【作者】 吴刚；

【导师】 叶庆富；

【作者基本信息】 浙江大学 ， 生物物理学， 2011， 博士

【摘要】 本文建立了一种新型的动物源性食品中农药多残留检测的前处理方法,选择乙腈作为提取溶剂,采用加速溶剂萃取,凝胶渗透色谱净化,是一种自动化程度高、溶剂用量少、简单可靠的前处理方法,适用于样品的批量处理。由此建立了动物源性食品中有机氯、有机磷、氨基甲酸酯和拟除虫菊酯等109种农药(含同分异构体)残留量的气相色谱-质谱联用(GC-MS)检测方法,每种农药选择一个定量离子和两个定性离子,采用选择离子监测(SIM)模式,d0o-毒死蜱作为内标,内标法定量。该方法的重现性较好,方法灵敏度高,最低检测低限(LOD)为0.1μg·kg-1,最大LOD为32.3μg·kg11;最低定量检测低限(LOQ)为0.3μg·kg-1,最大LOQ为108μg·kg-1。以添加浓度分别为0.05、0.1和0.2 mg·kg-1的牛肉样品来验证方法的回收率和精密度,结果表明,添加回收率为64.3±1.7-97.4±9.5%,相对标准偏差不超过21.1%(n=6),在0.05～1.5μg·mL-1浓度范围内,标准曲线的线性相关性较好(r≥0.99)。此外,还建立了动物源性食品中55种有机磷和氨基甲酸酯类农药的分析方法,基于超高效液相色谱-串联质谱(UPLC-MS/MS)特有优势,采用多反应离子监测模式(MRM),双离子对检测(一组定量离子对,一组定性离子对),外标法定量。具有检测速度高效、准确、灵敏度高的特点,仅需10min即可完成对55种有机磷与氨基甲酸酯类农药的检测,相对GC-MS方法而言,该方法对于动物源性食品中农药多残留的检测,灵敏度有了数量级的提高,方法最低LOD为0.006μg·kg-1,最大LOD为0.087μg·kg-1;方法最低LOQ为0.019μg·kg-1,最大LOQ为0.286μg·kg-1。以添加浓度分别为0.05、0.1和0.2mg·kg-1的牛肉样品来验证方法的回收率和精密度,结果表明,添加回收率为62.36±5.39-102.79±8.34%,相对标准偏差不超过12.76%(n=6),在0.01～0.1μg·mL-1浓度范围内,标准曲线的线性相关性较好(r≥0.90)。两种检测方法都具有检测效率高,操作简便,杂质干扰少,测定结果准确可靠,灵敏度高等特点,适合高通量样品的快速检测,检测低限均低于欧盟(EU)、日本肯定列表、WHO/FAO、国际食品法典委员会(CAC)等国际组织制定的动物源性食品中农药残留的限量标准,能够满足国际标准和国家标准GB 2763-2005等法规对动物源性食品中农药残留量最大残留限量(MRLs)与检测要求。利用建立的动物源性食品中农药多残留快速检测与筛选方法检测市售的牛肉、猪肉、鱼肉和鸡肉样品,结果发现50个样品中16%的样品含有农药残留。6个样品检测到p,p’-滴滴依的含量从0.022～0.103 mg·kg-1,1个鱼肉样品中发现含有γ-六六六、克百威、α-硫丹和β-硫丹。p,p’-滴滴依和γ-六六六是样品中最普遍存在的残留农药。此外,在牛肉和猪肉中还检测到毒死蜱和蝇毒磷。表明本文所建立的农药多残留检测方法能够同时检测动物源性食品中有机氯(α-硫丹,β-硫丹,p,p’-滴滴依,γ-六六六)、有机磷(毒死蜱,蝇毒磷)和氨基甲酸酯(克百威)等多类农药,说明该方法适用于动物源性食品中农药多残留的检测。基于以上结果,选取克百威和蝇毒磷,研究它们在水溶液和动物源性食品中的辐照降解(简称辐解)。通过以14C苯环标记的克百威和蝇毒磷为示踪剂,综合运用放射性同位素示踪技术以及LSC和LC-MS/MS等现代仪器分析技术,系统研究了克百威和蝇毒磷分别在水溶液和动物源性食品中的辐解动态,并对可能的辐解产物进行了鉴定,对降解机理进行了解析。研究结果表明：农药的降解率随着辐照剂量的增大而增加。对于不同浓度的相同农药,要达到相同的降解率,高浓度农药需要的辐照剂量往往高于低浓度农药所需的辐照剂量。原因可能是水溶液中农药的辐解大多表现为间接作用,主要依靠辐照水分子产生的自由基OH、H和eaq-与农药基团的断裂反应,生成小分子物质,辐照剂量一定时,溶液中产生的活性离子数目基本上相同,与溶质分子反应是一个常数,溶液的浓度越高,则溶质分子增大,其降解率也就降低。不同农药在水溶液中辐解情况也有差异,克百威在水溶液的辐解过程中,H起最主要作用,OH次之,eaq-起的作用最小,它们对克百威的降解速率常数的比率为k.H：k.OH:keaq-=25:7:6,H、OH、和eaq-的量子效率为η·H:η·OH:η1eaq-=12.7：1：2.6。然而,蝇毒磷水溶液的辐解过程中,eaq-起最主要作用,OH次之,H起的作用再次之,它们对蝇毒磷的降解速率常数的比率为keaq.:k·OH:k·H=3：2.8：1,量子效率为ηeaq-:η·OH:η·H=2.4:1:2.2。鱼肉中初始浓度为1mg·kg-1的克百威在6。Co-γ射线下辐解的半衰减剂量(D50)为72.95kGy,而相同浓度的克百威在水溶液中的D50则为6.73kGy。牛肉中初始浓度1mg·kg-1的蝇毒磷在60Co-γ射线下辐解的D50为29.74kGy,而相同浓度的蝇毒磷在水溶液中的D50仅为0.73kGy。同样辐照剂量下,对农药在动物源性食品和水溶液的辐解率进行比较,表明动物源性食品中农药的辐解率远低于水溶液中辐解率,要产生相同的辐解,动物源性食品中的辐照剂量显著高于水溶液中的剂量；辐解的产物也有差异,水溶液中农药辐解产物种类往往多于动物源性食品中农药的辐解产物。这可能由于农药周围介质存在显著差异而造成的两者辐解作用方式不同。水溶液中克百威的辐解产物主要是：2,3-二氢-2,2二甲基-3-羟基-7-苯并呋喃基-N-羟甲基氨基甲酸酯、2,3-二氢-2,2-二甲基-3-羰基-7-苯并呋喃基-N-羟甲基氨基甲酸酯、2,3-二氢-2,2二甲基-3-羟基-7-苯并呋喃基-N-醛基氨基甲酸酯、3-羟基-呋喃丹(或2,3-二氢-2,2-二甲基-3-羟基-7-苯并呋喃基-N-甲基氨基甲酸酯)、3-酮基-呋喃丹(或2,3-二氢-2,2-二甲基-3-羰基-7-苯并呋喃基-N-甲基氨基甲酸酯)、3-酮基-呋喃酚(或2,3-二氢-2,2-二甲基-3-羰基-7-苯并呋喃酚)、呋喃酚(或2,3-二氢-2,2-二甲基-7-苯并呋喃酚)和苯并呋喃；而鱼肉中克百威的辐解产物主要是：3-羟基-呋喃丹、2,3-二氢-2,2-二甲基-3-羰基-7-苯并呋喃基-N-羟甲基氨基甲酸酯、2,3-二氢-2,2二甲基-3-羟基-7-苯并呋喃基-N-醛基氨基甲酸酯和3-酮基-呋喃丹。