【作者】 应永飞；

【导师】 汪以真；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2014， 博士

【摘要】 饲料中p-受体激动剂、抗菌类药物等非法添加以及饲料中霉菌毒素污染等三大影响饲料安全的最主要问题,影响到肉、蛋、奶等动物产品的品质,制约了畜牧业的可持续发展,并危害到消费者的健康。因此,建立饲料中三大类主要化学污染物的检测方法,在此基础上对饲料质量进行监测和控制具有重要意义。但是,由于饲料样品基质的复杂性,已建立的饲料中化学污染物检测方法存在前处理过程复杂、单组分检测费时费力、结果准确性差、检测效率低、前处理成本很高、有机试剂用量大等问题。本研究旨在建立饲料中p-受体激动剂、霉菌毒素、抗菌类药物等三大类主要化学污染物快速、高效、多组分同步检测技术,有效保障饲料质量安全。本论文以玉米赤霉烯酮类霉菌毒素(雷索酸内酯)、p-受体激动剂、磺胺类和喹诺酮类药物等为研究对象,深入研究了饲料中化学污染物多组分同步检测的样品前处理技术和液相色谱-串联质谱(LC-MS/MS)分析方法。在样品前处理方法研究中使用了近年来引起广泛关注的碳纳米管材料和分散固相萃取技术,有效提高了前处理效率。仪器分析过程中采用UPLC进行快速分离,用串联质谱进行定性、定量检测。同时,运用同位素稀释定量技术进行校准,提高了定量方法的准确度和精密度。主要研究内容及结果如下：1.β-受体激动剂质谱裂解机理研究本研究以克伦特罗、莱克多巴胺、沙丁胺醇等为代表,阐明p-受体激动剂在正离子软电离模式下的裂解机理。采用AB SCIEX5500QTRAP三重四级杆电喷雾质谱仪,运用IDA-EPI技术,对p-受体激动剂类化合物裂解规律进行研究解析。结果表明,由于β-受体激动剂均属于苯乙醇胺类化合物,但苯环上及氨基连接官能团有差异,所以它们的ESI-MS/MS行为存在着相似性和差异性。在ESI正电离模式下p-受体激动剂容易获得H+而形成[M+1]+准分子离子峰,该结构易丢失水分子(H20),继而与氨基结构相连的碳氮键(-C-N-)键发生断裂,形成与苯环共轭的(Ph-CH=CH-NH+-)离子结构。本研究为同类化合物的结构解析研究提供可靠的依据,为跟踪新型β-受体激动剂提供有力手段,具有较强的理论意义和实用价值。2.多壁碳纳米管分散固相萃取结合LC-MS/MS测定饲料中11种β-受体激动剂研究采用多壁碳纳米管(MWCNTs)为吸附剂,建立分散固相萃取(dSPE)净化、超高效液相色谱-串联质谱测定饲料中11种p-受体激动剂(克伦特罗、莱克多巴胺、沙丁胺醇、苯乙醇胺A、氯丙那林、妥布特罗、西马特罗、特布他林、马布特罗、班布特罗和喷布特罗)的方法。本研究对样品前处理方法进行了系统优化,包括MWCNTs用量和类型、提取液pH值、MWCNTs提取时间以及洗脱液体积等,以提高检测方法通量和灵敏度。以0.1%甲酸溶液和甲醇为梯度洗脱流动相,采用ESI+模式电离,在MRM模式下建立了LC-MS/MS测定方法,内标法校准定量。研究结果表明,11种p-受体激动剂在0.1-20μg/L浓度范围内呈良好的线性关系,相关系数r2均大于0.997,猪配合饲料、浓缩饲料和预混合饲料样品中LOQ为0.05-0.48μg/kg；添加水平为0.5-100μg/kg时,11种p-受体激动剂的回收率在89.6%-112.9%,RSD均小于15%。该检测方法与农业部1486号公告的方法进行了比较,两种方法检测结果一致,说明新建立的多壁碳纳米管分散固相萃取结合LC-MS/MS测定饲料中11种β-受体激动剂的方法操作简便、稳定性好、准确度和精密度高。3.多壁碳纳米管结合LC-MS/MS测定饲料中6种玉米赤霉烯酮类霉菌毒素研究以多壁碳纳米管(MWCNTs)为吸附剂,建立分散固相萃取(dSPE)净化、液相色谱-串联质谱测定饲料中6种玉米赤霉烯酮类霉菌毒素(α-玉米赤霉醇、β-玉米赤霉醇、α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉酮、玉米赤霉烯酮)的方法。本研究对样品前处理方法进行了优化,包括提取液pH值、MWCNTs提取时间与MWCNTs用量和类型以及洗脱液体积等,以提高检测方法通量和灵敏度。以水和乙腈为流动相进行梯度洗脱,采用ESI-模式电离,在MRM模式下建立LC-MS/MS测定方法,内标法校准定量。研究结果表明,6种玉米赤霉烯酮类霉菌毒素在1.0-500μg/L浓度范围内呈现良好的线性关系,相关系数r2均大于0.9991；LOD为0.12-0.27μg/kg,LOQ为0.40-0.90μg/kg;在1.0μg/kg-500μg/kg添加浓度水平上,猪配合饲料、浓缩饲料和预混合饲料样品中平均回收率在94.6%-107.2%,批内RSD在2.6%-8.5%之间,批间RSD在4.5%-9.2%之间。将本方法检测结果与农业行业标准NY/T2071的方法进行了比较,两种方法阳性样品检测结果RSD在15%以内,表明新建立的多壁碳纳米管结合LC-MS/MS测定饲料中6种玉米赤霉烯酮类霉菌毒素的方法简便、快速、准确度和精密度高。4.碳纳米管分散固相萃取结合LC-MS/MS测定饲料中22种磺胺类和喹诺酮类药物研究建立多壁碳纳米管(MWCNTs)为吸附剂的分散固相萃取(dSPE)净化、液相色谱-串联质谱测定饲料中22种磺胺类和喹诺酮类药物(磺胺醋酰、磺胺嘧啶、磺胺甲恶唑、磺胺噻唑、磺胺间二甲氧嘧啶、磺胺邻二甲氧嘧啶、磺胺二甲嘧啶、磺胺对甲氧嘧啶、磺胺甲氧哒嗪、磺胺间甲氧嘧啶、磺胺喹恶林、磺胺氯哒嗪、氟甲喹、诺氟沙星、环丙沙星、氟罗沙星、奥比沙星、洛美沙星、恩诺沙星、氧氟沙星、沙拉沙星、二氟沙星)的方法。本研究对样品前处理方法进行了系统优化,包括MWCNTs用量和类型、提取液pH值、MWCNTs提取时间以及洗脱液体积等,以提高检测方法通量和灵敏度。以0.1%甲酸溶液和乙腈为梯度洗脱流动相,采用ESI+模式电离,在MRM模式下建立了LC-MS/MS测定方法,内标法校准定量。研究结果表明,22种磺胺类和喹诺酮类药物在1.0-1000μg/L浓度范围内呈良好的线性关系,线性相关系数r2均大于0.992,LOD为3.0-10μg/kg, LOQ为10-25μg/kg,猪和鸡的配合饲料、预混合饲料和浓缩饲料中添加水平为0.05-50mg/kg时,其回收率在71.5%-114.7%之间,批内RSD在1.5%-12.5%之间。利用该方法对本中心保留的1份磺胺喹恶啉阳性饲料样品进行了检测,并与GB/T19542的方法进行了比较,两种方法检测结果一致,表明新建立的碳纳米管分散固相萃取结合LC-MS/MS测定饲料中22种磺胺类和喹诺酮类药物的方法检测效率高、成本低、结果准确可靠。更多还原

【Abstract】 Illegal use of banned drugs, irrational use of veterinary drugs and mycotoxin contamination are the three main factors affecting feed safety. Feed safety issues caused by the three main factors affect the quality of meat, eggs, milk and other animal products, and also harm to animal safety and human health. Therefore, it has important significance to establish analysis method of above three types of drugs and monitoring and control of feed quality based on these analysis mehtods. However, there are many difficulties and problems in the feed safety testing due to the complexity of the feed sample matrix, time-comsuing and laborious of single component determination, low analytical efficiency, high cost of pretreatment, large dosage of organic solvents, and so on. The purpose of