A MODIFIED MULTI-RESIDUE METHOD FOR ANALYSIS OF 150 PESTICIDE RESIDUES IN GREEN BEANS USING LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

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1 A MODIFIED MULTI-RESIDUE METHOD FOR ANALYSIS OF 150 PESTICIDE RESIDUES IN GREEN BEANS USING LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY Abd El-Moneim M.R. Afify 1, Emad R. Attallah 2, and Hassan A. El-Gammal 2 1 Department of Biochemistry, Faculty of Agriculture, Cairo University, Giza, Egypt 2 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 ABSTRACT Three methods of sample preparation and analysis of 150 pesticides in green beans were evaluated by LC- MS/MS detection. The mass spectrometry parameters were optimized to give the best sensitivity, and two multiple reaction monitoring modes (MRMs) were chosen for quantification and conformation, based on the optimized declustering potential and collision energy. The first Quick, easy, cheap, effective, rugged, and safe (QuEChERS) method for pesticide residue analysis entailed extraction with acetonitrile whereas the second one used extraction with ethyl acetate, and the third one with acetone and partitioning by dichloromethane:petroleum ether (3:1). The extracts of the 3 methods were redissolved in methanol:buffer(1:1) solution (ammonium formate 10 mm), ph 4 as modification to increase injection volume to 25 µl without losing good peak shape separation). Stabilities of tested pesticides in 5 different calibration mixtures (ph 3, 4, 5, 6 and 7) stored for 2 weeks were studied. The total 150 pesticides showed % recovery at ph 4 while the recovery rates of the different pesticides were ph-dependent proving that acidic ph was suitable for preparation and determination of multi-residue pesticides. The 150 pesticide signal intensities were studied at 3 different ph values and 4 concentrations to show the effect of mobile phase on pesticide intensity due to ionization power. The recovery rates at the 3 different concentration levels (0.01, 0.05 and 0.1 mg/kg) ranged from 70 to120% were carried out. KEYWORDS: multi-residue, liquid chromatography-tandem mass spectrometry, pesticide residues, green beans 1. INTRODUCTION Pesticides are used to protect agricultural 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 Maximum Residue Limits (MRLs) of pesticide residues as food standards are critical factors for allowing agriculture products consignments to enter the importing countries, especially EU countries as recorded by the National Food Administration Data Requirements Sweden Plant Protection Products 2011 [1] and international Codex Committee standards Codex 2010 [2]. Because of the wide variety of food matrices, the samples must initially be cleaned up before final analysis. That is why the analytical chemist is faced with the need of new methodologies for determining such residues in a single analytical run. To accomplish the goal, QuEChERS methodology has been developed, and liquid or gas chromatography analysis was used for pesticides analysis [3]. An analytical MRM for the simultaneous determination of various classes of pesticides in vegetables (pepper and tomato) was developed. Vegetable samples were extracted with acetone and the pesticides partitioned into ethyl acetate/cyclohexane. Final determination was made by GC analysis with nitrogen phosphorus detection. Confirmation analysis of pesticides was carried out by GC-MS in the selected ion monitoring (SIM) mode. The identification of compounds was based on retention time and comparison of the primary and secondary ions. Recovery studies were performed at 0.05, 0.1and 0.02 mg kg 1 fortification levels of each compound, and the recoveries obtained ranged from 70.1 to 128.5% with relative standard deviations <7% [4]. Solid-phase micro-extraction coupled on line with high resolution gas chromatography and mass spectrometry detection is described for the analysis of pesticides in environmental water samples. Experiments were performed in order to optimize the solid-phase micro-extraction conditions for selected pesticides including tiomethon, trichorfon, dimethoate, diazinon, malathion, dicofol, methidathion, 24

