Study and development of quechers gc / ms 3 sim technique to analyse multi pesticide residues in soil

owadays, more than1500 different kinds of pesticides have been used, classified based on chemical structure (chlorinated, phosphorus, Carbamate, Pyrethroid ...) or by application (insecticide, fungicide, herbicide ...) In Vietnam, more and more different pesticides are used in agriculture (increased from 189 substances in 2003 to 437 substances in 2010). Pesticides have been known to be contaminants that persist for a long time in the environment. So over time, there will be residues of many different pesticide in the soil. Pesticides accumulated in the soil can be transferred to humans via the food chain, being potentially harmful to human health. Thus, the level of pesticide residues in the agricultural soil needs to be monitored as farmers switch to organic farming (green and safe agriculture) Currently, pesticide residues in soil is determined separately for each substance group, with different procedures, so increasing costs, extend the analysis time and analysis process becomes more complicated. Therefore, the project "Study and development of QuEChERS GC / MS 3 SIM technique to analyze multi pesticide residues in soil" was carried out, in order to reduce the analysis time, minimize the number of analytical steps, use fewer reagents in smaller amounts and provide high recovery 4 2. Scope of thesis Develop a rapid and simple method for analysis of multi-residue pesticides, including organophosphate, organochlorine, carbamate, and pyrethroid compounds in soil, with sample preparation based on QuEChERS technique and determination by GC-MS. 3. Main contents of thesis - Survey and select optimal conditions to analyze multi pesticides on GC / MS system: injection mode, temperature program, parameters for mass spectrometry. - Investigate optimization of processing samples including extraction, cleaning and enrichment: solvent and extraction time, influencing factors, cleaning agents. - Verification of analytical methods: determination of linear range, calibration curve, detection limit and quantitative limit, recovery coefficient and repeatability of the method. - Apply the optimal procedure to analyze 30 soil samples and compare results with 04 laboratories at Vietnam and Korea 4. New contributions of the thesis - This is the first study in Vietnam to apply the QuEChERS method for multi-residues pesticides analysis in the soil - d-SPE has been applied instead of soxhlet extraction, so the sample preparation time has been reduced from 24 h to 25 minutes, consuming only 15 ml solvent (the popular methods consume at least 300ml of solvent) - With a single run, 103 pesticides in soil have been analyzed (the latest publication, only 42 pesticides in the soil were quantified) - In quantitative step, only GC/MS is used (in publications, GC/MS/MS is analytical method usually selected) 5 OVERVIEW CHAPTER 1. Pesticides (herbicides, fungicides or insecticides) are environmental pollutants often found in soil, water, atmosphere and agricultural products, and may exist in harmful levels, posing an environmental threat. Even low levels of pesticide can cause adverse effects on humans, plants, animals and ecosystems. The application of pesticides has increased appreciably during the past few decades, resulting in a potential risk for the human health. Over 95% of sprayed pesticides reach a destination other (usually soil environment) than their target. So, the determination of pesticide residues in soil has been rising in demand. The analysis of pesticide residues in soil consists of sample preparation and the instrumental determination. The aim of the sample preparation is to isolate the trace amounts of analytes from a large quantity of complex matrices and eliminate the interferences from the soil matrix as much as possible. Typical sample preparation steps include the homogenization, extraction, and