the study is to develop fast, efficient and multicomponent determination technology to effectively ensuring feed quality safety.The study thoroughly study the feed safety multicomponent analytical sample pretreatment technique and liquid chromatography tandem mass spectrometry (LC-MS/MS) analytical methodology with β-receptor agonists, resorcylic acid lactones, sulfonamides and quinolones antibiotics as research objects in this study. Carbon nanotubes materials and dispersive solid-phase extraction technology, which had aroused wide concern, have been used during development of sample pretreatment method. Therefore, the established pretreatment method effectively improves the efficiency of sample pretreatment. UPLC has been used for rapid chromatographic separation and tandem mass spectrometry has been used for qualitative and quantitative determination. Moreover, isotope standard dilution has been used to improve the accuracy and precision of the method. The main research contents and results are as follows:1. Study on mass spectral characteristics and fragmentation mechanism of β-receptor agonistsTo clarify pyrolysis mechanism of β-receptor agonists under postitive mode, representative drugs including clenbuterol, salbutamol and ractopamine had been chosen to study with AB SCIEX5500QTRAP triple quadrupole mass spectrometry and IDA-EPI technology. The results showed that there are some similarities and differences for ESI-MS/MS behavior of β-receptor agonists due to them belong to phenylethanolamine compounds, which have the same basic skeleton and similar structure features and different connection for the benzene ring and amino functional groups. Under ESI postivie mode, β-receptor agonists are easy to form [M+1]+. The strcture is easy to loss neutral molecules, such as H2O. Moreover, the C-N bond connected with amino group is easy to broken and produce conjugated ion structure with benzene. This study has important theory research significance and practical value, such as providing reliable basis for other β-receptor agonists structural analysis and providing powerful means to track newp-receptor agonists.2. Study on the analysis110-receptor agonists in feeds by LC-MS/MS coupled with dispersive solid phase extraction using multiwalled carbon nanotubes as absorbentA simple, sensitive and reliable analytical method was developed for determination of (3-receptor agonists (clenbuterol, ractopamine, salbutamol, phenylethanolamine A, clorprenaline, tulobuterol, cimaterol, terbutaline, mabuterol, bambuterol, penbuterol) in feeds by ultra high performance liquid chromatography-positive electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) using dispersive solid phase extraction with multi-walled carbon nanotubes. To improve sample throughout and sensitivity of analytical method, the systematic optimization of sample pretreatment method had been carried out including the type and amount of MWCNTs, the pH value of the sample, the sample extraction time, and the volume of eluent. Feed samples were extracted with formic acid solution. The eluate was directly analyzed by LC-MS/MS on an Acquity UPLC BEH C18column with a mixture of methanol and0.1%formic acid solution as the mobile phase. The samples were quantified with the internal standard calibration curve method. The results showed that good linearities were obtained for the β-receptor agonists at the concentration of0.1-20μg/L with the linear relative coefficient more than0.997. The recoveries for the β-receptor agonists in formular feed, concentrated feed and premix feed of swine were89.6%-112.9%at the fortified levels