Validation of Multiresidue Method for Determination of 340 ...curresweb.com/mejas/mejas/2016/418-429.pdf · Saudia Arabia, United Arab Emirates, Hungary, USA, UK and Germany (General

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.

Middle East J. Appl. Sci., 6(2): 418-429, 2016 ISSN 2077-4613

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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.

Reagents:

Acetonitrile from (Lab-scan) (HPLC, assay >99%), methanol 99.9% HPLC grade (Merck ).Formic acid

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


420

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

# Pesticide Name

0.01 mg/kg 0.05 mg/kg 0.1 mg/kg All levels

Mean % CV% Mean % CV% Mean % CV% Mean % CV%

1 Abamectin 88 3 102 9 99 4 96 9

2 Acephate 73 4 81 3 81 2 78 6

3 Acetamiprid 81 7 94 8 97 3 91 10

4 Aclonifen 94 9 86 9 99 5 93 9

5 Acrinathrin 120 18 102 12 91 7 105 18

6 Aldicarb 85 18 96 9 88 5 85 6

7 Aldicarb Sulfone 87 5 85 5 85 7 85 7

8 Aldicarb Sulfoxide 77 2 86 2 91 2 90 12

9 Ametryn 95 16 98 7 84 5 92 12

10 Amidosulfuron 98 12 95 11 89 8 94 11

11 Aminocarb 88 4 89 3 93 4 90 4

12 Anilofos 92 9 101 4 104 5 99 8

13 Atrazine 89 14 98 4 96 3 94 9

14 Azaconazol 75 6 100 8 90 3 88 13

15 Azamethiphos 82 12 100 7 90 4 91 11

16 Azimsulfuron 85 7 93 10 83 8 87 10

17 Azinophos-ethyl 82 6 92 6 89 6 88 7

18 Azinphos-methyl 85 12 102 6 92 2 93 11

19 Azoxystrobin 95 8 94 2 84 8 91 8

20 Beflubutamid 81 4 91 4 91 4 88 7

21 Benalaxyl 77 6 102 3 94 3 91 12

22 Bendiocarb 81 10 92 12 114 7 96 17

23 Bensulfuron-methyl 103 6 93 10 92 8 96 9

24 Benthiavalicarb isopropyl 104 10 109 10 92 4 102 11

25 Benzoximate 84 10 82 6 108 6 91 15

26 Bifenazate 93 7 101 3 98 4 97 6

27 Bispyribac 83 5 92 7 81 8 85 8

28 Bitertanol 75 13 89 14 96 4 86 15


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# Pesticide Name



29 Boscalid 77 6 92 9 94 5 88 11

30 Bromacil 85 15 102 7 93 6 94 12

31 Bromuconazole 66 9 109 7 93 8 89 22

32 Bupirimate 96 5 90 8 100 6 95 7

33 Buprofezin 86 4 100 2 97 3 94 7

34 Butachlor 84 4 98 3 98 1 93 8

35 Butocarboxim 83 19 87 10 105 8 86 7

36 Butocarboxim sulfoxide 78 1 91 3 89 1 92 16

37 Butralin 79 3 91 6 93 2 87 9

38 Butylate 96 9 97 5 98 3 97 6

39 Carbaryl 97 17 111 6 98 5 102 12

40 Carbendazim 77 12 87 6 81 3 81 9

41 Carbetamide 89 10 92 4 97 4 93 7

42 Carbofuran 109 5 109 10 121 4 113 8

43 Carbofuran-3OH 74 5 92 5 92 2 86 11

44 Carboxin 91 8 104 9 95 6 97 9

45 Chlorbromuron 107 18 94 18 115 6 105 16

46 Chlorbufam 111 12 118 11 110 8 113 10

47 Chlorfenvinphos 78 14 101 6 96 6 92 13

48 Chlorfluazuron 77 20 113 17 98 7 96 21

49 Chloridazon 71 8 92 6 100 4 88 16

50 Chlorpyrifos 91 9 93 8 97 3 94 7

51 Chlorpyrifos-methyl 90 19 103 9 104 2 99 13

52 Chlorsulfuron 73 16 101 11 87 8 87 17

53 Chlorthiophos 67 9 94 11 100 4 87 19

54 Chromafenozide 77 12 104 4 97 6 93 14

55 Cinidon Ethyl 76 5 106 10 92 7 91 16

56 Cinosulfuron 91 11 105 8 94 9 96 11

