Determinationof15pesticidesresiduesinricebyultraperformanceliquidchromatography-tandemmassspectrometry
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摘要: 建立一种超高效液相色谱-串联质谱同时检测大米中甲氨基阿维菌素苯甲酸盐等15种常用农药残留的方法，考察了基质效应、提取溶剂种类、提取方法以及不同净化方法对15种农药回收率的影响。样品经V (乙腈) : V (丙酮) : V (水) = 16 : 2 : 2混合溶液匀浆提取，分散固相萃取法净化，电喷雾正离子 (ESI+) 模式电离，多反应监测 (MRM) 模式检测，外标法定量。结果表明：在0.005~1 mg/L范围内，甲氨基阿维菌素苯甲酸盐等15种农药的质量浓度与对应的峰面积间线性关系良好，相关系数 (r) ≥ 0.99；在0.01、0.1、1和4 mg/kg 4个添加水平下，15种农药在大米中的平均回收率在82%~116%之间，相对标准偏差在1.2%~12%之间 (n = 5)。方法检出限为0.000 5~0.005 mg/kg，定量限为0.01 mg/kg。用该方法对上海市郊20个批次的大米样品进行测定，均未检出农药残留超标。该方法操作简单、快速、准确，适用于大米中甲氨基阿维菌素苯甲酸盐等15种常用农药残留的同时检测。
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关键词:
- 超高效液相色谱-串联质谱 /
- QuEChERS方法 /
- 大米 /
- 农药 /
- 多残留
Abstract: A method was established for the residue detection of 15 pesticides in rice by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The influences of the matrix effects, the types of extraction solvent, extraction methods and different purification methods were all investigated. After homogenized mixing, samples were extracted by V (acetonitrile): V (acetone): V (water) = 16 : 2 : 2 and cleaned up by disperse solid phase extraction. The samples were then detected by MS/MS with positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode and quantified by external standard method. The results showed that the correlation coefficient (r) of 15 pesticides including emamectin benzoate were higher than 0.99 in the concentration ranged from 0.005 mg/L to 1 mg/L. The mean spiked recoveries of 15 pesticides at 0.01, 0.1, 1 and 4 mg/kg spiked levels ranged from 82% to 116% with the relative standard deviations (RSD, n = 5) between 1.2% and 12%. The limits of detection were 0.000 5-0.005 mg/kg, and the limits of quantitation for the method were 0.01 mg/kg. Twenty batches of rice from the outskirts of Shanghai municipality were determined by the established method. Excessive pesticide residues were not detected in all the rice samples. This new method is practical, fast and accurate, which meets the requirements for the determination of 15 pesticides including emamectin benzoate in rice at the same time. -
图 1 0.1%甲酸-水/甲醇 (A) 和0.1%甲酸-水/乙腈 (B) 体系下吡蚜酮提取色谱图
Figure 1. Extraction chromatogram of pymetrozine in 0.1% formic acid-water/methanol (A) and 0.1% formic acid-water/acetonitrile (B)
图 2 15种农药总离子流图
1-吡蚜酮pymetrozine；2-呋虫胺dinotefuran；3-烯啶虫胺nitenpyram；4-噻虫嗪thiamethoxam；5-吡虫啉imidacloprid；6-噻虫胺clothianidin；7-三环唑tricyclazole；8-氯虫苯甲酰胺chlorantraniliprole；9-嘧菌酯azoxystrobin；10-噻呋酰胺thifluzamide；11A-多杀霉素A spinosad A；11D-多杀霉素D spinosad D；12-戊唑醇tebuconazole；13-甲氨基阿维菌素苯甲酸盐emamectin benzoate；14-茚虫威indoxacarb；15-噻嗪酮buprofezin。
Figure 2. Total ion chromatogram (TIC) of the 15 pesticides
图 3 不同提取溶剂下15种目标农药在大米中回收率对比结果 (添加水平为0.1 mg/kg)
1-吡虫啉imidacloprid；2-吡蚜酮pymetrozine；3-A多杀霉素A spinosad A；3-D多杀霉素D spinosad D；4-呋虫胺dinotefuran；5-甲氨基阿维菌素苯甲酸盐emamectin benzoate；6-氯虫苯甲酰胺chlorantraniliprole；7-嘧菌酯azoxystrobin；8-噻虫胺clothianidin；9-噻虫嗪thiamethoxam；10-噻呋酰胺thifluzamide；11-噻嗪酮buprofezin；12-三环唑tricyclazole；13-戊唑醇tebuconazole；14-烯啶虫胺nitenpyram；15-茚虫威indoxacarb。
