionisation (ESI) interface. Separation was performed on an Agilent ZORBAX Eclipse XDB C18 column 4.6 x 150 mm, 5 Micron particle size. Ultra-Turrax® T25 Digital.
98-100% (Riedel–de Haen).Ammonia solution, 33% (Riedel–de Haen). De-ionized water was produced by a mille Q unit (Mille Pore).
Ready-made QuECHERS kits from Agilent Technologies; reagent packet contains 4g of magnesium sulfate anhydrous, 1g of sodium chloride, 1g of trisodium citrate dehydrate and 0.5g of disodium hydrogencitrate sesquihydrate.
LC Mobile Phase: ammonium formate solution in methanol-water (1:9), pH 40.1. Standard preparation: Stock solution: 1000 ug/ml reference standard solution of each pesticide was
prepared in toluene. Intermediate solutions: Mixture of standards of 5 ug/ml of each pesticide was prepared by diluting stock
solution in toluene. Calibration solutions: Calibration mixtures of concentration levels 0.005, 0.01, 0.05and 0.1 ug/ml were
prepared in methanol.
Extraction Procedure: Ten grams (W) of sample were weighed in 50 ml PFTE tube, 10 ml Acetonitrile was mixed with the sample
by mechanical shaking for one minute. The buffer-salt-mixture was added and shaken immediately for one minute. The sample was centrifuged at 4000 rpm for 5 minutes. Portion of Acetonitrile layer was filtered using syringe filter and directly injected into LC-MS/MS system. LC-MS/MS analysis:
Separation was performed on a C18 column ZORBAX Eclipse XDB-C18 4.6 x 150 mm, 5 μm particle
size. The injection volume was 5 µl. A gradient elution program was used at 0.3 mL/min flow, in which one reservoir contained 10mM ammonium formate solution in methanol-water (1:9) and the other contained methanol.
The ESI source was used in the positive mode, and N2 nebulizer, curtain, and other gas settings were optimized according to recommendations made by the manufacturer; source temperature was 400oC, ion spray potential, 5500 V, declustering potential and collision energy were optimized using apparatus syringe pump by introducing individual pesticide solutions into the MS instrument to allow optimization of the MS/MS conditions. The Multiple Reaction Monitoring mode (MRM) was used in which one MRM was used for
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quantitation and the other was used for confirmation. Matrix-matched standards were used to correct for matrix effect.
Results and Discussion
Method validation: The selected parameters for in-house validation were mainly taken from Eurachem guideline on method
validation (Eurachem Guide, 2014). The acceptance criteria were taken from Guidance document on analytical quality control and method validation procedures for pesticides residues analysis in food and feed. (Document N° SANTE/11945/2015).
The performance of the QuEChERS method was tested by performing six replicates of spiked blank pepper samples at three different concentration levels (0.01, 0.05 and 0.1 mg/kg) for 360 pesticides using LC/MSMS. There were 340 pesticides showed accepted recovery and precision, while 20 pesticides had shown unacceptable recovery and precision due to lower sensitivity on LC-MSMS system. These pesticides are GC-amenable compounds (e.g pyrethroids and sulfamides), Validation study of about 210 GC-amenable pesticides will be presented in other work. Table 1: Recovery% & CV% for spike samples (n=6) at different three concentration levels on pepper
Middle East J. Appl. Sci., 6(2): 418-429, 2016 ISSN 2077-4613
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# Pesticide Name
0.01 mg/kg 0.05 mg/kg 0.1 mg/kg All levels
Mean % CV% Mean % CV% Mean % CV% Mean % CV%
323 Tolclofos-methyl 82 15 105 7 98 6 95 14
324 Tralkoxydim 84 4 104 3 92 3 94 10
325 Tri allate 82 7 97 4 98 4 92 9
326 Triadimefon 79 13 97 8 87 7 88 12
327 Triadimenol 103 18 104 13 76 7 94 20
328 Triasulfuron 75 8 112 6 103 6 97 18
329 Triazophos 98 10 97 7 90 5 95 8
330 Triazoxide 90 25 96 8 90 8 92 15
331 Trichlorfon 76 8 85 3 89 3 83 8
332 Triclopyr-2-butotyl 84 4 97 8 93 4 91 8
333 Tricyclazole 90 9 89 4 89 5 89 6
334 Trietazine 77 9 89 5 102 2 89 13
335 Trifloxystrobin 84 3 98 6 98 3 93 9
336 Triflumizole 83 7 94 5 96 3 91 8
337 Triflumuron 117 11 98 8 87 8 101 15
338 Triticonazole 68 9 99 7 97 3 88 18
339 Vamidothion 73 4 91 8 91 2 85 11
340 Zoxamide 87 11 99 3 105 5 97 10
The mean recovery for all levels was ranged between 74%-116% and reproducibility expressed as CV%
for all level ranged between 4%-24% which fulfill the requirements of the DG-SANTE for recovery (70%-120%) and reproducibility (CV < 20%). However, there were 12 pesticides that shown lower recovery (59%-69%) at the LOQ level (0.01 mg/kg) due to lower sensitivity (e.g Bromuconazole, Chlorthiophos, Cyhalothrin-L, Fenitrothion, Fluvalinate-τ, Hexaconazole, Imibenconazole, Mepanipyrim, Napropamide, Permethrin, Thiodicarb and Triticonazole). Table 1 shows the results of recovery tests.