of0.5-100μg/kg. The limits of quantitation of11β-receptor agonists were0.05-0.48μg/kg, and the relative standard deviations were less than15%. Compared with the method of Announcement No.1486of the Ministry of Agriculture in positive sample analysis, results which obtained with the two methods, were consistent.3. Simultaneous determination of zearalenone and analogues in feed using LC-MS/MS and multiwalled carbon nanotubes as dispersive solid phase extraction sorbent A simple and cost-effective pre-treatment procedure was developed for six zearalenone and analogues (α-zearalanol, β-zearalanol, a-zearalenol, β-zearalenol, Zearalanone, Zearalenone) in feed using dispersive solid phase extraction (dSPE) with multi-walled carbon nanotubes (MWCNTs). After extraction with acetonitriel/water (80:20, v/v) and dilution with water, a dSPE procedure was carried out with MWCNTs. The pH value of the extract, extraction time with MWCNTs, type and amount of MWCNTs and type of eluent were optimized to increase the sample throughput and sensitivity. The eluate was directly analyzed by LC-MS/MS on an Acquity UPLC BEH C18column with a mixture of water and acetonitrile solution as the mobile phase. The samples were quantified using zearalenone-D6as internal standard. Mean recoveries from fortified swine mixed feed samples ranged from94.6%to107.2%, with relative standard deviations2.6%-8.5%, the limits of detection and quantification for RALs were in the ranges of0.12-0.27μg/kg and0.40-0.90μg/kg, respectively. At the same time, compared the positive results of ZEN with agriculture standard NY/T2071-2011method, relative standard deviation obtained with two analytical methods is within15%.4. Using carbon nanotubes as dispersion solid phase extraction and LC-MS/MS determination of22sulfonamides and quinolones antibiotics in feedA simple and cost-effective pre-treatment procedure was developed for determination of22sulfonamides and quinolones (sulfacetamide, sulfadiazine, sulfamethoxazole, sulfathiazole, sulfadimethoxine, sulfadoxine, sulfamethazine, sulfamethoxydiazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfaquinoxaline, sulfachloropyridazine, flumequine, norfloxacin, ciprofloxacin, fleroxacin, orbifloxacin, lomefloxacin, enrofloxacin, ofloxacin, sarafloxacin, difluoxacin) in feed using dispersive solid phase extraction with multiwalled carbon nanotubes. The type and amount of MWCNTs, pH value of the extract, extraction time with MWCNTs, and type of eluent were optimized to increase the sample throughput and sensitivity. The eluate was analyzed by LC-MS/MS on a Waters Atlantis Cis column with0.1%formic acid solution and acetonitrile as the mobile phase with gradient elution, and determined in electrospray ionization positive mode (ESI+) with multiple reaction monitoring (MRM). The samples were quantified using internal standard calibration. The results showed that22sulfonamides and quinolones were linear in the concentration range of1.0-1000μg/L, the linear correlation coefficient(r2) were more than0.992. The limits of detection were3.0-10μg/kg, and the limits of quantification wei.;10-25μg/kg. When spiked level at0.05-50mg/kg in formular feed, concentrated feed and premix feed of swine and chicken, the recoveries of sulfonamides and quinolones were71.5%-114.7%, and the relative standard deviation were in the range of1.5%to12.5%. One positive feed samples with sulfaquinoxaline were tested with the method, and compared with the method of GB/T19542. The results showed that the result was (58.9±5.75) mg/kg (n=5) with our developed method, and was (61.2±6.25)mg/kg (n=5) with GB/T19542method.更多还原