57 Clethodim 92 9 93 17 115 12 100 16

58 Clodinafop-propargyl 101 5 90 8 89 5 93 8

59 Clofentazine 79 5 101 7 96 3 92 12

60 Clomazone 88 13 101 7 92 6 94 10

61 Cloquintocet mexyl 85 3 97 7 101 3 95 9

62 Clothianidin 81 9 97 6 92 4 90 10

63 Coumaphos 106 18 93 5 94 8 98 13

64 Coumatetralyl 88 5 110 3 96 5 98 10

65 Cyazofamid 105 11 119 6 103 5 109 10

66 Cyclohexamid 80 12 96 10 91 2 89 11

67 Cyflufenamid 79 9 101 5 100 4 94 12

68 Cyhalothrin-L 59 12 94 9 89 8 81 22

69 Cymoxanil 77 10 103 8 98 2 93 14

70 Cypermethrin 74 14 92 9 104 5 90 16


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# Pesticide Name



71 Cyproconazole 77 10 101 10 92 2 90 14

72 Cyprodinil 79 8 95 5 100 4 91 11

73 Deltamethrin 100 18 93 13 84 9 92 15

74 Demeton-S-methyl 71 8 91 7 98 9 87 16

75 Demeton-S-methylsulfone 80 6 104 6 101 8 95 13

76 Desmedipham 90 13 104 7 105 3 100 10

77 Diafenthiuron 118 9 111 6 118 3 116 7

78 Diazinon 81 5 94 3 94 2 90 8

79 Dichlofenthion 77 16 105 11 96 14 93 18

80 Dichlorvos 108 19 99 18 87 11 98 18

81 Diclofop methyl 122 5 102 9 95 4 107 13

82 Dicrotophos 74 4 91 4 94 4 86 11

83 Diethofencarb 85 7 102 4 105 5 97 11

84 Difenoconazole 102 11 98 8 91 5 97 9

85 Diflufenican 93 11 96 5 88 7 92 9

86 Dimethachlor 100 11 109 3 98 5 102 8

87 Dimethenamid 80 6 96 4 93 6 90 9

88 Dimethoate 83 6 92 4 93 4 89 7

89 Dimethomorph 87 8 113 4 102 6 101 12

90 Diniconazole 86 5 102 5 94 5 94 9

91 Dinotefuran 78 3 100 4 92 2 90 11

92 Diphacinone 94 10 105 9 96 8 99 10

93 Disulfoton sulfoxide 78 8 99 2 98 4 92 12

94 Disulfoton_Sulfone 84 7 96 9 99 5 93 10

95 Diuron 83 6 104 10 105 2 97 13

96 DMF 94 10 106 9 91 4 97 10

97 Dodemorph 88 10 114 4 98 3 100 13

98 Dodine 92 17 96 18 112 18 100 19

99 Edifenphos 77 6 102 5 92 4 90 13

100 Emamectin 93 17 99 8 94 4 95 11

101 EPN 101 15 90 12 109 10 100 14

102 Epoxiconazole 82 6 110 8 107 8 100 15

103 Ethiofencarb 97 13 102 4 94 4 89 12

104 Ethiofencarb Sulfone 75 6 95 6 96 2 89 8

105 Ethiofencarb Sulfoxide 81 5 92 7 93 3 97 8

106 Ethion 96 8 93 6 92 4 94 6

107 Ethirimol 83 9 110 3 96 3 96 13

108 Ethofumesate 87 8 108 10 90 3 95 13

109 Ethoprophos 107 6 97 4 88 5 97 10

110 Ethoxyquin 102 13 110 8 121 6 111 11

111 Etoxazole 82 4 103 4 94 3 93 10

112 Etrimfos 79 4 96 1 97 3 91 10


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# Pesticide Name



113 Famoxadone 84 19 103 18 82 12 90 19

114 Fenamidone 83 10 108 6 97 9 96 14

115 Fenamiphos 81 10 98 5 95 4 96 12

116 Fenamiphos sulfone 94 18 103 6 91 5 85 11

117 Fenamiphos Sulfoxide 85 11 85 17 85 5 91 10

118 Fenarimol 73 21 104 13 91 10 89 20

119 Fenbuconazole 77 11 113 5 101 5 97 17

120 Fenfuram 93 8 107 8 98 6 100 9

121 Fenhexamid 75 20 96 6 104 10 92 18

122 Fenitrothion 68 13 107 15 86 16 87 24

123 Fenoxaprop-p-ethyl 73 10 103 7 95 4 90 16

124 Fenoxycarb 87 9 94 7 86 5 89 8

125 Fenpropathrin 91 6 99 6 93 4 94 6

126 Fenpropidin 75 11 112 8 98 3 95 18

