4-04-IAEA CN-222-128-Cesio

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4-04-IAEA CN-222-128-Cesio Pesticide residues analysis in Bee products: a useful tool to assess food safety and agro ecosystems sustainability Silvina Niell, Natalia Gérez, Andres Perez, RosanaDiaz, YamanduMendoza HoracioHeinzen,María Verónica Cesio* *GACT: Pharmacognosy & Natural Products, Fac. de Quimica, UdelaR PAAP; Litoral Chemistry Department, CENUR, UdelaR 1 Apiculture in Uruguay • 3.000 beekeepers • 500.000 beehives • 12.000 ton honey/ year • 90% of the production is exported • U$S 35.000.000 • High social impact Toxic substances bees faced nowadays - Increased use of herbicides & agrochemicals in agriculture activities. - Increased use of “bee chemicals” Ee 2000 2005 2010 Herbicides 2.079.534 4.946.492 11.856.155 (kg active principle) Insecticides 503.356 1.057.895 1.732.906 (kg active principle) Changes in soil use Disminishing of polen & néctar sources. Beehive products important food components Bee and bee products as markers of the environmental status of an agroecosystem why? The hive is not only a source of food but also could give environmental information environment food THE ANALYSIS FOR PESTICEDES RESIDUES OF THE BEE PRODUCTS IS OF PARAMOUNT IMPORTANCE Beehive representative matrices Propolis Honey Wax Royal Pollen jellly From the analysis of hive matrices, information for both, food safety & environmental pollution can be gathered Strategy of the work Monitoring Pesticides Methodologies Statistical development Samples selection analyzed analysis 10 Validation parameters • Lineality • % Recovery • Repeteability (RSD r) • Intermediate precision (RSD wR ) •Reporting Límit • Matrix effect Method validation and quality control procedures for pesticide residues analysis in food and feed. Document 11 No. SANCO/ 12571 /2013 Propolis has a complex chemical composition 80% of the chemicals in propolis are Flavonoids, Aromatic resins They have similar physicochemical properties to the most common insecticides Clean-up through MSPD Dispersant for propolis: aluminium salt Al 2(SO4)3 OH OH O O O OH O OH O +3Al Al+3 Complexation reaction: Sequesters interferring compounds from the matrix changing its polarity, allowing a better chromatographic separation Pesticides in propolis: following the clean up process PEREZ-PARADA ANDRÉS; COLAZZO M.; N. BESIL; GEIS ASTEGIANTE, L; REY F; HEINZEN, H Determination of coumaphos, chlorpyrifos and ethion residues in propolis... Journal of Chromatography - A, v.: 1218 34, p.: 5852 - 5857, 2011 Simultaneous determination of fluvalinate & coumaphos using GC-MS(SIM) Pesticide t R m/z rec.