克百威在鱼肉中的主要辐解产物均在水溶液的辐解中产生,但是2,3-二氢-2,2-二甲基-3-羰基-7-苯并呋喃基-N-羟甲基氨基甲酸酯、3-酮基-呋喃酚、呋喃酚和苯并呋喃只在水溶液中辐解产生。水溶液中蝇毒磷辐解产物主要是：O,O-二乙基-O-(3-氯-4-甲基香豆素-7)磷酸酯、O,O-二乙基-O-(4-甲基香豆素-7)磷酸酯、O-乙基-O-羟基-O-(3-氯-4-甲基香豆素-7)硫逐磷酸酯、O,O-二乙基-O-(3-氯-4-甲基香豆酸-7)磷酸酯和O,O-二乙基-O-(3-氯-4-甲基香豆酸-7)硫逐磷酸酯。而牛肉中蝇毒磷辐解产物主要是：O,O-二乙基-O-(3-氯-4-甲基香豆酸-7)硫逐磷酸酯、O,O-二乙基-O-(3-氯-4-甲基香豆素-7)磷酸酯和O-乙基-O-羟基-O-(3-氯-4-甲基香豆素-7)硫逐磷酸酯。O,O-二乙基-O-(3-氯-4-甲基香豆酸-7)磷酸酯和O,O-二乙基-O-(4-甲基香豆素-7)磷酸酯是蝇毒磷在水溶液中的辐解产物,不在牛肉中辐解产生；而蝇毒磷在牛肉中的辐解产物均在水溶液的辐解中产生。可见,动物源性食品中农药残留的辐解情况比较复杂。为了降低食品中农、兽药残留对人类的危害,首先应该加强源头控制,降低食品的污染：同时需要对辐解过程、降解产物和产物毒理进一步研究,以促进辐解技术在食品安全应用中的发展。更多还原

【Abstract】 A novel multi-pesticide residues sample preparation method for the foods of animal origin was developed. Acetonitrile was selected for accelerated solvent extraction (ASE) for effectively extracting the pesticides from the fatty samples. The cleanup was performed with an automated gel permeation chromatography (GPC) cleanup system. It is a simple and reliable pre-treatment method for batch processing of samples with a high degree of automation and less solvent.A new analytical method was developed to simultaneously determine the residues of 109 pesticides (including isomers) in the foods of animal origin, included organochlorines, organophosphates, carbamates and pyrethroids. The prepared samples were analyzed with GC-MS in the selected ion monitoring (SIM) mode using one target and two qualitative ions for each analyte. Chlorpyrifos-d10 was used as an internal standard. The lowest limit of detection (LOD) was 0.1μg·kg-1, the largest LOD was 32.3μg·kg-1. The lowest limit of quantification (LOQ) was 0.3μg·kg-1, the largest LOQ was 108μg·kg-1. The recoveries and relative standard deviations (RSDs) were checked by spiking untreated samples with pesticides at 0.05,0.1 and 0.2 mg·kg-1. The average recoveries of most pesticides were from 64.3±1.7 to 97.4±9.5%. The precision values expressed as RSDs were all≤21.1% (n=6). Good linearity (r≥0.99) was observed between 0.05 and 1.5μg·mL-1.Another analytical method was developed to simultaneously determine residues of 55 organophosphate and carbamate pesticides in the foods of animal origin. The prepared samples were analyzed within 10 min with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in the multiple reaction monitoring (MRM) mode using a set of quantitative ions pair and a set of qualitative ions pair for each analyte. The lowest LOD was 0.006μg·kg-1, the largest LOD was 32.3μg·kg-1. The lowest LOQ was 0.019μg·kg-1, the largest LOQ was 0.286μg·kg-1. The recoveries and RSDs were checked by spiking untreated samples with pesticides at 0.05,0.1 and 0.2 mg·kg-1. The average