Figure 3. The comparison of the recoveries of 15 pesticides in rice under the different solvent conditions (spiked 0.1 mg/kg)
图 4 不同提取方法下15种目标农药在大米中回收率对比结果 (添加水平为0.1 mg/kg)
1-吡虫啉imidacloprid；2-吡蚜酮pymetrozine；3-A多杀霉素A spinosad A；3-D多杀霉素D spinosad D；4-呋虫胺dinotefuran；5-甲氨基阿维菌素苯甲酸盐emamectin benzoate；6-氯虫苯甲酰胺chlorantraniliprole；7-嘧菌酯azoxystrobin；8-噻虫胺clothianidin；9-噻虫嗪thiamethoxam；10-噻呋酰胺thifluzamide；11-噻嗪酮buprofezin；12-三环唑tricyclazole；13-戊唑醇tebuconazole；14-烯啶虫胺nitenpyram；15-茚虫威indoxacarb。
Figure 4. The comparison of the recoveries of 15 pesticides in rice under the different extractionconditions (spiked 0.1 mg/kg)
图 5 5种不同净化方法对15种目标农药回收率的影响 (添加水平为0.1 mg/kg)
1-吡虫啉imidacloprid；2-吡蚜酮pymetrozine；3-A多杀霉素A spinosad A；3-D多杀霉素D spinosad D；4-呋虫胺dinotefuran；5-甲氨基阿维菌素苯甲酸盐emamectin benzoate；6-氯虫苯甲酰胺chlorantraniliprole；7-嘧菌酯azoxystrobin；8-噻虫胺clothianidin；9-噻虫嗪thiamethoxam；10-噻呋酰胺thifluzamide；11-噻嗪酮buprofezin；12-三环唑tricyclazole；13-戊唑醇tebuconazole；14-烯啶虫胺nitenpyram；15-茚虫威indoxacarb
Figure 5. The effect of five different purification methods on the recoveries of 15 pesticides (spiked 0.1 mg/kg)
图 6 大米实际样品中吡虫啉和三环唑的总离子流图
Figure 6. Total ion chromatogram of imidacloprid and tricyclazole in a real rice sample
表 1 15种农药质谱采集参数
Table 1. Mass spectrum acquisition parameters of 15 pesticides
农药
Pesticide母离子
Precursor ion, m/z子离子
Product ions, m/z碰撞电压
Fragmentor/V碰撞能量
Collision energy/V吡蚜酮 pymetrozine 218.3 105.2* 80 23 79.0 80 23 呋虫胺 dinotefuran 203.1 129.0* 90 12 114.0 90 10 烯啶虫胺 nitenpyram 271.0 237.0* 100 15 224.0 100 15 噻虫嗪 thiamethoxam 292.0 211.0* 80 10 181.0 80 20 吡虫啉 imidacloprid 256.1 209.0* 80 10 175.0 80 10 噻虫胺 clothianidin 250.0 169.0* 90 5 132.0 90 5 三环唑 tricyclazole 189.7 135.7 30 26 162.7* 30 22 氯虫苯甲酰胺 chlorantraniliprole 484.0 453.0* 120 17 286.0 120 23 嘧菌酯 azoxystrobin 404.1 372.0* 120 10 344.0 120 15 噻呋酰胺 thifluzamide 526.8 168.0* 100 25 148.0 100 25 多杀霉素 A spinosad A 732.5 142.1* 140 35 98.0 140 55 多杀霉素 D spinosad D 746.5 142.1* 140 35 98.0 140 55 戊唑醇 tebuconazole 308.2 124.8* 120 30 70.0 120 40 甲氨基阿维菌素苯甲酸盐 emamectin benzoate 886.2 158.2* 200 40 126.1 200 50 茚虫威 indoxacarb 528.0 249.0* 120 15 218.0 120 20 噻嗪酮 buprofezin 306.2 201.1* 120 15 116.1 120 10 *定量离子 (Quantitative ion)。 
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表 2 溶剂和基质标准样品的标准曲线、检出限及斜率比值
Table 2. Standards curves and LODs of 15 pesticides in solvent and matrix and their slope ratios
农药 Pesticide 溶剂 Solvent 基质 Matrix 斜率比
Ratio of slope检出限
LOD/(mg/kg)线性方程
Linear equation相关系数
r线性方程
Linear equation相关系数