2 ethion, bromopropylate and pyrazophos from spiked water solutions [5]. A method based on matrix solid-phase dispersion and liquid chromatography electrospray ionization mass spectrometry was used to analyze 15 fungicide residues in fruits and vegetables. The method required only 0.5 g of sample, and C 18 -bonded silica was used as dispersant and sorbent, and ethyl acetate was used as eluting solvent. Fortified recoveries in apple, orange, banana, lettuce, grape and tomato samples ranged from 71 to 102%, and relative standard deviations were less than 13% with fortified levels of mg kg 1 as described by [6]. A liquid chromatographic/electrospray ionizationtandem mass spectrometric method was developed and validated to determine 142 pesticides in fruit- and vegetable based infant foods, including apples, apples and bananas, pears, bananas, apple juice, peas, sweet potatoes, creamed corn, squash, and carrots. Pesticides were extracted from infant foods by QuEChERS method. Quantification was based on matrix-matched standard calibration curves with the use of an isotopically labeled standard, or a chemical analogue as the internal standard, to obtain method accuracy. The overall recoveries for spiking levels of 10, 50, and 80 µg/kg in a range of % [7]. The applications of ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry in the determination of 138 pesticides in fruit- and vegetable-based infant foods were also investigated extracting pesticides by using QuEChERS method, and high sensitivity proved to be an ideal tool for screening of a large number of pesticides in a single analysis [8]. In a follow-up study [9], its effectiveness was demonstrated for >200 pesticides in lettuce, orange, and several other matrices using GC/MS and LC/MS/MS analyses. Degradation of base-sensitive pesticides, such as captan, folpet, dichlofluanid and chlorothalonil, was also observed, especially in non-acidic matrix such as lettuce. Problems in the analysis of these pesticides are well known in the pesticide community and, typically, they are merely screened in multiclass/multi-residue methods requiring specialized techniques for more accurate analysis. A new method used sodium acetate as extraction step to overcome the degradation of problematic pesticides [10] and could decrease the efficiency of the clean-up step using PSA, while [11] used disodium citrate and trisodium citrate as buffering mixture. A validated method for the multiresidue analysis of 82 pesticides in grapes at 25 ng/g levels was described by [12]. Analyses of 105 pesticides with GC/SQ-MS and 46 pesticides with HPLC/IT-MS after extraction with QuECheRS method in 4 matrices (grape, lemon, onion and tomato) were also described [13] as well as LC-MS/MS method for the quantitative determination of 44 pesticide residues with hydrolysable functional group [14]. In the present study, we used the simple, rapid and reliable multi-residue method QuEChERS for determination of pesticides in green beans using acetonitrile for extraction and LC-MS/MS determination with comparison by two other methods of extraction as described by [15], and stabilized LC-MS/MS parameters to increase performance of the individual pesticides in multi-residual mixture analysis. 