recoveries of most pesticides were from 62.36±5.39 to 102.79±8.34%. The precision values expressed as RSDs were all≤12.76% (n =6). Good linearity (r≥0.90) was observed between 0.01 and 0.1μg·mL-1.Both the two analysis methods were suitable for rapid detection of high-throughput samples with high efficiency and easy operation, less interference of impurities, accurate, reliable and high sensitivity. The LODs of these methods were both lower than the developed maximum residue limits (MRLs) for the pesticide residues in foods of animal origin by European Union (EU), Japan’s positive list system, WHO/FAO, Codex Alimentarius Commission (CAC) and other international organizations. Therefore, these methods meet the the lowest LODs and testing requirements of the international standards and China’s national standard of GB 2763-2005 and other regulations on pesticide residues in foods of animal origin.Samples of pork, beef, chicken and fish from local markets were prepared and analyzed using the developed sample preparation, cleanup and analysis methods. Pesticide residues were detected in 16% of the total samples (or 8 out of 50 samples). The residues were found in the pork, beef, chicken and fish samples, p, p’-DDE was detected in 6 samples at concentrations ranging from 0.022 to 0.103 mg·kg-1, whileγ-BHC, carbofuran,α-endosulfan, and (β-endosulfan were each found in one fish sample. The most common pesticide residues found were p, p’-DDE and y-BHC. Chlorpyrifos or coumaphos was found in the beef and pork samples. The diversity of pesticide classes, including organochlorine (α-endosulfan,β-endosulfan, p, p’-DDE,γ-BHC), organophosphorus (chlorpyrifos, coumaphos) and carbamate (carbofuran), showed that the proposed method was versatile and sensitive for the determination of multi-residues of pesticides in samples of animal origin.Based upon the results, carbofuran and coumaphos were selected as the research objects. Theγrays radiation-induced degradation of them in aqueous solvent and foods of animal origin were performed. The degradation dynamics of carbofuran and coumaphos in aqueous and foods of animal origin were systematically investigated by using the 14C labeled benzene ring of carbofuran and coumaphos as radioisotope tracer and the modern equipments, included liquid scientillation counting (LSC) and LC-MS/MS. The structures of the degradation products were derived and identified. As a result, theγrays radiation-induced degradation mechanisms of carbofuran and coumaphos in aqueous and foods of animal origin were resolved.The results showed that the degradation rate of pesticides in aqueous solution increased with the increase of irradiation dose. However, in order to