clean-up. Due to the low concentration levels of pesticide in soil, a technique strong enough to extract bound residues is necessary. The most common of these techniques are mechanical agitation by shaking, sonication, microwave energy, and liquid-solid extraction (e.g.: Soxhlet extraction; accelerated solvent extraction, pressurized liquid extraction, and, supercritical fluid extraction). The most popular clean-up methods are based on the solid phase extraction technique using florisil cartridges. These established methods are effective, yet time consuming (taking as long as 1,5day), complicated and expensive. 6 As a result, the development of new analytical methods for the determination of multi residue pesticides in soil samples is currently a high-interest research area. Many innovations have occurred in analytical methods for the extraction of organic compounds from different matrices that reduce the analysis time, minimize the number of analytical steps, use fewer reagents in smaller amounts and provide high recovery. In 2003, Michelangelo Anastassiades developed a method for the multi- class, multi-residue extraction of pesticides in fruits and vegetables. This method was called QuEChERS, which stands for Quick, Easy, Cheap, Rugged and Safe, and it is based on dispersive solid phase extraction (d-SPE). In d-SPE, pesticides are extracted with an aqueous miscible solvent with a high amount of salt, in order to induce liquid phase separation. The QuEChERS method is particularly popular for the determination of wide range of chemical residues, mostly pesticides in various food matrices, because of its simplicity, low cost, and high efficiency with a minimal number of steps. QuEChERS approach is very flexible and it serves as a template for modification depending on the analyte properties, matrix composition, equipment and analytical technique available in the laboratory. Unfortunately, the application of the d-SPE technique in the analysis of pesticides in agricultural soils is very rare, with a limited number of pesticides analyzed. Currently, in Vietnam, QuEChERS method has only been applied for pesticide analysing in food and medicinal plant sample. The level of pesticide residues in soil, still determined by GC/ECD or GC/NPD, with Shoxlet extraction. 7 EXPERIMENT CHAPTER 2. Materials and apparatus 2.1 Stock standard solutions of 2.00 mg/g of each pesticide were prepared in MeCN, stored in −20◦C. Intermediate mixture standard solution (10 mg/kg) was prepared by diluting the stock standard solutions with MeCN. Gas chromatography mass spectrometry system MS-QP 2010 (Shimadzu), DB-5MS capillary column Research content 2.2 2.2.1 Selection of pesticides 103 different group pesticides , have been widely used in agricultural cultivation in Vietnam as well as in the world and have suitable chemical and physical properties for analysis on GC / MS has been selected. 2.2.2 Survey and select optimal conditions to analyze multi pesticides on GC / MS system - Survey the injection temperature and injection speed, sample volume and carrier gas - Survey temperature program - Select ion mass for quantitative 2.2.3 Investigate optimization of processing samples - Solvent and extraction time, - Influencing factors: pH, ionic strength, organic, - Absorbent agents Develop analytical process. 2.3 From the research results obtained, develop an analytical procedure including: sample processing and setting on GC / MS equipment. Verification of analytical methods: determination of 8 linear range, calibration curve, detection limit and quantitative limit, recovery coefficient and repeatability of the method. Analysis of real samples 2.4 Apply the optimal procedure to analyze 30 soil samples (were collected in Nam Dinh, Nghe An, and Hanoi with different pH, ion exchange and organic matter characteristics) and compare results with 04 laboratories at Vietnam and Korea RESULTS AND DISCUSSION CHAPTER 3. Survey and select