2. MATERIALS AND METHODS 2.1. Apparatus LC MS/MS was performed with an Agilent 1200 Series HPLC instrument coupled to an API 4000 Qtrap MS/MS from Applied Biosystems with electrospray ionization (ESI) interface Reagents Acetonitrile (Lab-Scan; HPLC, assay >99%), methanol (Merck, 99.9% HPLC grade), ethyl acetate (Lab-Scan, Dublin, Ireland; pesticide grade), acetone, dichloromethane and petroleum ether (Lab-Scan, Pestiscan), formic acid, (Riedel de Haen; %), ammonia solution, (Riedel de Haen; 33%), sodium chloride (Riedel de Haen; 99%), disodium hydrogencitrate sesquihydrate (Fluka, 99%), trisodium citrate dihydrate (Fluka), sodium chloride and anhydrous magnesium sulphate (Merck), anhydrous sodium sulphate (Riedel-deHaen) were used for experiments, and de-ionized water was produced by a Milli-Q unit (Millip Corporation, USA). Pesticide reference standards (purity >95%) were from Dr. Ehrensdorfer (Augsburg, Germany) Stock solutions Reference standard solutions (1000 µg/ml) of all the analyzed pesticides were prepared in methanol and kept at -20 ± 2 o C [15] Intermediate mixture solutions Mixtures with 10 µg/ml from all compounds were prepared as intermediate stock solutions in methanol used for spiking and also kept at -20 ± 2 C [16] Calibration mixture solutions Calibration mixtures of concentration levels 0.005, 0.01, 0.05, 0.1 and 0.5 µg/ml were prepared in methanol:ammonium formate buffer 10 mm ph 4 (1:1) and kept at -20 ± 2 o C Pesticide stability mixtures The calibration mixture was prepared at 5 different ph values (3, 4, 5, 6 and 7) but the same solution (methanol:ammonium formate buffer 10 mm, 1:1) and concentration (0.5 ppm ) to check pesticide stabilities for 2 weeks. Then, 4 different calibration mixture solutions were injected after storage at -20 ± 2 o C for 2 weeks, and calibration mixture 0.5 ppm in methanol as reference standard solution was freshly prepared. 25

3 2.7. LC-MS/MS conditions LC - Mobile Phase Ammonium formate was prepared as follows: Ammonium formate (10, 5, 1 & 0.1 mm) was adjusted to ph 4 as well as ammonium formate (1 mm) to ph values 3.5 and 3.0, all with formic acid dissolved in methanolwater (1: 9) LC-MS/MS conditions All pesticide sensitivities were tested by 3 different ammonium formate buffer concentrations (0.1, 5 and 10 mm at ph 4) and 3 different ph values (3, 3.5 and 4) using the same gradient and flow-rate by injecting calibration mixtures. Separation was performed on a C18 column ZOR- BAX Eclipse XDB-C x 150 mm, 5 µm particle size. The injection volume was 25 µl. A gradient elution program at 0.3 ml/min flow, in which one reservoir contained 10 mm ammonium formate solution in methanol-water (1:9) and the other one methanol, was used. 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 400 o C, ion spray potential 5500 V, decluster potential and collision energy were optimized using a Harvard apparatus syringe pump by introducing individual pesticide solutions into the MS instrument to allow optimization of the MS/MS conditions (shown in Table 1). MRM was used, one mode for quantification and the other one for confirmation Extraction procedure QuEChERS method [17] Green bean sample (10 g) was transferred to a polyethylene (PFTE) 50-ml tube, 10 ml acetonitrile was added and shaken vigorously for 1 min. Then, buffer-salt-mixture (4±0.2 g of magnesium sulfate anhydrous, 1±0.05 g of sodium chloride, 1±0.05 g of trisodium citrate dehydrate and 0.5±0.03 g of disodium hydrogencitrate sesquihydrate) was added and shaken immediately for 1 min. Centrifugation was carried out at 4,000 rpm for 5 min. Supernatant (4 ml) of the clear solution