achieve the same degradation rate for the different concentrations of the same pesticides, the higher concentration of pesticides often need higher necessary radiation dose than that required for the lower concentrations of pesticide. It was probably due to the y rays radiation-induced degradation of pesticide in aqueous solution induced in most of the performance indirect effects, mainly relying on the cleavage reaction to generate small molecules by the free radicals of’OH,·, and eaq-produced by irradiation of water molecules with the pesticide groups. When the radiation dose keeps constant, the number of active ions in the solution is essentially constant. If the concentration of the solution is increased, the solute molecules will increase, too. Consequently, the degradation rate will decrease.The y rays radiation-induced degradation of pesticide in aqueous solution was different from different pesticides. The contribution to carbofuran degradation by the radicals was in the order as follows, H> OH> eaq-. The rate of degradation rate constant for carbofuran was as follows, kH:k.OH:keaq-=5:7:6. The quantum efficiency ratios of H, OH and eaq- for the degradation of carbofuran were calculated as 12.7:1:2.6. However, the contribution to coumaphos degradation by the radicals was in the order as follows, eaq- >’OH>H. The rate of degradation rate constant for coumaphos was as follows, keaq.:k.OH:k.H=3:2.8:1. The quantum efficiency ratios of eaq-,OH and H for the degradation of coumaphos were calculated as 2.4:1:2.2.The half decay dose (D50) was 72.95 kGy for carbofuran in fish at the initial concentration of 1 mg·kg-1 by 60Co-γrays irradiation, while the D50 of the same concentration of carbofuran in aqueous solution was 6.73 kGy. The D50 was 29.74 kGy for coumaphos in beef at the initial concentration of 1 mg·kg-1 by 60Co-y rays irradiation, while the D50 of the same concentration of coumaphos in aqueous solution was 0.73 kGy.The degradation rate of pesticide in the foods of animal origin was far less than that in aqueous at the same irradiation dose. In order to gain the same degradation rate for the same pesticide in different media, the irradiation dose for pesticide in the foods of animal origin was significantly higher than that in aqueous solution.There are also differences in the products of radiation degradation for the same pesticide. The degradation products in aqueous were much more than those in the foods of animal origin. This may be due to the significant difference of the surrounding medium between the pesticides to cause the different degradation mode.The main products of radiation degradation for carbofuran in aqueous were as follows,3-hydroxy carbofuran (or 2,3-dihydro-2,2-dimethyl-3-hydroxy-7-benzofuranyl N-hydroxymethyl carbamate),2,3-dihydro-2,2-dimethyl-3-oxo-7-benzofuranyl N-hydroxymethyl carbamate,2,3-dihydro-2,2-dimethyl-3-hydroxy-7-benzofuranyl N-al carbamate,3-hydroxy carbofuran (or 2,3-dihydro-2,2-dimethyl-3-hrdroxy-7- benzofuranyl N-methylcarbamate),3-keto-carbofuran (or 2,3-dihydro-2,2-dimethyl-3-oxo-7-benzofuranyl N-methylcarbamate),3-keto-7-phenol (or 2,3-dihydro-2,2-dimethyl-3-oxo-7-benzofuranol), carbofuran-7-phenol (or 2,3-dihydro-2,2-dimethyl-7-benzofuranol) and 2,3-dihydro-benzofuran. Meanwhile, the main products of radiation degradation for carbofuran in fish were as follows,3-hydroxy carbofuran, 2,3-dihydro-2,2-dimethyl-3-oxo-7-benzofuranyl N-hydroxymethyl carbamate, 2,3-dihydro-2,2-dimethyl-3-hydroxy-7-benzofuranyl N-al carbamate and 3-keto-carbofuran. The main products of radiation degradation for carbofuran in fish were also generated in the aqueous solution. However,2,3-dihydro-2,2-Dimethyl-3-hydroxy-7-benzofuranyl N-hydroxymethyl carbamate, and 3-keto-7-phenol, carbofuran-7-phenol and 2,3-dihydro-benzofuran were only produced in the aqueous solution.The main products of radiation degradation for coumaphos in aqueous were as follows,O,O-diethyl-O-(3-chloro-4-methyl-7-hydroxy-coumarin) phosphate,O, O-diethyl-O-(4-methyl-7-hydroxy-coumarin) phosphate, O-ethyl-O-hydroxyl-(3-chloro-4-methyl-7-hydroxy-coumarin) phosphorothioate, O, O-diethyl-O-(3-chloro-4-methyl coumarate-7) phosphate and O, O-diethyl-O-(3-chloro-4-methyl coumarate-7) phosphorothioate. Meanwhile, the main products of radiation degradation for coumaphos in beef were as follows, O, O-diethyl-O-(3-chloro-4-methyl coumarate-7) phosphorothioate,0,0-diethyl-O-(3-chloro-4-methyl-7-hydroxy-coumarin) phosphate and O-ethyl-O-hydroxyl-(3-chloro-4-methyl-7-hydroxy-coumarin) phosphorothioate. However,O, O-diethyl-O-(3-chloro-4-methyl coumarate -7) phosphate and O, O-diethyl-O-(4-methyl-7-hydroxy-coumarin) phosphate were only produced in the aqueous solution rather than in beef byγirridiation. All the products of y rays rays radiation-induced degradation of coumaphos in beef were produced in the aqueous solution, too.The result shows that theγrays radiation-induced degradations of pesticide residues in foods of animal origin may be much more complex. In order to reduce the harmful to humans from the residues of pesticide and veterinary drug, source control should be strengthened to reduce harmful contamination of food at first. At the same time, in order to promote the application of theγrays radiation-induced degradations in food safety, the radiation degradation process, degradation products and their toxicology should be further studied.更多还原