optimal conditions to analyze multi 3.1 pesticides on GC / MS system 3.1.1 Survey the injection temperature and injection speed With the slow injection mode, the sensitivity of pesticides surveyed was 50% lower than that of the fast and medium mode. In addition, the stability is not high, most % RSD> 30%. The sensitivity has been significantly improved, and there is not so much difference between the fast and medium sample injection modes. However, the fast injection mode is more stable,% RSD is <10% at all survey temperatures. The result also show that the sensitivity of the substances is relatively uniform at different temperatures and reaches a maximum at 260 0 C (except for permethrine at 210 0 C). However, at 260 0 C and in the fast injection mode the highest stability with relative standard deviation% RSD is around 2-4%. From the research results show that the optimal injection port temperature for pesticide analysis is 260oC and the sample injection speed is set to "Fast". 9 3.1.2 Sample injection volume and carrier gas speed The effect of carrier gas velocity complies with the van Deemter equation. According to the calculation results, the sample injection volume is suitable for MeCN from 1.0 to 1.2 L. From the theoretical calculation, combined with the carrying gas speed, we conducted experiments to find the sample injection volume of 1.0 L and the carrier gas rate of 1.7 mL / minute, which is suitable for the analysis of pesticides. 3.1.3 Temperature program 06 temperature programs have been run. The results show that program No. 6 has the best separation. Specifically, the initial temperature of 60 ° C, hold for 1 minute; increased 20oC/min to 180oC; increased 10oC/min to 190oC; increased 3oC/min to 240oC; increased 10oC/min to 300oC, and held for 5 minutes. Figure 3.9. Standard chromatogram at different temperature program 50 100 150 200 250 300 350 0 5 10 15 20 25 30 35 10 Program 1 Program 2 Program 3 Program 4 Program 5 Program 6 Terbufos & Quintozene Diazinon, Etrimfos & BHC-delta Chlorpyrifos, Fenthion & Parathion Fludioxonil & Isoprothiolane pp’-DDE, Oxadiazone & op-DDD pp’-DDD, op-DDT & Ethion Figure 3.10. Resolution of some pesticides at different temperature programs 11 3.1.4 Select ion mass for quantitative The selection of a main fragment for quantitation is based on the following priority criteria:  Main fragment of pesticide (M +: Mother ion);  If there is no mother ion, select the segment with the highest signal. Priority should be given to the fragment having m / z greater than 100 to avoid the effect of fragments formed by solvent;  Avoid using identical forming fragments of pesticides with adjacent elution times. On the basis of the resulting chromatograms, the main and sub- fragments have been established. Investigate optimization of processing samples 3.2 3.2.1 Selection of extraction solvent Table 3.6 shows that MeCN has the best recovery and repeatability among the researched solvents and is used as the solvent for sample extraction. Table 3.6. Recovery, standard deviation of pesticide at different extraction solvents Solvent Recovery (%) RSD (%) MeCN 84 - 111 3 - 17 MeOH 40 - 142 3 - 26 EtOAc 72 - 111 1 - 30 Acetone 59 - 126 1 - 45 3.2.2 Selection of extraction time Extraction time of 1, 3, 5, 10 and 20 minutes using QuEChERS technique has been surveyed. The results show that the extraction time 3-5 minute is most appropriate (Table 3.8). 12 Table 3.8. Results of the effect on sample extraction time Extraction time 1’ 3’ 5’ 10’ 20’ Recovery (%) 47-114 69-110 69-111 67-119 63-111 % RSD 2-44 1-19 2-18 1-16 1-22 Pesticides with recovery < 50% 1 0 0 0 0 Pesticides with recovery 50-60% 5 0 0 0 0 Pesticides with recovery 60-70% 11 1 2 4 7 Pesticides with recovery >70% 86 102 101 99 96 3.2.3 Effect of absorbents Interferences have maximum absorption in the wavelength range of 190 - 230 nm, including fats ((max = 205 - 233 nm), sugar compounds ((max = 190 nm); and triaglycerol ((max = 210 nm). Figure 3.14. Efficient removal of interferences by adsorbents (a) florisil; (b) C18; (c) PSA; and (d) GCB Experimental results (Figure 3.14) show that florisil, C18, PSA with content of 20 mg / mL and GCB with content of 10 mg / L are all capable of cleaning. 