was transferred to a 50-ml roundbottom flask and evaporated with rotary evaporator at 40 o C. Residues were re-dissolved in 4 ml (methanol:ammonium format buffer 10 mm ph 4; 1:1). Injection of 25 µl of the sample into LC-MS/MS system was carried out Luke et al. method [18] Green beans sample (50 g) was added to 100 ml acetone and blended for 2 min at medium speed; the homogenized sample was filtered through a Buchner funnel containing a Whatman no.1 filter paper fitted to a Buchner flask. The blender jar was rinsed with 50 ml acetone and filtered again on the same funnel, and the extracted volume was recovered. A 40-ml sample extract was transferred to a 500-ml separator funnel, 50 ml petroleum ether and 50 ml dichloromethane were added to be shaken vigorously for 2 min, then transferring the lower aqueous layer to a graduated cylinder and the upper organic layer by passing through anhydrous sodium sulfate supported on washed cotton in a funnel to the receiving flask; about 2 g sodium chloride was added to the aqueous phase which was shaken vigorously for 1 min until most of the sodium chloride was dissolved. Then, it was transferred to the same separator funnel, 50 ml dichloromethane was added, and it was shaken for 1 min. The lower dichloromethane layer was filtered through sodium sulphate, the water layer was taken and the last dichloromethane partitioning step repeated. Then, sodium sulfate was rinsed with 25 ml dichloromethane, the received solution was rotary-evaporated to about 2ml at C. Evaporation was continued by air just to dryness, and the residue was re-dissolved in 10 ml methanol: ammonium formate buffer 10 mm ph 4 (1:1) and filtered through a 0.45-µm syringe filter. The clear filtrate was injected directly into LC/MS/MS system Ethyl acetate method [19] Green bean sample (50 g) was added to 10 ml ethyl acetate in a 50-ml PTFE centrifuge tube and blended for 1 min. An aliquot of 4 ml was rotary-evaporated at 40 o C just to dryness. The residue was re-dissolved in 4 ml methanol:ammonium formate buffer 10 mm ph 4 (1:1) and filtered through a 0.45-µm syringe filter. The clear filtrate was injected directly into LC-MS/MS system. 3. RESULTS AND DISCUSSION 3.1. Injection volume For comparison between the 3 different injection solvents (acetonitrile, methanol and methanol/buffer 1:1), an injection volumes of 25 µl for 3 pesticides (acetamiprid, aldicarb and aminocarb) were used to show the relationship between the 3 injection solvents and peak shape (Figs. 1ac). It is clear that peak symmetry of the pesticides in methanol/buffer is much better compared to acetonitrile. Methanol had a stronger elution power than the mobile phase; therefore, pesticides are not retained completely at the beginning of the column which led to unsymmetrical peaks. This result was in agreement with Agilent technology recommendation stating that a strong injection solvent may cause poor peak shape, and injecting a solvent stronger than the mobile phase can cause peak shape problems, such as peak splitting or broadening [20], and weakly retained analytes under aqueous mobile phase conditions [21] inducing strong solvent-like effects [22] 3.2. The signal intensity The signal intensity of pesticides in LC-MS/MS can be influenced by the mobile phase composition. In order to optimize the signal intensity, standard mixtures in methanol were injected into the LC-MS/MS system, using different mobile phase compositions. Three different buffer constituents were tested: ammonium format (0.1, 5 and 10 mm) at 3 ph values (3, 3.5 and 4). Evaluation was done by recording the MS/MS signal enhancement (SE) 26

4 FIGURE 1a - Chromatogram of acetamiprid injected in acetonitrile, methanol and methanol/buffer (25 µl). 27

