游离棉酚及其代谢产物棉酚酮在动物体内残留和组织分布研究

发布时间：2018-03-24 11:13

本文选题：游离棉酚　切入点：棉酚酮　出处：《新疆医科大学》2017年硕士论文

【摘要】：目的:1.本实验通过对实验大鼠、鲫鱼、鸡喂食含有棉酚的自制棉籽饲料,模拟动物进食过程,实验周期内定期取实验动物各组织、器官,建立HPLC检测实验样品中游离棉酚及其代谢产物(棉酚酮)残留的方法。并采用高灵敏度的UPLC-MS/MS分析游离棉酚及其代谢产物在大鼠体内的组织分布。2.建立牛乳中游离棉酚及其旋光异构体的HPLC检测方法。并根据上述方法对市售的动物源性食品进行游离棉酚及其代谢产物残留的检测。方法:1.用自制的含定量棉酚的棉籽饲料饲养实验动物一定周期,定期取实验动物各组织器官(心、肝、脾、肺、肾、肉、血浆),将各样品匀浆,加入提取剂(0.2%甲酸乙腈溶液)及甘草次酸内标液,经除蛋白、离心等前处理方法后,采用HPLC及UPLC-MS/MS法确定动物体内各个组织器官中游离棉酚及其代谢产物的残留和组织分布。2.采用以(R)-(-)-2-胺基-1-丙醇为手性拆分试剂、甘草次酸为内标物的柱前衍生高效液相色谱法,对牛乳中游离棉酚及其旋光异构体含量进行检测。结果:1.HPLC及UPLC-MS/MS测定动物体内游离棉酚及其代谢产物的分析方法良好。HPLC法测定大鼠和鸡体内各组织器官中游离棉酚及棉酚酮的线性范围为0.5-20μg/mL,鲫鱼为0.1-10μg/mL,检出限0.03μg/mL。UPLC-MS/MS法检测大鼠体内各组织器官中游离棉酚及棉酚酮的线性范围均为0.01-10μg/mL,检出限为0.002μg/mL。UPLC-MS/MS分析表明大鼠体内游离棉酚及棉酚酮的含量随饲养时间延长而蓄积增加,体内棉酚含量依次为:肝脏、血浆、脾脏、肺脏、心脏、睾丸(雄性)、肾脏、肌肉,且肝脏中蓄积最多,动物体内代谢物棉酚酮仅在肝脏中发现。通过对雌、雄大鼠进行对比分析,结果显示棉酚在雌性大鼠体内的蓄积更高。2.牛乳中棉酚及其旋光异构体浓度线性范围均为2-10μg/mL。3.运用所建立的检测方法,对市售动物源性食品中游离棉酚及其代谢产物进行检测,包括鱼、鸡、猪、羊(肝、肉)共58份样品,检出率为51.7%。结论:本研究建立了动物体内和乳制品中游离棉酚及棉酚酮HPLC检测方法,为市场上动物源性食品中游离棉酚及棉酚酮的检测提供了数据支持。同时通过UPLC-MS/MS法提高了高灵敏度,进一步证实了棉酚在动物体内的代谢产物为棉酚酮,为体内棉酚代谢产物的研究提供实验基础。
[Abstract]:Objective 1. In this experiment, the experimental rats, crucian carp and chickens were fed with self-made cottonseed feed containing gossypol to simulate the feeding process of the animals. The tissues and organs of the experimental animals were taken regularly during the experiment cycle. A HPLC method for the determination of free gossypol and its metabolites (gossypol ketone) in experimental samples was established. The tissue distribution of free gossypol and its metabolites in rats was analyzed by high sensitivity UPLC-MS/MS. 2. Free gossypol and its metabolites in milk were established. HPLC method for detection of gossypol and its optical isomers. Determination of free gossypol and its metabolite residues in animal-derived foods sold on the market based on the above method. Methods: 1. Feeding with cotton seed feed containing quantitative gossypol. Experimental animals have a certain period, The tissues and organs (heart, liver, spleen, lung, kidney, meat, plasma) of experimental animals were regularly taken. The samples were homogenized and added with the extract of 0.2% acetonitrile formate solution, and the internal standard solution of glycyrrhetinic acid was added. The residual and tissue distribution of free gossypol and its metabolites in animal tissues and organs were determined by HPLC and UPLC-MS/MS methods. A precolumn derivatization high performance liquid chromatography (HPLC) was developed, which was used as chiral resolution reagent and glycyrrhetinic acid as internal standard. The content of free gossypol and its rotatory isomers in milk was determined. Results 1. The determination of free gossypol and its metabolites in animals by HPLC and UPLC-MS/MS. The method of HPLC was good for the determination of free gossypol in tissues and organs of rats and chickens. The linear range of gossypol and gossypol was 0.5-20 渭 g / mL and 0.1-10 渭 g / mL, respectively. The detection limit of 0.03 渭 g/mL.UPLC-MS/MS was 0.01-10 渭 g / mL for free gossypol and gossypol in various tissues and organs of rats. The detection limit of free gossypol and gossypol was 0.002 渭 g/mL.UPLC-MS/MS. The accumulation increased with the increase of feeding time. The contents of gossypol in vivo are as follows: liver, plasma, spleen, lung, heart, testis (male, kidney, muscle, and liver). The metabolite gossypol is found only in the liver. The results of comparative analysis in male rats showed that gossypol accumulated higher in female rats. The linear range of gossypol and its optical isomers in milk was 2-10 渭 g / mL 路3.Using the established method, A total of 58 samples of free gossypol and its metabolites, including fish, chicken, pig, sheep (liver, meat), were detected in animal food. Conclusion: this study established a HPLC method for the detection of free gossypol and gossypol in animal and dairy products. It provides data support for the detection of free gossypol and gossypol in animal food on the market. At the same time, the high sensitivity of gossypol in animal body is improved by UPLC-MS/MS method, which further proves that the metabolite of gossypol in animal body is gossypol. To provide experimental basis for the study of gossypol metabolites in vivo.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R99

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