13 Figure 3.16. The soil sample chromatogram is purified by various absorbents 14 However, when observing the baseline as well as the intensity of impurities in the chromatogram (Figure 3.16), we can see: - For florisil: baseline rise to 150,000 and decrease to baseline until 18 minutes. Impurities are very much with high intensity. - For C18 and GCB: baseline rises to 100,000 and after 11 minutes decreases to baseline - For PSA: the baseline only rises to 60,000 to the baseline (50,000) after 8 minutes Therefore, absorbents PSA (500mg) and GCB (10mg) were selected for the cleaning of pesticides in soil samples. 3.2.4 Effect of sample matrix - Effect of pH: pH can affect the chemical and physical properties of pesticides as well as the efficiency of sample extraction. With agriculture soil pH ranges from 5.5 to 8.5. Experimental results show that pH from 5 to 9 has no significant effect on the extraction process. The recovery is not much different and is in the range of 75 - 110% with % RSD ranging from 1 - 18%. - The influence of ionic strength The ionic level of the mixture, making it easier to extract pesticides. However, some metals with the ability to form complexes such as Co, Cd and Cu will reduce the ability to extract some highly polarized pesticides. Studies with Cu content of 1000 mg / kg showed no effect on the extraction of pesticides in soil. The recovery ranges from 69 - 110% with % RSD from 1 - 17%. 15 - Influence of sample size and organic matter content: Studies conducted with coarse-grained (<2 mm), fine- grained (<0.05 mm) and organic matter content (10%) showed that the sample was crushed through a 2 mm sieve and organic matter content not exceeding 10% will not affect sample extraction performance. Develop analytical process 3.3 3.3.1 Sample preparation procedure Sample preparation is summarized in Figure 3.18 Figure 3.18. Sample preparation diagram 3.3.2 Analytical procedure on the GC/MS system a) GC setting Injection port temperature: 260 oC; Injection volume: 1,0 L; Air flow rate: 1.7 mL / minute; 10 g sample 2ml aliquot /tube 5ml Take 0,5ml analysis 10ml H2O + IS. Shake 1 min. Wait 30 min 4g MgSO4 và 1g NaCl 10ml MeCN Shake 3 min Centrifuge for 5 min at 4.000 rpm 150mg MgSO4, 50mg PSA, 10mg GCB Shake 30 second Centrifuge for 5 min at 4.000 rpm 16 GC temperature program: the initial temperature of 60 ° C, hold for 1 minute; increased 20oC/min to 180oC; increased 10oC/min to 190oC; increased 3oC/min to 240oC; increased 10oC/min to 300oC, and held for 5 minutes Sampling mode: splitless mode with sampling time is 1 minute. b) MS setting - Temperature: ion source: 200oC; interface 280oC. - In Scan mode, m/z starts at 50, m/z ends at 700 with a scan speed of 1428. - Retention time, main fragment (quantitative) and sub-fragments (qualitative) are presented in Table 3.10 Verification of method 3.4 3.4.1 Determination of recovery coefficient 10 grams of soil (absent of studied pesticides) passed through a 2mm sieve, were spiked standards to achieve concentrations of 50, 100 and 500 µg/kg. Results showed that the mean percentage recoveries of all pesticide residues studied are from 73 to 115% ( n=5). There was not difference in recovery between fist and second extraction. 