127 Fenpropimorph 81 8 99 5 95 3 92 10

128 Fenpyroximate 82 4 100 6 91 2 91 9

129 Fenthion 75 9 99 4 98 4 95 18

130 Fenthion Oxon Sulfone 86 24 91 11 109 10 91 12

131 Fenthion Oxon Sulfoxide 87 8 83 2 103 8 89 10

132 Fenthion Sulfone 87 13 91 11 89 7 93 13

133 Fenthion sulfoxide 92 19 97 9 89 10 91 14

134 Fenvalerate 93 7 88 5 108 5 97 11

135 Flamprop 80 5 94 5 98 2 91 10

136 Flonicamid 101 18 72 6 94 4 89 18

137 Fluazifop-p-butyl 87 4 95 3 92 4 91 5

138 Flubendiamide 71 17 97 9 84 10 84 17

139 Flucythrinate 89 19 83 19 82 16 85 17

140 Flufenacet 77 20 88 9 95 6 87 14

141 Flufenoxuron 79 2 99 11 97 5 92 13

142 Flumetsulam 75 15 98 11 103 6 92 17

143 Flumeturon 91 18 106 10 109 6 102 13

144 Fluopicolide 89 7 96 8 97 8 94 8

145 Fluquinconazole 111 17 100 10 93 11 102 15

146 Fluroxypyr 83 18 99 8 109 8 91 12

147 Fluroxypyr meptyl 79 8 97 10 97 3 97 16

148 Flusilazole 80 10 110 7 93 3 94 15

149 Flutolanil 86 13 112 4 100 3 100 13

150 Flutriafol ND ND 104 14 87 8 95 15

151 Fluvalinate-tau 69 6 96 11 100 5 88 17

152 Foramsulfuron 99 8 103 12 100 12 101 10

153 Forasulam 76 10 93 9 95 2 88 12

154 Formetanate 89 12 110 6 100 3 99 11


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# Pesticide Name



155 Fosthiazate 83 4 102 3 104 4 96 11

156 Fuberidazole 80 11 91 9 90 6 87 10

157 Furathiocarb 84 3 94 6 97 5 92 8

158 Halosulfuron methyl 79 12 108 10 90 6 92 16

159 Haloxyfop Ethyl 82 9 106 3 105 4 98 13

160 Heptenophos 71 18 111 6 113 7 98 22

161 Hexaconazole 68 7 104 9 96 4 89 19

162 Hexazinone 81 10 98 6 95 6 91 11

163 Hexythiazox 89 8 96 6 98 4 94 7

164 Imazalil 85 14 113 5 97 5 99 14

165 Imazamethabenz-methyl 79 4 97 7 92 6 90 11

166 Imazamethpyr 93 8 92 6 90 5 92 6

167 Imibenconazole 67 10 109 10 93 5 90 21

168 Imidacloprid 77 11 89 4 97 4 88 11

169 Indoxacarb 105 18 104 13 98 8 102 13

170 Iprobenfos 72 13 95 7 100 3 89 16

171 Iprodione 84 11 93 9 89 7 89 10

172 Iprovalicarb 88 7 104 7 106 5 99 10

173 Isofenphos 82 6 96 6 97 7 92 10

174 Isofenphos-methyl 115 5 95 3 95 3 102 10

175 Isofenphos-oxon 82 9 92 3 89 5 88 7

176 Isoprothiolane 80 7 102 5 86 4 90 12

177 Isoproturon 87 8 104 4 99 2 97 9

178 Isoxaben 87 6 111 7 100 7 99 12

179 Karbutilate 80 5 97 5 99 7 92 11

180 Kresoxim-methyl 91 9 94 7 93 2 93 6

181 Lenacil 85 7 104 7 98 7 96 11

182 Linuron 100 10 99 7 99 9 99 8

183 Lufenuron ND ND 112 8 86 22 99 20

184 Malaoxon 91 10 105 7 93 3 97 10

185 Malathion 85 10 102 6 95 2 94 10

186 Mandipropamid 90 10 105 6 104 6 100 10

187 Mecarbam 88 5 107 8 99 5 98 10

188 Mefenacet 79 5 101 2 92 5 91 11

189 Mefenpyr diethyl 80 7 98 4 92 5 90 10

190 Mepanipyrim 67 20 99 5 96 6 88 20

191 Mepronil 79 5 101 3 88 4 89 11

192 Metaflumizone 75 13 73 21 99 14 82 21

193 Metalaxyl 93 3 105 6 102 2 100 6

194 Metamitron 85 8 93 6 89 5 89 7

195 Metazachlor 97 4 106 7 103 4 102 6

196 Metconazole 77 5 105 5 108 3 97 16


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# Pesticide Name