5 FIGURE 1b - Chromatogram of aldicarb injected in acetonitrile, methanol and methanol/buffer (25 µl). 28

6 FIGURE 1c - aminocarb injected in acetonitrile, methanol and methanol/buffer (25 µl). 29

7 for each pesticide with a calculation based on 5 mm, ph 4. The mobile phase during this test was composed of 50% buffer and 50% methanol (1:1). Results showed that there is no variation in SE >4% between all tested mobile phases, except of 10 mm at ph 4 which caused increase of 26 compounds >15% as shown in Table 1. Twelve pesticides were increased in SE from 15 to 20 % while 14 pesticides were increased in SE, such as 20% (cyanophos) to 38 % (tetraconazole). Therefore, higher ion strength contributes to a more stable system, both for retention and SE with acidic ph ranging from 4.0 to 4.2. Thus, we concluded to choose buffer strength of 10 mm (ph 4) as compromise for 57 pesticides [23] The total ion chromatogram The total ion chromatogram for the 150 pesticides injected into LC-MS/MS system is illustrated in Fig. 2 A. It seems that the pesticide peaks are not resolved but, in fact due to the high selectivity of the MS-MS system, the peaks can be resolved easily (Figs. 2B, C and D). All the 150 pesticides could be analyzed by a single chromatographic run of 33 min and each MRM could be separated as single peak in a chromatogram as shown in Figs. 2B, C and D for fenpropathrin, dichlofuanid and fenhexamide pesticides [24]. It is clear that although dichlofuanid (Fig. 2 C) has the same molecular weight as fenpropathrin (Fig. 2B) (absence of cross talk) and the same TABLE 1 - Comparison between pesticide sensitivity using 10 mm buffer compared to 5 mm buffer. Pesticides SE Pesticides SE Pesticides SE Pyrazophos 15% Cyanophos 20% Carboxin 24% Thifensulfuron_Me 15% Flamprop 20% Thiocyclam HO 27% Pyrimethanil 15% Chlorpyrifos-Me 20% Bensulfuron-Me 28% Methoxyfenozide 16% Carbofuran-3OH 21% Aldicarb 29% Thiobencarb 17% Diafenthiuron 21% Myclobutanil 30% Triadimifon 17% Tolylfluanid 21% Nuarimol 32% Butralin 19% Isoproturon 21% Diuron 33% Pyrifenox 19% Linuron 22% Tetraconazole 38% Prochloraz 19% Phenthoate 23% - - SE :Signal enhancement in 10 mm compared to 5 mm. FIGURE 2 - Chromatogram of 150 pesticides as A = 300 MRM, B = fenpropathrin, C = diclofuanidnid, D = fenhexamide. 30

8 retention of fenhexamide (Fig. 2 D), but it is easily resolved from both compounds Stability of 150 standard pesticides A standard solution of the 150 pesticides was prepared (concentration 0.5 µg/ml) and kept in a freezer for 15 days at -20±2 o C and compared to freshly prepared standard solution. The stability of these pesticides at different ph values (3, 4, 5, 6, 7) are shown by calculating percentage of recovery (Table 2b and Fig. 3). All 150 pesticides showed % recovery at ph 4 while the recovery of the different pesticides were ph-dependent proving that acidic ph was suitable for preparation and determination of multi-pesticides. The degradation rates of pesticides at phs 3, 5, 6 and 7 were investigated and recorded. The pesticides which had lost more than 10% of their TABLE 2 - Degradation of pesticides at different ph values of 3, 5, 6 and 7. Pesticides ph 3 ph 5 ph 6 ph 7 Triflumizole 56% a a a Fenoxaprop-ethyl 50% a a a Aldicarb Sulfoxide 30% a a a Thiophanate -methyl 22% a a a Metribuzin 20% a a a Propamocarb -HCl 20% a a a Thiocyclam -HO 18% a a a Acephate 16% a a a Diazinon 14% a a a Metosulam 14% a a a Diclorovs 13% a a a Omethoate 12% a a a Edifenophos 12% a a a Butachlor 12% a a a Pymetrozine 11% a a a Diafenthiuron 25% 43% 45% 37% Diniconazole 12% 12% 11% 18% Parathion-ethyl a a 24% 28% Flamprop a a 17% 17% Piperonyl-butoxide a a 13% 14% Dimethomorph a a a 35% Thiobencarb a a a 12% Diflufenican a a a 24% a =Accepted stability of pesticide at tested ph (90%). TABLE 2b - Effect of ph on stability of pesticide standard solution. Pesticides Recovery ph 3 ph 4 ph 5 ph 6 ph % % % <70% Pesticides Stability <70% 70-80% 80-90% % Compounds ph 3 ph 4 ph 5 ph 6 ph 7 Tested ph FIGURE 3 - Pesticide stability at ph 3, 4, 5, 6 and 7. 31

