【作者】 崔淑华；

【导师】 王开运；

【作者基本信息】 山东农业大学 ， 植物检疫与农产品安全， 2012， 博士

【摘要】 大葱是传统的调味品蔬菜,更是我国重要的出口蔬菜之一。随着大葱种植面积的不断扩大和复种指数的提高,大葱病虫草害发生逐年加重,使用化学农药防治仍是控制其病虫杂草的最主要手段。近年来,我国出口鲜葱因农药残留超标被退运或销毁的事件屡见发生。选择残留风险较低的农药进行合理的化学防治及建立农药多残留的快速检测方法均是杜绝农药残留超标的关键环节,也是控制农药残留、保证食品安全和避免国际间有关贸易争端的基础。本研究通过对样品提取净化等前处理步骤进行优化,建立了简单、快速、净化效果好、灵敏度高的大葱中GC-MS分析方法；并针对近几年应用较多的一些新型农药、热不稳定、高极性农药,建立了大葱中多种农药的LC-MS/MS检测方法；同时针对在出口大葱基地推荐使用的农药品种,研究了其在春季大葱中的多残留特性。为这些农药在大葱中的合理使用提供理论支持,并为更好地应对国外技术壁垒奠定技术支撑。研究结果如下：1.通过对样品提取、净化步骤的优化,研究建立了大葱中193种农药残留的气相色谱-质谱(GC-MS)检测方法。样品经1%乙酸乙腈振荡超声提取,C18/PSA固相萃取柱净化,乙腈洗脱浓缩后,采用气相色谱-质谱法选择离子监测模式测定和确证,以磷酸三苯酯(TPP)为内标定量。结果表明,193种农药在10-1000μg/L范围内呈线性关系,相关系数均大于0.9967,方法检出限为0.04μg/kg-8.11μg/kg,方法定量限为0.13μg/kg～27.03μg/kg;在加标水平为10、20、50μg/kg时,193种农药的平均回收率为73.3%-108.8%,相对标准偏差(RSD, n=6)为2.8%-11.9%。该方法净化效果好、准确、灵敏、简便,适合用于大葱中农药残留的日常实际检测。2.针对热不稳定和强极性农药,参照QuEChERS前处理技术的分散固相萃取技术,研究建立了大葱中36种重点监控农药及新型农药残留的高效液相色谱-串联质谱(HPLC-MS/MS)检测方法。样品用含1%乙酸的乙腈溶液振荡超声提取,提取液经PSA和C18基质分散净化,用LC-MS/MS检测和确证,采用基质空白标准溶液外标法定量。结果表明,36种农药在1-100μg/L范围内具有良好的线性关系,相关系数为0.9990-0.9999,方法检出限为0.07μg/kg-2.94μg/kg,方法定量限为0.22μg/kg-9.80μg/kg;在加标水平为5、10、20μg/kg时,36种农药的平均回收率为82.3%-107.4%,相对标准偏差(RSD)为2.0%-10.0%。3.研究建立了大葱中灭蝇胺残留量测定方法。样品经乙腈提取,PSA净化,采用HPLC-MS/MS多反应监测(MRM)正离子模式测定,外标法定量。结果表明,灭蝇胺在2-200μg/L范围内呈良好线性关系,相关系数为0.9995,方法检出限为0.58μg/kg,定量限为1.94μg/kg,在加标水平为10、20、50μg/kg时,平均回收率为80.6%-87.2%,相对标准偏差(RSD,n=6)为2.8-6.1%。4.在春季大葱灰霉病发生季节,采用田间残留试验,研究了嘧霉胺和乙霉威在春季大葱中的残留消解动态和最终残留量。试验结果表明,嘧霉胺和乙霉威在大葱中消解较快,半衰期分别为4.1d和3.1d,药后14d均消解90%以上；26%嘧霉·乙霉威WP按推荐高剂量1800g/hm2,12(嘧霉胺180g a i/hm2,乙霉威288g a i/hm2)和加倍剂量3600g/hm2(嘧霉胺360gai/hm2,乙霉威576gai/hm2)喷雾处理大葱2-3次,末次施药后7d、14d、21d大葱中嘧霉胺残留量为0.007-0.560mg/kg.乙霉威残留量为ND-0.394mg/kg,低于同本、美国、欧盟等大葱主要出口国的MRL值,满足出口国要求。5.在大葱疫病和霜霉病发生季节,采用罔间残留试验,研究了烯酰吗啉在春季大葱巾的残留消解动态和最终残留量。试验结果表明,烯酰吗啉在大葱中消解较快,消解半衰期为3.5d。喷药14d烯酰吗啉降解95%以上。50%烯酰吗啉WP按推荐剂量300g ai/hm2和加倍剂量600g a i/hm2喷雾处理大葱2-3次,末次施药后7d、14d、21d大葱中烯酰吗啉残留量为0.0094-0.681mg/kg,低于同本、美国、欧盟等大葱主要出口国的MRL值。6.在大葱锈病和褐斑病发生季节,采用田间残留试验,研究了腈菌唑在春季大葱中的残留消解动态和最终残留量。试验结果表明,腈菌唑在大葱中消解较快,消解半衰期为3.4d。喷药14d烯酰吗啉降解90%以上。12.5%腈菌唑EC按推荐剂量62.5mg a.i./kg和加倍剂量125.0mg a.i./kg对水喷雾2-3次,末次施药后7、14d(大葱收获)大葱中腈菌唑残留量为0.003-0.017mg/kg,低于日本、美国、欧盟等大葱主要出口国MRL值。7.在大葱移栽时,采用田间残留试验,研究了二甲戊灵在春季大葱中的残留消解动态和最终残留量。试验结果表明,二甲戊灵在大葱中消解较快,消解半衰期为3.8d,降解90%需12.7d。33%二甲戊灵EC按推荐剂量742.5g a i/hm2和加倍剂量1485gai/hm2封闭处理土壤80d后,即在大葱收获时,大葱中二甲戊灵残留量均低于其检测限(0.88μg/kg),满足日本、美国和欧盟要求。8.在大葱移栽时,采用田间残留试验,研究了灭蝇胺在春季大葱中的残留消解动态和最终残留量。试验结果表明,灭蝇胺在大葱中消解较快,消解半衰期为8.8d,降解90%需29.1d。50%灭蝇胺WP按推荐剂量450g a i/hm2和加倍剂量900g ai/hm2顺葱灌根,于大葱收获时(施药后80d)采集大葱测定其灭蝇胺残留量分别为0.027mg/kg和0.019mg/kg,低于日本、美国、欧盟等大葱主要出口国MRL值。更多还原

【Abstract】 Welsh onion, a traditional condiment vegetable, which is one of China’s the most important export vegetables. The risk of pathogens, pests and weeds epidemic when crops are grown under such intensive regimes and the increase of the multiple crop indexes. The use of chemical pesticides is still one of the most important instruments for pest control. However, in recent years, welsh onion for export often encounters with the foreign technical barriers because that pesticides residues exceed maximum residue limit. Choosing the low-risk pesticides and developing rapid detection of pesticide residues are both the key of preventing pesticides residues exceed maximum residue limit. They are also the basic of controlling pesticides residues, ensuring food safety and avoiding trade dispute between countries.This research, optimized the steps before samples extract and purification, developed a simple, rapid and high sensitiveness analysis method of gas chromatography-mass spectrometric (GC/MS) in welsh onion. Established an analysis method of liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) in welsh