197 Methabenzthiazuron 93 11 109 7 96 5 99 10

198 Methacrifos 94 16 100 18 106 6 100 14

199 Methamidophos 84 12 75 11 92 7 83 13

200 Methidathion 103 9 120 4 108 4 110 9

201 Methiocarb 90 5 106 6 99 3 91 9

202 Methiocarb Sulfone 81 4 97 6 93 2 91 8

203 Methiocarb Sulfoxide 81 4 93 5 97 2 99 8

204 Methomyl 86 6 98 6 96 4 93 8

205 Methoprotryne 73 9 105 3 94 4 91 16

206 Methoxyfenozide 103 19 114 7 92 7 103 15

207 Metobromuron 91 11 118 8 101 5 103 13

208 Metolachlor 90 7 95 6 96 4 93 6

209 Metosulam 111 8 86 9 97 9 98 13

210 Metoxuron 74 18 96 12 85 6 85 16

211 Metribuzin 88 6 99 9 101 2 96 8

212 Mevinphos 104 6 91 7 98 14 98 11

213 Molinat 77 16 100 8 110 4 96 17

214 Monocrotophos 82 5 94 5 93 3 89 7

215 Monolinuron 106 15 100 2 98 1 101 9

216 Monuron 85 4 91 6 94 4 90 6

217 Myclobutanil 115 15 100 5 86 4 100 16

218 Napropamide 69 8 99 7 100 1 89 17

219 Neburon 80 10 97 3 98 7 92 11

220 Nicosulfuron 117 13 105 8 101 4 108 11

221 Nitenpyram 84 12 95 5 88 5 89 9

222 Novaluron 86 16 112 8 90 11 96 16

223 Nuarimol 80 17 107 11 93 6 93 16

224 Ofurace 82 10 107 5 100 3 96 12

225 Omethoate 71 3 86 4 88 3 82 10

226 Oxadiargyl 107 18 86 8 105 17 99 18

227 Oxadiazon 77 7 91 7 93 3 87 10

228 Oxamyl 86 5 79 5 96 4 87 9

229 Oxasulfuron 100 16 117 10 96 5 104 14

230 Oxycarboxin 76 11 92 5 92 4 87 11

231 Oxydemeton methyl 77 6 85 6 87 6 83 8

232 Paclobutrazol 82 5 105 7 87 3 91 12

233 Paraoxon-ethyl 91 5 100 5 97 5 96 6

234 Paraoxon-methyl 91 20 98 13 93 5 94 13

235 Parathion-ethyl 88 10 85 9 103 16 92 15

236 Penconazole 110 11 101 8 98 5 103 10

237 Pencycuron 75 7 85 6 91 4 83 10

238 Pendimethalin 85 7 99 10 97 3 94 10


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# Pesticide Name



239 Permethrin 69 8 87 6 87 3 81 12

240 Phenmedipham 85 6 104 6 104 3 98 11

241 Phenthoate 96 11 97 4 99 5 97 7

242 Phorate 79 8 90 6 99 2 94 11

243 Phorate sulfone 87 6 106 5 90 3 95 4

244 Phorate sulfoxide 92 5 97 4 95 1 90 11

245 Phosalone 90 13 102 5 100 7 97 10

246 Phosmet 85 7 120 5 109 5 105 15

247 Phosphamidon 80 7 94 4 97 4 90 10

248 Phoxim 85 4 104 6 96 4 95 10

249 Picolinafen 96 15 118 5 94 3 103 14

250 Picoxystrobin 95 7 93 5 91 4 93 5

251 Piperonyl butoxide 86 5 95 6 98 3 93 7

252 Pirimicarb 76 11 112 6 99 6 90 5

253 Pirimicarb desmethyl 86 3 93 4 91 3 96 18

254 Pirimiphos-ethyl 92 4 99 4 96 2 96 5

255 Pirimiphos-methyl 78 2 97 5 95 5 90 10

256 Prochloraz 83 12 89 6 107 8 93 14

257 Profenofos 72 18 92 8 94 5 86 15

258 Profoxydim-Li 76 4 108 9 100 5 94 16

259 Promecarb 84 18 97 9 101 6 94 13

260 Prometon 88 11 112 3 103 2 101 12

261 Prometryn 90 8 99 7 93 5 94 8

262 Propachlor 88 4 101 3 96 4 95 7

263 Propamocarb HCl 75 3 79 3 80 3 78 4

264 Propanil 82 22 91 13 84 5 86 15

265 Propaquizafop 88 9 107 4 94 6 96 10

266 Propargite 87 6 96 6 97 4 93 7

267 Propazine 90 11 110 10 89 4 96 13

268 Propazine-2-hydroxy 78 9 81 6 82 4 80 7