9 concentration are shown by degradable percentage. Triflumizole showed a degradation of 56% at ph 3, and degradation of the remaining pesticides was ph-depending. This result was in agreement with the US-EPA (EPA Pesticide Fact Sheet 10/91) studies [25] on triflumizole showing hydrolysis studies of phenyl-labeled carbon 14 triflumizole at 5 ppm, which was degraded in sterile aqueous 0.01 M buffered solutions with half-lives of 7-15 days at ph 5, >30 days at ph 7 and 3-17 days at ph 9, while fenoxapropethyl showed degradation of 50% below ph 4.6 [26] Effect of extraction methods Different types of extraction procedures were tested as described in materials using three methods, e.g. Luke, QuEChERS and ethyl acetate. Extraction was done on green bean samples at spiking levels of 0.5 mg/kg. Blank samples and standard were dissolved in solvent and injected in parallel to spiked samples and the same run. The results of recovery tests on green beans were discussed and evaluated by the pesticides with accepted recovery rates >60 % (Table 3). Results showed that propamocarb- HCl is an example for a highly polar compound which had a low recovery by ethyl acetate extraction (11%) and was not recovered in the partitioning step of Luke method [18]. Luke method was significantly more effective for the extraction of non-polar and medium-polar compounds. Therefore, the best recoveries for polar compounds were achieved by QuEChERS and ethyl acetate methods. QuEChERS was the only method that provided an overall recovery value of 60 70% for none, medium and polar compounds The optimized LC-MS/MS parameters The optimized LC-MS/MS parameters and the best extraction procedure were used to study the method performance by carrying out recovery tests at different levels on some representative agricultural product samples. Six replicates of recovery tests were done at concentration levels of 0.01, 0.05 and 0.1 mg/kg on grapes [15] and green beans. The results in Table 4 showed that the 150 pesticides could be determined at 0.01 mg/kg with accepted recovery and precision. The injection of 25 µl of acetonitrile into the LC system leads to non-symmetrical peak shapes; therefore, acetonitrile was evaporated and sample re-dissolved in methanol-water solution. This step improved the pesticide peak shapes and lowered the matrix effect due to precipitation of some insoluble substances as discussed above. The recovery of most pesticides (143 pesticides) is in the range %, the recovery rates of 7 pesticides (chlorfluazuron 24±17, 30±5, 24±16 at concentrations of 0.01, 0.05 and 0.5 mg/kg, L-cyhalothrin, deltamethrin, diafenthiuron, flufenoxuron, lufenuron and pymetrozine) were lower than 60% due to the evaporation of acetonitrile and re-dissolving in methanol-water solution [12]. Therefore, QuEChERS method was introduced for the determination of a wide range of pesticides including very polar, moderately polar, non-polar and basic compounds; for pesticides belonging to various chemical classes from oranges, red wine, red grapes, raisins and wheat flour [8, 11, 13], and 160 selected multi-class pesticides were determined within a 33-min run time. TABLE 3 - Recovery tests on green bean samples using different extraction methods at spiking level of 0.05 mg/kg. Pesticides Luke Ethyl-acetate QuEChERS Acephate 57% a a Butralin 54% 41% a Chlorfluazuron 34% 25% 26% Cyhalothrin-L 31% 23% 29% Cyprodinil a 58% a Deltamethrin 19% 24% 25% Diafenthiuron 12% 17% 20% Diniconazole 54% a a Fenpropathrin 48% 55% 53% Fenpyroximate 50% 41% a Flufenoxuron 37% 30% 34% Hexythiazox 56% a a Lufenuron a 41% 47% Methamidophos 31% a a Pendimethalin 56% 51% a Propamocarb -HCl 11% 1% a Pymetrozine 57% 58% a Thiocyclam-HO 46% 35% a Total a =Accepted pesticides recovery )>60.(% 32