onion depending on some new kinds of pesticides that were widely used, thermally unstable and highly polar in recent years. Meantime, the characters of multi-pesticides residues in spring welsh onion were also determined. The results of this research would be the theoretical basis of rationally use of pesticides in welsh onion and better deal with the foreign technical barriers. The results are as follows:1. A GC/MS method was developed by optimizing the procedure of extraction and purification for the determination of193pesticide residues in welsh onion. The analytes were extracted from the samples using1%acetic acid acetonitrile. The extracts were cleaned up by SPE column filled with C18/PSA, eluted using acetonitrile, and followed by determination with GC-MS at selected ion monitor mode (SIM) using TPP as internal standard. All of the tested pesticides showed good linear relationship (r≥0.9967) between10μg/L to1000μg/L The limits of detection (LOD) and limits of quantification (LOQ) of the method were0.04μg/kg-8.11μg/kg,0.13μg/kg-27.03μg/kg. The mean recoveries for welsh onion, spinach, strawberry and apple samples at three spiked concentrations levels of10,20,50μg/kg were73.3%-108.8%, with relative standard deviations (RSD, n=6) was2.8%-11.9%. The method was simple, sensitive and accurate, and could be used for the routine analysis of193pesticide residues in welsh onion.2. A method was established to determine36pesticides that were thermal unstable or/and high polar residues in welsh onion. The analytes were extracted from the samples using1%acetic acid acetonitrile, and purified by dispersion solid phase extration using C18and PSA as solidphase, then analyzed by HPLC/MS/MS with multiple-reaction monitoring (MRM), quantified using an external standard. The interference of matrix was reduced by the matrix-matched calibration standards curve. A good linear of the calibration curve developed were exhibited over a concentration range of1μg/L to100μg/L for the36pesticides with a correlation coefficient of0.99960to0.9999.The limits of quantification were0.22μg/kg to9.80μg/kg and the limits of detection were0.07μg/kg to2.94μg/kg. The mean recoveries for welsh onion, spinach, strawberry and apple samples at three spiked concentrations levels of5,10,20μg/kg were82.3%-107.4%with relative standard deviations (RSD, n=6) of2.0%-10.0%.3. A method was established to determination of cyromazine residuse in welsh onion. The samples were extracted with acetonitrile and purified by dispersion solid phase extraction using PSA as solid phase. Then analyzed by HPLC-MS/MS with mulpitle-reaction monitoring (MRM) under positive ion mode of electrospray ionization (ESI), quantified by the external standard. The results showed that the good linearity was achieved in the range of2-200μg/L for cyromazine, the linear correlation coefficient was0.9995. The fortified recovery at level of10,20,50μg/kg ranged from80.6%to87.2%, with relative standar deviation of2.8%～6.1%. The limit of detection for cyromazine in welsh onion was0.58μg/kg and the limit of quantification was1.94μg/kg。