(%) RSD(%) Coumaphos 12.68 334 106 1 362 364 τ-Fluvalinate 13.9 181 96 1 250 252 Chemical complexity of the different matrices 16 Honey QuEChERS citrate Sample+H 2O+MeCN+ CEN Dispersive Clean-up (MgSO 4, PSA); Freeze out Instrumental Analysis NEILL, S.; M.V. CESIO; HEPPERLE J; ROUX D; KOBERG D; KEISH L; ANASTASIADES, M; HEINZEN, H . Analisis multiresiduo de 41 pesticidas en miel por LC-MS/MS: evaluación de 18 dos métodos de Clean-up. Agrociencia (Uruguay), v.: 17 1, p.: 101 - 107, 2013 Honey QuEChERS citrate LC-MS/MS Pesticides analyzed in (+) mode Pesticides analyzed in (-) mode 19 QuEChERS citrate validation 0,1 mg/kg %rec RSD Trifluralin 89 19 Atrazina 87 11 Clorotalonil 78 10 Diazinon 95 11 Clorpirifos-metil 58 5 Malation 73 7 Clorpirifos 61 11 0,1 mg/kg 0,05 mg/kg 0,01 mg/kg Clorfenvinfos 81 13 % rec RSD % rec RSD % rec RSD Fipronil 82 4 Endosulfan a 131 3 109 14 85 11 Metidation 93 11 Endosulfan b 81 4 97 3 54 10 Endosulfan nd nd Ciflutrina 77 5 96 3 76 6 p,p-DDE 69 12 Cipermetrina 215 5 301 4 127 3 Etion 81 11 Tau- Endosulfan sulfato 81 6 Fluvalinato 87 4 74 4 92 5 p,p-DDT 76 13 Deltametrina 79 5 93 6 79 7 Acetamiprid 75 4 Bromopropilato 78 8 Azinfos-metil 80 11 Cumafos 83 7 Ciflutrina 97 14 Cipermetrina nd nd Tau-Fluvalinato 82 14 20 Deltametrina 84 13 Analytic al Method for candies with honey and propolis • 10 g crushed candies + 1.5g NaCl + 8g MgSO4 + 10 mL ethyl acetate + 10mL distilled water. Extraction • 5 minutes vigorous manual agitation • 15 minutes of centrifugation at 3000 rpm . • 5.0 mL supernatant + 750 mg MgSO4 • 150 mg PSA + 100 mg GCB d-SPE • Vortex and centrifugation Clean-up • 4.0mL to dryness under reduced pressure. • 1,00 mL of TPP (Internal Standard) in EtAc is added and analized GC-MS. GC-MS Some analytical parameters of the validated methodology Abundance Abundance m/z 334 85 600 m/z 314 550 m/z 362 75 500 m/z 258 65 450 m/z 364 400 55 m/z 286 350 300 45 Coumaphos Chlorpyrifos 250 35 200 150 25 100 15 50 0 5 26.60 27.00 27.40 27.80 28.20 28.60 29.00 Time (min) 14.20 14.60 15.00 15.40 V samples of both honey and propolis cointaining candies were analyzed. VCoumaphos and chlorpyrifos residues were found in propolis candies. VNo pesticides were detected in honey candies. Pesticide residues in propolis and honey candies Final methodology for beeswax Niell S, Cesio M.V, Hepperle J, Doerk D, Kirsch L, Kolberg D, Scherbaum E, Anastassiades M, Heinzen H. J Agric Food Chem. 2014 Apr 30;62(17):3675-83. first Second Confirmation Quantification ion PesticidePesticideTranssision SelectedDP ions (m/z) CE Transsision DP CE m/z>m/z m/z>m/zIon (m/z) (m/z) AcetamipridAtrazina 223,2>126,1200, 55 215,217 25 223,2>99,2215 55 200 47 AmitrazAzinfos-metil 294>163,1 160, 100 132,125 17 294,3>122,1125 100 160 37 AzoxistrobinBromopropilato 404,1>372,1341, 343, 72 339, 183 19 404,1>344343 72 183 31 Boscalid 343,1>139,8 89 24 343,1>112,2 89 57 Cipermetrina 163, 181, 165, 209 181 163 Carbaril 202,2>145 68 12 202,2>127,1 68 35 Clorfenvinfos 267, 269, 323 323 267 Carbendazim 192,1>160 80 23 192,1>132,1 80 42 Clorotalonil 264, 266, 268, 231 264 266 Clorpirifos-etil 349,9>198,1 80 23 349,9>97 80 38 ClotianidinClorpirifos-metil 250>169 286, 56 288, 125 17 250>132 125 56 286 19 CumafosCiflutrina 