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动物性食品中兽药残留定量和确证分析关键技术研究
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摘要
食品安全问题关系到国民的身体健康,已经成为人类共同关心的问题。兽药残留是影响动物性食品安全的主要原因之一,因此兽药残留分析方法是保障动物性食品安全的有效手段。兽药残留分析主要包括:样品前处理和分析检测技术两个方面。由于食品中兽药残留物水平低,样品基质复杂,使得复杂食品基质中兽药残留分析需要更为有效的前处理方法和灵敏的检测技术。目前,动物性食品中兽药残留定量和确证方法中存在的主要问题是:样品前处理方法,操作繁琐,耗时较长,试剂需要量大,同时分析药物种类少,提取样品的种类少。色谱分析方法,同时分离测定的药物种类少,检测灵敏度低,分离度差,基质干扰严重。因此,有必要对动物性食品中兽药残留定量和确证方法中样品前处理和色谱分析等关键技术进行研究。
     本论文主要研究内容是:研究简便、快速、低廉和有效的样品前处理技术,研究快速、灵敏的多组分同时分析的液相色谱和色谱-质谱联用技术,用于动物性食品中兽药残留检测。本论文以糖皮质激素类、苯并咪唑类和机胂类药物为研究目标,探讨动物性食品中兽药残留定量和确证方法中的样品前处理技术和色谱分析技术,建立动物性食品中糖皮质激素类、苯并咪唑类和机胂类药物残留检测方法。本论文的研究,可以为动物性食品中兽药残留的检测研究提供技术支持和理论依据,对动物性食品安全评价也具有指导意义和参考价值。
     1、动物性食品中糖皮质激素类药物多残留分析方法研究。
     建立高效液相色谱-串联质谱(HPLC-MS/MS)检测猪、牛、羊肌肉和肝脏、鸡肌肉、鸡蛋和牛奶动物性食品中的糖皮质激素类药物方法(泼尼松、泼尼松龙、氢化可的松、地塞米松、倍他米松、氟氢可的松、甲基泼尼松、倍氯米松)。样品前处理采用碱水解,乙酸乙酯提取,硅胶固相柱萃取净化。采用碱水解,缩短了样品水解时间。采用硅胶固相萃取柱用于各种基质中糖皮质激素药物的萃取净化,简化了多种萃取柱多步净化的方法,样品操作简便,净化效果好。采用C18色谱柱,乙腈和水(0.2%甲酸)为流动相,成功地解决了地塞米松和倍他米松2种同分异构体分离的难题。质谱检测采用电喷雾离子源,多反应选择监测模式,方法具有灵敏度高,特异性好等特点。糖皮质激素类药物在肝脏中定量限为1.0μg/kg-4.0μg/kg,在牛奶中定量限为0.2μg/g-0.8μg/kg,在肌肉和鸡蛋中定量限为0.5μg/kg-2.0μg/kg。本方法已成功用于2008年北京奥运会动物性食品中糖皮质激素类药物监控。本方法简便、快速,适用于动物性食品中糖皮质激素类药物的定性和定量分析。
     2、动物性食品中苯并咪唑类药物多残留分析方法研究
     建立苯并咪唑类21种药物在猪、牛、羊、鸡肌肉和肝脏、鱼肌肉、鸡蛋和牛奶动物性食品中的高效液相色谱-紫外(HPLC-UV)和HPLC-MS/MS的定量和确证分析方法。样品前处理采用固相萃取技术和加速溶剂萃取技术,详细探讨了这两种提取净化技术的提取效果和影响因素。采用固相萃取净化,样品前处理简单,有机试剂用量少。采用快速溶剂萃取,样品前处理简便,快速,有机试剂用量少,可以实现自动化,减少有毒试剂对人体的污染。用固相萃取技术和加速溶剂萃取技术,解决了多种样品基质中,多种苯并咪唑类药物分析中的样品前处理技术的难题。采用C18色谱柱梯度洗脱,解决了21种苯并咪唑类药物采用HPLC测定时色谱分离的难题,建立了测定21种苯并咪唑类药物HPLC方法,在猪、牛、羊、鸡和鱼的肌肉、鸡蛋和牛奶中定量限为10gg/kg,猪、牛、羊和鸡肝脏中定量限为20μg/kg。优化了色谱和质谱参数,建立测定21种苯并咪唑类药物HPLC-MS/MS方法,在动物性食品中定量限为0.02μg/kg-0.5μg/kg,方法灵敏度高,特异性好。
     3、动物性食品中有机胂类药物多残留分析方法研究
     建立阿散酸、洛克沙胂、硝苯胂酸、卡巴胂4种有机胂类药物在猪、鸡肌肉和肝脏、鱼肌肉和鸡蛋动物性食品中的HPLC-UV和HPLC-MS/MS方法。样品前处理采用超声振荡萃取技术,以甲醇、乙腈和三氯乙酸混合溶液为提取剂,可以同时提取动物性食品中4种有机胂类药物;采用强阴离子交换(SAX)固相萃取柱净化,样品前处理简单,有机试剂用量少,基质干扰少。HPLC-UV方法采用Hypersil ODSC18色谱柱,乙腈和1%乙酸为流动相。HPLC-MS/MS方法采用HILIC Silica色谱柱,乙腈和乙酸铵为流动相。本方法采用HPLC-UV和HPLC-MS/MS同时测定动物源性食品中4种有机胂类药物,检测灵敏度高、特异性好。本方法可以广泛应用于动物源性食品中多种有机胂类药物的监测,对动物性食品中多残留分析方法研究具有指导意义,为动物性食品安全评价提供技术支持。
Food safety concerns everyone's health and has attracted more attention around the world. Veterinary drug residues in food of animal origin are one of the main impacts on food safety. Hence, the development of analysis methods for the determination of veterinary drugs residues in food is necessary to ensure food sasfety. There are two prominent problems in veterinary drug residues analysis, one is the pretreatment means, and the other is the separation and deterniantion method. Since the food matrices are complex and veterinary drug residues are of trace level, more effective sample pretreatment and sensitive determination method should mathch the requirement of the veterinary drug residues analsis in food. Hence, the study on the sample preparation technology and chromatographic analysis is crucial to determination of veterinary drug residues in food.