4. The field residue dynamics study and final residue trials of pyrimethanil and diethofencarb in spring welsh onion growing of gray mold in Shandong province, China were designed. The field residue dynamics study showed that the half-lives of pyrimethanil and diethofencarb in welsh onion was4.1d and3.1d, respectively. It is shown that pyrimethanil and diethofencarb belonged to non-persistent pesticides (Ti/2<30d). When the welsh onion was sprayed of26%pyrimthanil-diethofencarb WP for2～3successive times at application dose (pyrimthanil180g ai/hm2, diethofencarb288g ai/hm2) and at double dose(pyrimthanil360g ai/hm, diethofencarb576g ai/hm), at7,14and21th day after the last application, the final residues of pyrimethanil and diethofencarb in welsh onion were between0.007～0.560mg/kg and ND～0.394mg/kg, respectively, which were below the MRLs of Japan, the United States of America (USA) and European Union (EU), which are the major importing country of welsh onion.5. The field residue dynamics study and final residue trials of dimethomorph in spring welsh onion growing of blight and/or downy mildew in Shandong province, China were designed. The field residue dynamics study showed that the half-lives of dimethomorph in welsh onion was3.5d. It’s shown that dimethomorph belonged to non-persistent pesticides (T1/2<30d). When the welsh onion was sprayed of50%dimethomorph WP for2-3successive times at application dose (360g ai/hm2) and at double dose(720g a i/hm2), at7th,14th and21th day after the last application, the final residues of dimethomorph in welsh onion were between0.0094-0.681mg/kg, which were below the MRLs of Japan, the United States of America (USA) and European Union (EU), which are the major importing country of welsh onion.6. The field residue dynamics study and final residue trials of myclobutanil in spring welsh onion growing of rust disease and/or brown patch in Shandong province, China were designed. The field residue dynamics study showed that the half-lives of myclobutanil in welsh onion was3.4d. It’s shown that myclobutanil belonged to non-persistent pesticides (T1/2<30d). When the welsh onion was sprayed of12.5%myclobutanil EC for2-3successive times at application dose (62.5mg a.i./kg) and at double dose (125.0mg a.i./kg), at7th and14th day after the last application, the final residues of myclobutanil in welsh onion were between0.003-0.017mg/kg, which were below the MRLs of Japan, the United states of America (USA) and European Union (EU), which are the major importing country of welsh onion.7. The field residue dynamics study and final residue trials of phendimethalin in spring welsh onion when transplanting in Shandong province, China were designed. The field residue dynamics study showed that the half-lives of phendimethalin in welsh onion was3.8d. It’s shown that phendimethalin belonged to non-persistent pesticides (T1/2<30d). When the soil of planting welsh onion was handled by33%phendimethalin EC at application dose (742.5g ai/hm2) and at double dose (1485g ai/hm2), at80th day after application, namely time for harvesting the final residues of phendimethalin in welsh onion were below the limit of detection for phendimethalin in welsh onion (0.88μg/kg), which were below the MRLs of Japan, the United States of America (USA) and European Union (EU), which are the major importing country of welsh onion.8. The field residue dynamics study and final residue trials of cyromazine in spring welsh onion when transplanting in Shandong province, China were designed. The field residue dynamics study showed that the half-lives of cyromazine in welsh onion was8.8d. It’s shown that cyromazine belonged to non-persistent pesticides (T1/2<30d). When the root of welsh onion was poured by50%cyromazine WP at application dose (450g ai/hm2) and at double dose(900g ai/hm2), at80th day after application, namely time for harvesting the final residues of cyromazine in welsh onion were below the limit of detection for cyromazine in welsh onion (0.88μg/kg), which were below the MRLs of Japan, the United States of America (USA) and European Union (EU), which are the major importing country of welsh onion.更多还原