363,1>227,1163, 165, 100 206, 226 33 363,1>307 226 100 163 22 DimetoatoDeltametrina 230,1>198,9181, 253, 50 251, 255 13 230,1>125,1251 50 181 28 HaloxifopDiazinon metil 376,1>316,1179, 137, 85 304, 152 22 376,1>288,1 304 85 179 36 HexitiazoxEndosulfan alfa 353,1>228,1241, 237, 70 195, 239 23 353,1>168,1237 70 195 34 EndosulfanImazalil beta 297>159195, 237, 130 241, 207 32 298,9>161,1 241 130 195 30 EndosulfanImazapir sulfato262>217,2272, 274, 137 387, 237 28 262>220,1 387 137 272 24 Imidacloprid 256,1>209,1 86 22 256,1>175,1 86 23 Etion 231, 153, 384 384 231 Iprodione 330,1>245,1 64 21 330,1>288 64 16 Fipronil 367, 369, 213 213 367 Metomilo 163,1>88 44 12 163,1>105,9 44 14 Lambda Cialotrina 181, 197, 208 208 181 MetsulfuronMalation metil 382,1>167,1173, 125, 62 127, 2193 382,1>140,9 127 62 173 29 Paration-metilMetidation 265>129,1 145, 85, 38 125, 302 11 265>229,2302 38 145 13 Piraclostrobinp,p-DDE 388,1>194,2246, 67 248, 318 17 388,1>163,1 318 67 246 39 Tebuconazolp,p-DDT 308,1>70,3235, 85 237, 165 40 308,1>125 165 85 235 45 TiaclopridTau-Fluvalinato 253,1>126 98 28 255>128 98 25 250, 252, 181, 251 181 25027 Tiametoxam 292>211,1 88 15 292>181,2 88 43 Trifluralin 306, 290, 335 335 306 QuEChERS LC-MS/MS /VALIDATION 0.2 mg/kg 0.1 mg/kg 0.01 mg/kg RL Rec(%) RSD (%) Rec (%) RSD (%) Rec (%) RSD (%) (mg/kg) Amitraz 73 2 77 4 85 13 0,01 Atrazina 89 4 94 4 97 13 0,01 Azoxistrobin 110 3 1110.2 mg/kg 4 112 0.1 14 mg/kg 0,01 0,.01 mg/kg RL Boscalid 98 6 103Rec (%) 8 RSD (%) 105 Rec (%) 24 RSD (%) 0,1 Rec (%) RSD (%) (mg/kg) Carbarilo 104Iprodione 4 111 108 5 27 165 123 6 24 0,1 nd 0,2 Linuron 96 6 99 5 107 15 0,01 Carbendazim 72 4 78 6 100 15 0,01 Metalaxil 107 3 109 5 113 15 0,01 Carboxin 104 4 108 6 107 9 0,01 Metomilo 111 8 127 4 113 12 0,01 Clorpirifos-etil 92 6 105 6 218 15 0,1 Metoxifenozide 107 4 111 5 146 7 0,1 Clodinafop-propargil 104Metolaclor 1 105 96 5 3 104 103 14 2 0,01 120 17 0,01 Cumafos* 144Metribuzin 3 229 102 4 7 1679 111 8 7 0.1 nd 0,1 Cihalotrina-lambda 92Ometoato 16 100 96 9 11 nd 104 8 0,1 111 6 0,01 Dimetoato Pendimetalin102 6 106 81 6 19 107 83 7 19 0,01 nd 0,1 Epoxiconazol 94Pirimicarb 4 106 108 7 3 nd 114 4 0,1 111 11 0,01 Fenpropatrin Profenofos95 7 102 89 11 6 159 94 15 6 0,1 92 18 0,01 Flutriafol Piraclostrobin101 11 114 102 15 4 nd 106 5 0,1 112 14 0,01 Hextiazox Tebuconazol182 4 323 98 5 11 2800 99 3 5 - 103 27 0,1 Imidacloprid Tebufenozide108 7 106 96 8 4 100 99 9 5 0,01 104 15 0,01 Tetraconazol 103 7 104 6 111 23 0,1 Tiacloprid 113 7 120 5 108 18 0,01 Tiametoxam 103 14 103 7 106 15 0,01 Tiodicarb 90 5 85 7 107 12 0,01 Tiofanato-metil 94 5 91 7 97 1528 0,01 Triflumuron 91 5 98 10 104 12 0,01 QuEChERS Matrix Effect lineal range: 0.005-0.25 mg/kg LC 0.012-0.24 mg/kg GC Moderate Matrix effect (<13%) for most of the pesticides •azoxistrobin (24%) •carbendazim (32%) •imazapir (-55%) •cumafos (24%) •hexitiazox (-41%).
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