3.4.2 Determination of method detection limit Spiked soil samples with concentrations of 5 µg/kg and 10 µg/kg were analyzed according to the described procedure above. The value of S/N were determined. Based on S/N values to calculate the method detection limit (LOD) and method quantification limit (LOQ). S/N ≥ 3 will be LOD and S/N ≥ 10 will be LOQ (table 3.11) 17 Table 3.10. Retention time, main fragment (quantitative) and sub-fragments (qualitative) (m/z) of pesticides Pesticides Retention time main fragment sub- fragments Alachlor 12,064 188 160, 146 Aldrin 13,357 263 265, 293 Benalaxyl 19,78 148 206, 234 BHC-alpha 9,881 219 181, 109 BHC-beta 10,341 183 219, 109 BHC-delta 10,545 181 219, 109 BHC-gamma 10,441 109 109, 181 Bifenthrin 23,118 181 166 Bitertanol 27,325 170 112, 141 Bromacil 12,79 207 164, 190 Buprofezin 17,224 105 172, 305 Cadusafos 9,674 159 127, 270 Pesticides Retention time main fragment sub- fragments Captan 14,938 79 149, 117 Carbofenothion 19,919 157 342, 199 Chlordane-cis 15,959 373 377, 272 Chlordane-trans 15,461 373 272, 237 Chlorfenapyr 17,626 59 247, 408 Chlorfenvinphos 14,69 267 323, 295 Chlorobenzilate 18,333 251 139, 111 Chlorpropham 9,413 127 213, 171 Chlorpyrifos 13,218 197 314, 258 Chlorpyrifos-methyl 11,874 286 125, 199 Cyfluthrin 28,569 163 226, 206 Cyhalothrin 25,481 181 208, 180 18 Pesticides Retention time main fragment sub- fragments Diazinon 10,692 179 137, 304 Dichlofluanid 12,953 123 224, 167 Dichlorobenil 7,131 171 173, 136 Dichlorvos 6,379 109 185, 145 Dimethenamid 11,735 154 230, 203 Dimethipin 10,323 118 124, 76 Diniconazole 18,47 268 281, 232 Dithiopyr 12,401 354 286, 237 Edifenphos 20,06 173 109, 310 Endosulfan-alpha 15,459 241 195, 265 Endosulfan-beta 15,961 241 195, 265 Endosulfan-sulfate 20,065 272 387, 229 Endrin 17,863 263 265, 245 EPN 22,841 157 185, 141 Pesticides Retention time main fragment sub- fragments Esprocarb 13,005 222 162, 91 Ethion 18,768 231 153, 97 Etrimfos 11,074 292 277, 181 Fenamidone 23,448 238 268, 281 Fenitrothion 12,737 277 125, 260 Fenobucarb 8,976 121 150, 207 Fensulfothion 18,35 293 308, 141 Fenthion 13,36 278 169, 125 Fipronil 14,447 367 369, 213 Fludioxonil 16,551 248 127, 182 Flusilazole 17,176 233 206, 165 Flutolanil 16,445 173 281, 145 Folpet 15,147 260 295, 262 Hexaconazole 16,485 214 234, 175 19 Pesticides Retention time main fragment sub- fragments Iprobenfos 11,315 204 91, 123 Isofenphos 14,645 213 121, 185 Isoprocarb 8,454 121 136, 103 Isoprothiolane 16,626 118 162, 189 Kresoxim-methyl 17,331 116 206, 131 Malathion 13,011 125 173, 158 Mepanipyrim 16,086 222 223, 111 Methidathion 15,405 145 85, 125 Methoprene 15,334 73 111, 153 Metolcarb 7,945 108 106, 90 Mevinphos 7,668 127 192, 109 Molinate 8,546 126 187, 98 Myclobutanil 17,052 179 150, 206 Napropamid 16,303 72 128, 100 Pesticides Retention time main fragment sub- fragments o,p’-DDD 17,135 235 281, 165 o,p’-DDT 18,755 235 165, 199 Oxadiazon 16,983 175 258, 302 Parathion 13,35 291 139, 109 Parathion-methyl 12,041 263 125, 109 Penconazole 14,535 248 159, 213 Pendimethalin 14,312 252 191, 162 Permethrin 27,547 183 163, 127 Phenamiphos 16,282 303 288, 154 Phosalone 24,555 182 367, 121 Pirimicarb 11,269 166 238, 72 Pirimiphos-ethyl 13,941 318 333, 304 Pirimiphos-methyl 12,658 290 305, 276 pp'-DDD 18,681 235 236, 165 20 Pesticides Retention time main fragment sub- fragments pp'-DDE 16,887 246 318, 176 pp'-DDT 20,406 235 165, 199 Pretilachlor 16,635 238 262, 202 Probenazole 12,867 130 159, 103 Procymidone 15,005 96 283, 67 Profenofos 16,739 339 374, 208 Prometryn 12,348 241 226, 184 Propanil 11,789 161 217, 162 Propoxur 8,993 110 152, 81 Pyridaben 27,726 147 117, 309 Pyridaphenthion 22,43 340 199, 188 Pesticides Retention time main fragment sub- fragments Quintozene 10,439 237 249, 295 Tebuconazole 21,063 250 125, 163 Terbufos 10,591 231 153, 186 Terbuthylazine 10,579 214 173, 130 Tetraconazole 13,563 336 171, 101 Thiobencarb 13,277 100 257, 125 Tokuthion 16,554 309 267, 162 Tolclofos-methyl 12,074 265 250, 125 Triadimenol 15,332 112 168, 128 Trifluralin 9,397 306 264, 290 Vamidothion 15,957 87 145, 109 21 Table 3.1. Method detection limit No. Pesticides LOD µg/kg LOQ µg/kg 1. Alachlor 4 11 2. Aldrin 1 4 3. Benalaxyl 2 7 4. BHC-alpha 4 13 5. BHC-beta 3 10 6. BHC-delta 4 14 7. BHC-gamma 3 10 8. Bifenthrin 2 7 9. Bitertanol 11 33 10. Bromacil 7 23 11. Buprofezin 3 10 12. Cadusafos 3 8 