10 TABLE 4 - Recovery tests on green bean samples at 0.01 mg/kg, 0.05 mg/kg and 0.1 mg/kg. Green beans Green beans mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Pesticides (%) (%) (%) Pesticides (%) (%) (%) Abamectin 74 ± ±11 68 ±13 Malaoxon 89 ±15 93 ±5 87 ±4 Acephate 94 ±19 75 ±6 78 ±5 Malathion 95 ±18 97 ±3 81 ±6 Acetamiprid 91 ±12 92 ±4 78 ±3 Metamitron 85 ±14 92 ±6 84 ±3 Aldicarb 90 ±14 91 ±4 77 ±6 Methamidophos 73 ±18 78 ±3 77 ±2 Aldicarb Sulfoxide 81 ±31 84 ±6 83 ±3 Methiocarb 80 ±15 98 ±9 91 ±7 Aldicarb Sulphone 98 ±14 90 ±4 83 ±3 Methiocarb Sulfoxid 93 ±12 90 ±3 77 ±3 Ametryn 96 ±13 96 ±2 78 ±4 Methiocarb Sulphon 89 ±16 91 ±5 86 ±3 Aminocarb 86 ±9 85 ±4 73 ±4 Methomyl 95 ±22 90 ±5 89 ±3 Anilofos 84 ±12 92 ±1 77 ±4 Methoxyfenozide 82 ± ±1 83 ±4 Atrazine 91 ±15 92 ±4 80 ±3 Metosulam 87 ±12 93 ±4 86 ±4 Azinophos-ethyl 85 ±16 97 ±10 82 ±6 Metribuzin 98 ±25 86 ±6 81 ±4 Azinphos-methyl 97 ± ±9 79 ±8 Metsulfuron-methyl 85 ± ±3 87 ±4 Azoxystrobin 98 ±14 96 ±4 78 ±2 Monocrotophos 89 ±22 87 ±5 87 ±4 Benalaxyl 95 ±14 93 ±3 76 ±4 Myclobutanil 92 ±17 95 ±5 85 ±4 Bendiocarb 93 ±11 91 ±3 78 ±2 Nuarimol 78 ±20 89 ±4 81 ±2 Bensulfuron-Me 105 ±14 92 ±4 79 ±3 Omethoate 82 ±24 81 ±5 84 ±2 Bromuconazole 87 ±12 90 ±6 81 ±4 Oxadiargyl 75 ±16 84 ±4 77 ±7 Bupirimate 85 ±11 93 ±4 75 ±4 Oxadiazon 71 ±17 85 ±5 73 ±10 Buprofezin 98 ±18 94 ±4 74 ±6 Oxamyl 99 ±16 89 ±5 80 ±4 Butachlor 81 ± ±5 66 ±13 Oxycarboxin 87 ±17 92 ±5 87 ±2 Butralin 63 ±19 68 ±7 79 ±6 Oxydemeton-methyl 91 ±24 83 ±3 86 ±4 Carbaryl 93 ±13 93 ±2 79 ±2 Paraoxon-ethyl 89 ±15 91 ±3 86 ±4 Carbendazim 89 ±11 90 ±3 74 ±3 Parathion-ethyl 88 ± ±12 80 ±20 Carbofuran 99 ±16 93 ±2 80 ±2 Penconazole 78 ±16 89 ±3 85 ±5 Carbofuran-3OH 90 ±15 87 ±5 87 ±4 Pencycuron 71 ±16 84 ±2 81 ±7 Carboxin 76 ±15 67 ±18 73 ±5 Pendimethalin 61 ±19 74 ±1 62 ±11 Chlorfluazuron 24 ±17 30 ±5 24 ±16 Phenmedipham 88 ±18 88 ±2 86 ±3 Chlorpyrifos 65 ±15 76 ±6 68 ±4 Phenthoate 87 ±19 91 ±4 86 ±5 Chlorpyrifos-methyl 80 ±16 82 ±10 77 ±3 Phosalone 77 ±19 82 ±3 80 ±9 Clodinafop-propargyl 82 ±13 90 ±3 68 ±4 Phosphamidon 90 ±15 94 ±5 93 ±7 Clothianidin 93 ±12 85 ±4 82 ±3 Piperonyl butoxide 77 ±23 90 ±2 85 ±4 Cyanophos 95 ±11 94 ±8 72 ±6 Pirimicarb 91 ±18 97 ±3 89 ±1 Cyhalothrin-L 25 ±27 32 ±12 31 ±17 Pirimiphos-ethyl 84 ±18 99 ±3 87 ±4 Cymoxanil 97 ±12 88 ±4 79 ±4 Pirimiphos-methyl 83 ± ±8 88 ±6 Cyprodinil 80 ±10 89 ±3 68 ±4 Prochloraz 86 ±18 87 ±2 72 ±11 Deltamethrin 23 ±22 29 ±5 24 ±14 Profenofos 75 ±19 85 ±4 82 ±5 Demeton-S-methylsulphon 95 ±15 94 ±4 83 ±5 Promecarb 88 ±20 95 ±3 87 ±4 Diafenthiuron 11 ±30 6 ±35 42 ±64 Prometryn 83 ±16 91 ±3 83 ±4 Diazinon 96 ± ±4 82 ±4 Propamocarb-HCl 81 ±23 73 ±5 81 ±3 Dichlofuanid 87 ±16 69 ±12 53 ±4 Propargite 78 ±19 71 ±4 64 ±12 Diclorvos 113 ±19 69 ±16 79 ±6 Propiconazole 78 ±25 89 ±3 86 ±4 Difenoconazole 86 ±17 86 ±4 74 ±4 Propoxur 87 ±15 92 ±4 86 ±3 Diflufenican 64 ±12 79 ±4 68 ±5 Pymetrozine 59 ±28 68 ±8 56 ±5 Dimethoate 96 ±14 91 ±4 77 ±3 Pyrazophos 73 ±20 91 ±3 83 ±5 Dimethomorph 99 ±13 94 ±4 85 ±24 Pyrazosulfuron-ethyl 82 ±32 96 ±2 85 ±14 Diniconazole 92 ±14 90 ±3 80 ±7 Pyrethrins 62 ±20 86 ±4 61 ±15 Diuron 100 ±10 95 ±3 77 ±2 Pyrifenox 96 ±9 95 ±4 78 ±8 Edifenphos 