r吡蚜酮 pymetrozine y = 827 150x + 1 623 0.999 0 y = 714 658x + 1 753.8 0.999 9 0.864 0 0.001 呋虫胺 dinotefuran y = 35 666x + 759.09 0.995 8 y = 19 349x + 183 0.999 7 0.542 5 0.005 烯啶虫胺 nitenpyram y = 67 218x + 1 604 0.995 0 y = 83 265x + 1 422 0.998 9 1.239 0 0.005 噻虫嗪 thiamethoxam y = 50 155x + 1 215 0.994 4 y = 37 501x + 602 0.998 8 0.747 7 0.005 吡虫啉 imidacloprid y = 46 688x + 382 0.998 2 y = 29 524x + 8.158 0.999 9 0.632 4 0.005 噻虫胺 clothianidin y = 27 074x + 232 0.997 9 y = 14 451x + 21 0.999 8 0.533 8 0.005 三环唑 tricyclazole y = 78 356x + 1 409 0.996 1 y = 78 982x + 412 0.999 9 1.008 0 0.005 氯虫苯甲酰胺
chlorantraniliproley = 16 318x + 285 0.994 2 y = 15 142x + 171 0.998 7 0.927 9 0.005 嘧菌酯 azoxystrobin y = 454 071x + 9 051 0.995 2 y = 313 989x + 3 264 0.999 5 0.691 5 0.001 噻呋酰胺 thifluzamide y = 3 751x + 62 0.997 3 y = 1 901x – 11 0.999 1 0.506 8 0.005 多杀霉素 A spinosad A y = 968 735x + 10 362 0.998 0 y = 1 000 000x + 2 030.8 0.999 9 1.032 0 0.005 多杀霉素 D spinosad D y = 172 804x + 190 0.999 6 y = 192 931x – 1 210.4 0.999 7 1.117 0 0.005 戊唑醇 tebuconazole y = 330 633x + 1 192 0.999 2 y = 201 355x – 1 202 0.999 7 0.609 0 0.005 甲氨基阿维菌素苯甲酸盐
emamectin benzoatey = 213 488x + 3 785 0.996 7 y = 382 367x – 118 0.999 9 1.791 0 0.005 茚虫威 indoxacarb y = 3 803x + 146 0.995 4 y = 1 987x + 9 0.999 1 0.522 5 0.005 噻嗪酮 buprofezin y = 2 000 000x + 39 237 0.997 8 y = 2 000 000x + 29 888 0.999 9 1.000 0 0.000 5
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表 3 15种农药在大米中的平均添加回收率和相对标准偏差
Table 3. The average recoveries and relative standard deviations (RSDs) of 15 pesticides in rice at 4 spiked levels
农药
Pesticide添加水平 Spiked level/(mg/kg) 0.01 0.1 1 4 回收率
Recovery /%相对标准偏差
RSD/%回收率
Recovery /%相对标准偏差
RSD/%回收率
Recovery/%相对标准偏差
RSD/%回收率
Recovery/%相对标准偏差
RSD/%吡蚜酮 pymetrozine 102 7.2 84 4.1 89 2.2 99 3.3 呋虫胺 dinotefuran 103 8.6 101 2.5 105 1.8 99 8.4 烯啶虫胺 nitenpyram 84 7.5 82 3.5 86 3.3 99 5.6 噻虫嗪 thiamethoxam 112 5.9 107 3.3 109 1.6 100 4.4 吡虫啉 imidacloprid 106 4.5 110 4.5 112 1.2 96 1.4 噻虫胺 clothianidin 109 5.9 108 4.6 100 1.7 99 2.3 三环唑 tricyclazole 98 7.6 107 2.7 106 2.3 106 6.4 氯虫苯甲酰胺 chlorantraniliprole 106 11 112 2.9 105 4.8 84 6.9 嘧菌酯 azoxystrobin 95 7.8 112 1.9 113 1.7 102 5.9 噻呋酰胺 thifluzamide 106 9.0 111 12 110 2.1 113 5.6 多杀霉素 A spinosad A 100 6.5 112 2.6 107 1.7 105 5.4 多杀霉素 D spinosad D 107 8.9 113 4.9 108 1.7 102 5.7 戊唑醇 tebuconazole 101 9.1 109 4.3 114 1.3 93 3.3 甲氨基阿维菌素苯甲酸盐 emamectin benzoate 106 4.8 108 4.6 116 3.8 97 7.4 茚虫威 indoxacarb 106 12 102 10 109 4.5 104 6.7 噻嗪酮 buprofezin 111 4.5 112 5.7 104 2.2 102 2.7
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表 4 15种目标农药在大米实际样品中的残留量
Table 4. Analytical results of 15 pesticides residues in rice samples
农药
Pesticide检出浓度
Concentration/(mg/kg)最大残留限量[21]
MRLs/(mg/kg)吡蚜酮 pymetrozine - 0.2 呋虫胺 dinotefuran - 1 烯啶虫胺 nitenpyram - 0.1 噻虫嗪 thiamethoxam - 0.1 吡虫啉 imidacloprid 0.023~0.043 0.05 噻虫胺 clothianidin - 0.2 三环唑 tricyclazole 0.011~0.023 2 氯虫苯甲酰胺 chlorantraniliprole - 0.5 嘧菌酯 azoxystrobin - 0.5 噻呋酰胺 thifluzamide - 3 多杀霉素 A spinosad A - 1 多杀霉素 D spinosad D - 1 戊唑醇 tebuconazole - 0.5 甲氨基阿维菌素苯甲酸盐 emamectin benzoate - 0.02 茚虫威 indoxacarb - 0.1 噻嗪酮 buprofezin - 0.3 -: 未检出 (not detected)。
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