13. Captan 2 7 14. Carbophenothion 8 24 15. Chlordane-cis 3 8 16. Chlordane-trans 3 8 17. Chlorfenapyr 0,3 1 18. Chlorfenvinphos 10 31 19. Chlorobenzilate 2 5 20. Chlorpropham 4 12 21. Chlorpyrifos 5 15 22. Chlorpyrifos-methyl 4 12 23. Cyfluthrine 11 34 24. Cyhalothrin 6 18 25. Diazinon 4 13 22 No. Pesticides LOD µg/kg LOQ µg/kg 26. Dichlofluanid 3 10 27. Dichlorobenil 3 9 28. Dichlovos 3 11 29. Dimethenamid 2 7 30. Dimethipin 6 17 31. Diniconazole 9 28 32. Dithiopyr 2 7 33. Edifenphos 8 25 34. Endosulfan-alpha 9 28 35. Endosulfan-beta 7 22 36. Endosulfan-sulfate 7 21 37. Endrine 7 20 38. EPN 6 18 39. Esprocarb 1 4 40. Ethion 7 22 41. Etrimfos 9 27 42. Fenamidone 4 12 43. Fenitrothion 7 22 44. Fenobucarb 2 7 45. Fensulfothion 7 21 46. Fenthion 3 8 47. Fipronil 5 15 48. Fludioxonil 6 19 49. Flusilazole 8 25 50. Flutolanil 2 7 51. Folpet 4 11 23 No. Pesticides LOD µg/kg LOQ µg/kg 52. Hexaconazole 5 16 53. Iprobenfos 3 10 54. Isophenphos 7 22 55. Isoprocarb 4 11 56. Isoprothiolane 3 8 57. Kresoxim-methyl 2 6 58. Malathion 5 15 59. Mepanipyrim 3 10 60. Methidathion 4 12 61. Methoprene 2 5 62. Metolcarb 2 8 63. Mevinphos 10 30 64. Molinate 2 5 65. Myclobutanil 12 35 66. Napropamid 6 18 67. op-DDD 2 6 68. op-DDT 2 7 69. Oxadiazone 3 9 70. Parathion 2 7 71. Parathion-methyl 8 23 72. Penconazole 9 27 73. Pendimethalin 3 10 74. Penthoate 6 17 75. Permethrine 2 5 76. Phenamiphos 8 26 77. Phosalone 5 15 24 No. Pesticides LOD µg/kg LOQ µg/kg 78. Pirimicarb 1 4 79. Pirimiphos-ethyl 6 17 80. Pirimiphos-methyl 4 11 81. pp-DDD 3 8 82. pp-DDE 3 8 83. pp-DDT 2 6 84. Pretilachlor 4 11 85. Probenazole 7 20 86. Procymidone 2 6 87. Profenofos 6 17 88. Prometryn 4 12 89. Propanil 4 11 90. Propoxur 3 10 91. Pyridaben 2 7 92. Pyridaphenthion 8 24 93. Quintozene 5 15 94. Tebuconazole 13 38 95. Terbufos 4 12 96. Terbuthylazine 3 10 97. Tetraconazole 4 11 98. Thiobencarb 1 4 99. Tokuthion 3 10 100. Tolclofos-methyl 2 7 101. Triadimenol 10 29 102. Trifluralin 5 14 103. Vamidothion 4 11 25 Method evaluation by real sample analysis 3.5 3.5.1 Application to real samples 30 soil samples (provided by the Agricultural Environment Institute) were analyzed. The pesticides were detected in 310l samples. Among 10 positive samples, seven samples were found positive for organochlorine pesticides (DDT). 3.5.2 Compare results with other laboratories 30 soil samples were sent to 02 analytical center in Korea: Korea Research Institute of Analytical Technology (ANAPEX) and Power Chemical Analysis Management (PCAM). These laboratories are capable of analyzing more than 100 pesticides. 05 soil samples / 10 positive samples were sent to 02 pesticide analytical in Vietnam. Include: Center for Standards, Measurement and Quality 1 (QUATEST): 37 substances and National fertilizer quality testing center: 26 substances. Analysis results show similarities between laboratories. CONCLUSION CHAPTER 4. The thesis has developed a process for analysis of 103 pesticide residues of different groups (chlorinated, phosphorus, carbamate and pyrethroid) in soil on gas chromatography mass spectroscopy system by new and modern method (QuEChERS GC / MS 3 SIM). Inside: 1. The extraction process is carried out only one step including extracting and cleaning with a total time of 25 minutes, 15 ml of acetonitrile solvent and reaches a recovery efficiency of 70% - 120% (current methods, it takes at least 02 soxhlet extraction processes with 24 - 32 hours, 300 ml - 500 ml of solvent) 26 2. Analysis on GC/MS with a single run for 103 pesticides, the total time is 40 minutes, 3. The procedure is simple, easy to apply and suitable for facilities in the analytical laboratories 4. The method detection limit is in the range of 5ppb - 30ppb (adaptable to Vietnam's standard QCVN 15/2008 / BTNMT on the pesticide residues in soil), the recoveries are from 80 to 110% (only one substance 73%) 5. The procedure was applied analysis of pesticides for 30 soil samples with different cha