269 Propetamphos 108 18 101 8 102 11 104 13

270 Propiconazol 77 13 94 8 89 6 87 12

271 Propoxur 100 10 86 10 101 2 96 11

272 Propyzamide 78 8 106 5 91 4 92 14

273 Proquinazid 76 4 99 4 93 3 90 12

274 Prosulfocarb 89 4 94 2 94 2 92 4

275 Prothioconazole 82 21 84 10 90 7 85 13

276 Pyraclostrobin 91 6 110 7 99 3 100 10

277 Pyraflufen Et 111 23 98 10 96 7 101 17

278 Pyrazofos 84 5 98 7 95 5 92 9

279 Pyrazosulfuron-ethyl 78 7 93 6 91 9 87 11

280 Pyrethrins 86 5 105 5 98 2 96 9


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# Pesticide Name



281 Pyridaben 82 2 100 7 94 3 92 9

282 Pyridalyl 89 11 97 5 88 14 91 11

283 Pyridaphenthion 76 13 101 6 90 6 89 14

284 Pyridate 74 2 75 5 73 3 74 4

285 Pyrimethanil 87 12 102 5 96 5 95 10

286 Pyriproxyfen 76 4 98 8 99 3 91 13

287 Pyroxsulam 85 11 99 7 89 5 91 10

288 Quinalphos 73 4 99 4 100 4 91 15

289 Quinmerac 76 5 80 4 89 4 82 8

290 Quinoxyfen 100 9 99 5 98 2 99 6

291 Quizalofop-ethyl 87 11 98 4 89 2 91 8

292 Rimsulfuron 98 11 99 14 107 6 102 11

293 Rotenone 102 23 96 6 82 3 94 17

294 Sebuthylazine 70 19 93 12 101 7 101 20

295 Sebuthylazine desethyl 84 4 102 23 116 14 88 19

296 Simazine 94 11 118 4 95 7 102 13

297 Simetryn 84 8 105 4 93 3 94 11

298 Spinetoram 88 13 104 9 87 11 93 13

299 Spinosad 100 19 84 14 91 12 92 16

300 Spirodiclofen 88 5 97 7 92 2 92 6

301 Spiroxamine 83 6 106 3 102 2 97 11

302 Sulfotep 84 9 85 8 84 2 84 7

303 Tebuconazole 107 16 101 5 97 5 102 11

304 Tebufenozide 94 8 90 7 91 7 91 7

305 Tebufenpyrad 85 7 97 7 90 6 91 8

306 Tebutam 97 6 90 7 97 5 95 7

307 Tebuthiuron 80 4 100 7 95 3 92 11

308 Tepraloxydim 84 11 95 14 89 9 90 12

309 Terbufos 85 6 95 3 94 2 92 6

310 Terbumeton 83 5 105 3 93 2 94 10

311 Terbuthylazine 82 8 98 8 90 4 90 10

312 Terbutryn 96 2 101 5 93 3 97 5

313 Tetrachlorvinphos 92 11 89 14 85 8 89 11

314 Tetramethrin-NH4 88 12 105 6 89 5 94 11

315 Thiabendazole 77 11 92 7 96 2 88 11

316 Thiacloprid 84 7 93 6 96 2 91 7

317 Thiamethoxam 79 4 91 6 89 6 87 8

318 Thifensulfuron-methyl 82 10 98 4 99 7 93 11

319 Thiobencarb 81 4 95 4 99 3 92 9

320 Thiodicarb 69 15 102 6 95 7 89 18

321 Thiofanox 98 24 74 20 100 15 91 23

322 Thiophanate-methyl 100 7 99 9 88 6 96 9


428

# Pesticide Name



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

Round: 19193 Sweet pepper Lab No: 26 Report date: 9/7/2015

Carbofuran 131 118 -0.5 Deltamethrin 68.3 110 2.8 Fenarimol 29.2 32 0.4 Flutriafol 181 147 -0.9 Phosalone 137 133 -0.1 Pyrazophos 123 130 0.3

Round:19172 Sweet pepper Lab No: 28 Report date: 28/7/2014

Carbendazim 61.2 51 -0.8 Chlorfenpyr 54.4 48 -0.5 Chlorpyrifos me 122 135 0.5 Clofentezine 43.4 30 -1.4 Fenitrothion 76.5 78 0.1 Heptenophos 70.4 93 1.5 Omethoate 47.9 48 0 Terbuthylazin 121 91 -1.1 Thiabendazole 40.2 33 -0.8 Triadimenol 42.2 33 -1


429

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.

http://www.eurachem.org/

Documents

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