     The main study content of this thesis was to develope the sample preparation technology and chromatographic analysis method to determine veterinary drugs residues in food. The main purpose of this thesis was to resolve the questions of sample preparation technology and chromatographic analysis, in order to esbilish methods for determination of residues of glucocorticoids, benzimidazoles and organic arsenic in food. The study provides technical and thesis support for controlling veterinary drug residues in food and evaluating on animal food safety.
     1. Development of a method for simultaneous determination of residues of glucocorticoid in animal derived food.
     A confirmatory and quantitative method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the presence of eight glucocorticoids (prednisone, prednisolone, hydrocortisone, methylprednisolone, dexamethasone, betamethasone, beclomethasone, and fludrocortisone) in edible tissues of swine, cattle, sheep and chicken, egg and milk is described. After deconjugation in alkali media, samples were extracted with ethyl acetate for glucocorticoids followed by solid-phase extraction clean-up and reconstitution in the LC mobile phase. The hydrolysis procedure with sodium hydroxide was used to reduce handling time. A single-step solid-phase extraction method was optimized which is suitable for the clean-up of the compounds of interest in many diverse tissue matrices. LC separations were performed on a C18column with gradient elution using acetonitrile and water (containing0.2%formic acid) and the two epimers betamethasone and dexamethasone were successfully separated. LC-electrospray ionization (ESI)-MS/MS in negative mode with selected reaction monitoring (SRM) mode was performed to improve method sensitivity and reduce matrix interference. Two SRM transitions were used for each compound. The optimized procedure was successfully applied to monitor the food at the2008Summer Olympics Games in Beijing, China, demonstrating the method to be simple, fast, robust, and suitable for identification and quantification of glucocorticoids residues in foods of animal origin.
     2. Development of a method for simultaneous determination of residues of benzimidazole and its metabolites in animal derived food.
     A confirmatory and quantitative method using HPLC-UV and HPLC-MS/MS have been established for simultaneous determination benzimidazole and its metabolites in the muscles and livers of swine, cattle, sheep, and chicken, muscle of fish, eggs, and milk. Sample prepatation techniques including solid phase extraction, and accelerated solvent extraction (ASE) were applied in extracting benzimidazole and its metabolites in animal derived foods. The sample pretreatment has been simplified for using the ultrasonic extraction and solid phase extraction in the method. The volume of the organic solvent was decreased using the acceleratet solvent extraction method, avoiding the toxicity solvent using, and realizing sanple-handling automatism in the method. HPLC-UV analysis was performed on C18column eluted and applied to simultaneous determination benzimidazoles. HPLC-MS/MS analysis was performed on C18column eluted by a mobile phase of acetonitrile spiked with a buffer solution consisting of5mmol l-1formic ammonium, and the analytes were detected by LC-ESI-MS/MS in positive mode with selected reaction monitoring (SRM) mode. The method has been successfully applied to monitoring real samples containing benzimidazole and its metabolites, which demonstrated that it is a simple, fast and robust method, and could be used as a regulatory tool to determine the residues of benzimidazole and its metabolites in animal-origin foods.
     3. Development of a method for simultaneous quantification of residues of organic arsenic in animal derived food.
     HPLC-UV and HPLC-MS/MS have been established for simultaneous determination of4organic arsenic (arsanilic Acid, roxarsone, nitarsone, carbarsone) in the muscles and livers of swine and chicken, the muscle of fish and egg. And some conditions such as sample extraction, purification and sample solvent have been deeply investigated in this experiment. Samples were extracted with methanol-acetonitrile-trichloroacetic acid solution in an ultrasonic bath, and purified by solid phase extraction on strong anion exchanger cartridges. The sample pretreatment has been simplified for using the ultrasonic extraction and solid phase extraction in the method. The volume of the organic solvent was decreased using the sample pretreatment method. This simply and little contaminative method with the familiar materials can be applied on monitoring the use of multi-organic arsenic in animal food. The samples were analyzed on an Hypersil ODS C18liquid chromatography column using a gradient program with methanol and1%acetic acid by HPLC-UV. The samples were analyzed on a HILIC Silica liquid chromatography column using a gradient program with acetonitrile and ammonium acetate by HPLC-MS/MS with electrospary ionization method. HPLC-UV and HPLC-MS/MS have been firstly established for simultaneous determination of organic arsenic in animal dereived foods. The methods have good sensitivity, specificity, and which can be appilied to monitor the possible misuse of organic arsen residues in animal derived foods. The experiment can be taken as an instruction to do studies on analysis methods for determination of multi-residues in animal tissues and provide technical support for evaluation on animal food safety.
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