82 ±11 92 ±3 77 ±4 Pyrimethanil 87 ±17 87 ±3 81 ±4 Ethion 64 ±16 79 ±5 68 ±4 Pyriproxyfen 65 ±17 80 ±1 71 ±8 Ethoprophos 86 ±31 91 ±6 80 ±7 Quizalofop-Et 72 ±17 85 ±3 74 ±7 Famoxadone 68 ±14 79 ±6 69 ±6 Spinosad-A 78 ±17 84 ±5 61 ±13 Fenamiphos 81 ±9 86 ±5 72 ±3 Spinosad-D 87 ±6 92 ±8 69 ±11 Fenarimol 78 ±14 90 ±5 77 ±4 Tebuconazole 76 ±21 95 ±13 84 ±9 Fenhexamid 88 ±7 88 ±7 58 ±5 Tebufenozide 88 ±17 89 ±6 90 ±7 Fenoxaprop-P-ethyl 112 ±20 81 ±6 117 ±17 Terbuthylazine 82 ±21 93 ±2 86 ±4 Fenpropathrin 48 ±17 60 ±7 52 ±7 Tetraconazole 92 ±20 93 ±3 87 ±5 Fenpyroximate 66 ±19 65 ±6 60 ±8 Thiabendazole 86 ±16 92 ±6 81 ±3 Fenthion 82 ±16 88 ±9 67 ±4 Thiacloprid 84 ±16 90 ±4 82 ±3 Fipronil 99 ±13 87 ±6 85 ±7 Thiamethoxam 87 ±21 84 ±4 91 ±4 Flamprop 86 ±13 84 ±5 76 ±3 Thifensulfuron-methyl 86 ±20 99 ±3 89 ±2 Flufenoxuron 29 ±17 39 ±7 34 ±15 Thiobencarb 77 ±20 86 ±3 87 ±4 Flumetsulam 105 ±14 91 ±4 78 ±5 Thiocyclam-OH 75 ±23 66 ±4 68 ±4 Fluroxypyr 104 ±20 88 ±9 77 ±5 Thiodicarb 95 ±12 96 ±2 73 ±3 Flusilazole 92 ±14 92 ±6 75 ±4 Thiometon 115 ±35 90 ±19 86 ±12 Flutolanil 94 ±12 99 ±4 76 ±3 Thiophanate-methyl 71 ±35 83 ±19 79 ±30 Hexaconazole 97 ±16 91 ±4 78 ±4 Tolclofos-methyl 84 ±18 82 ±6 76 ±8 Hexythiazox 68 ±16 74 ±5 79 ±5 Tolylfluanid 76 ±21 89 ±6 80 ±9 Imazalil 113 ±20 98 ±4 74 ±3 Triadimefon 84 ±19 89 ±3 87 ±3 Imazamethabenz-methyl 98 ±14 90 ±2 78 ±3 Triadimenol 97 ±23 87 ±8 80 ±4 Imidacloprid 85 ±16 93 ±5 85 ±4 Triazophos 88 ±19 92 ±2 89 ±2 Indoxacarb 70 ±22 90 ±3 74 ±10 Triclopyr-butotyl 68 ±16 84 ±2 77 ±8 Isoprothiolane 86 ±18 94 ±2 90 ±3 Trifloxystrobin 73 ±19 86 ±2 85 ±6 Isoproturon 90 ±17 90 ±3 90 ±2 Triflumizole 93 ±21 85 ±4 76 ±8 Linuron 77 ±16 94 ±3 88 ±4 Triforine 53 ±18 87 ±10 76 ±39 Lufenuron 43 ±18 55 ±18 42 ±18 Triticonazole 85 ±19 89 ±3 84 ±4 33

11 4. CONCLUSION In the last few years, the Egyptian exports of some agricultural products were considered to be of great concern, due to the international market demand, especially when exporting to EU. Multi-residue determination method of 150 pesticides is developed at 0.01 mg/kg limit of determination which fulfills the EU MRLs for organic agricultural products and baby foods. The mass spectrometry parameters were optimized to give the best sensitivity, and two MRMs were chosen for quantification and conformation; the selected MRMs were based on the optimized declustering potential and collision energy. The use of mobile phase ammonium format buffer concentration of 10 mm at ph 4 is the best condition for sensitivity and to separate all tested pesticides. The injection of the sample dissolved in methanol/buffer increased the sensitivity with regard to acetonitrile. The QuEChERS method followed by LC-MSMS was found to be the best combination for determination of the 150 pesticides in terms of high recovery rates, short time of analysis, low costs and safety. ACKNOWLEDGEMENT This work was carried out in the Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Foods, Agricultural Research Center, Ministry of Agriculture and Land Reclamation, incorporation with Fac. of Agriculture Dept of Biochemistry, Cairo University. The authors express their deep thanks to Prof. Dr. Sohair Ahmed Gadalla Ahmed for supporting our research. REFERENCES [1] The National Food Administration Data Requirements (7).Sweden Plant Protection Products. ns_en.htm) [2] CODEX Codex alimentarius commission Pesticide residues in food and feed. [3] Wilkowska, A. and Biziuk, M. ( 2011) Determination of pesticide residues in food matrices using the QuEChERS methodology.food Chemistry, 125, ( 3), 1, [4] Fenoll, J., Hell, P., Carmen, M., nez, M., M., Miguel, F, P. 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