The LOQ for all pesticides was 0.01 mg/kg which fulfill the EU MRLs for almost all agricultural products. Only two pesticides couldn’t be detected at the 0.01 mg/kg which were flutriafol and lufeneron and their LOQs were considered to be 0.05 mg/kg. However, the LOQ of 0.05 mg/kg will fairly fulfill the high EU MRLs of 1.0 mg/kg for flutriafol and lufenuron on pepper.
Proficiency Testing: The method accuracy has been confirmed by participating in proficiency testing (PT) programs organized
by FAPAS, UK. The following table (2) shows accepted z-scores (z = ± 2) for FAPAS PT rounds, questionable result (z =
2.8) was observed for deltamethrin due to calculation in matrix effect corrections. Table 2: Assigned values, lab results and z-scores for FAPAS PT rounds
FAPAS PT program Pesticide Assigned value Result z-score
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Conclusion
In the last few years, the Egyptian exports of pepper were considered of great concern, due to the international market demand.
In the present study, multiresidue method of determination of 340 pesticides was validated at three different concentration levels, the limit of determination of 0.01 mg/kg which fulfills the EU MRLs for organic agricultural products and baby foods.
The QuEChERS method followed by LC-MSMS was found to be the best combination for determination of the 340 pesticides in terms of accepted recovery, short time of analysis, low cost and safety. The validated method could be used to control the contamination levels of pesticide residues in fresh pepper. The current study covered LC- amenable pesticides of polar and moderately polar pesticides. Validation of GC-amenable pesticide, about 210 pesticides on pepper will be presented in other publication.
References
Agriculture Pesticide Committee (APC). 2015. APC approved pesticide list. http://www.apc.gov.eg/EN/ Anastassiades, M., S.J. Lehotay, D. Stajnbaher, F.J. Schenck, 2003. 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(2): 412-431.
Attallah, E.R., M.E. Amer, A.M. Gomaa and A.A. El Gohary, 2012. QuEChERS analytical method for determination of 93 pesticide residues in apples and potatoes using LC–MS/MS. Journal of Applied Sciences Research, 8(12): 5690-5697.
Banerjee, K., P. Dasharath, O.S. Dasgupta, S.B. Patil, S.H. Patil, R. Savant, P.G. Adsule, 2007. 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(1-2): 98-109.
Casanova, J., 1996. Use of Solid-Phase Extraction Disks for Analysis of Moderately Polar and Nonpolar Pesticides in High Moisture Foods. J. AOAC. Int., 79: 936-940.
CommissionRegulation (EU) No 1021/2014.Official Journal of the European Union, (2014) L 283/32 - L 283/39.
DG-SANTE, European Commission Directorate-General for Health and Food Safety, 2015. Guidance document on analytical quality control and method validation procedures for pesticides residues analysis in food and feed. Document N° ANTE/11945/2015.
Eurachem Guide, B. Magnusson and U. Ornemark, 2014. (eds.): The Fitness for Purpose of Analytical Methods. A Laboratory Guide to Method Validation and Related Topics, (2nd ed. 2014). ISBN 978-91-87461-59-0.Available fromwww.eurachem.org.”
General Organization for Export & Import Control (GOEIC).http://www.goeic.gov.eg/en/index_r.asp Jansson, C., T. Pihlström, B. Österdahl, K.E. Markides, 2004. A new multi-residue method for analysis of
pesticide residues in fruit and vegetables using liquid chromatography with tandem mass spectrometric detection. J. Chromatography A 1023(1): 93-104.
Kmellar, B., P. Fodor, L. Parejab, C. Ferrer, M.A. Martnez-Uroz, A. Valverde, A.R. Fernandez-Alba, 2008. . Validation and uncertainty study of a comprehensive list of 160 pesticide residues in multi-class vegetables by liquid chromatography–tandem mass spectrometry. J. Chromatography A 1215(1-2): 37-50.
Lehotay, S.J., A. De-Kok, M. Hiemstra, P. van-Bodegraven, 2005. Validationof a fast and easy method for the determination of residues from 229 pesticides in fruits and vegetables using gas and liquid chromatography and Mass spectrometric detection. J. AOAC. Int., 88(2): 595-614.
Lesueur, C., P. Knittl, M. Gartner, M. Mentler, M. Fuerhacker, 2008. Analysis of 140 pesticides from conventional farming foodstuff samples after extraction with the modified QuECheRS method. J. Food Control., 19(9): 906-914.