选择性雄激素受体调节剂（SARMs）检测方法的建立及应用

发布时间：2018-04-24 10:52

本文选题：液质联用 + 飞行时间质谱　；参考：《北京体育大学》2015年硕士论文

【摘要】：目的本论文以选择性性激素受体调节剂(SARMs)为主要研究对象,利用液相色谱-串联质谱、液相色谱-串联飞行时间质谱来探索、建立人尿中选择性性激素受体调节剂及其代谢物的检测和评估方法,并使用建立的检测方法检测运动员样品,以证明检测方法的有效性,能保证出国运动员不出现兴奋剂问题。方法采用液液萃取(LLE)方法:取1ml尿液加入10μL×5 ppm的甲睾作为内标,50μL葡萄糖醛酸甙酶,0.5 mL磷酸盐缓冲盐,震荡混匀,于55℃水浴1 h,加0.5 mL碱性缓冲盐,加4 mL甲基叔丁基醚,萃取,保留有机相,常温氮气流吹干,用初始流动相溶解定容至100μL,进样分析;采用固相萃取(SPE)方法对代谢物进行研究:Waters Sep-Pak C18固相萃取小柱(6CC,500mg)经3mL甲醇活化、3mL去离子水洗涤,加入1ml大鼠尿液(或细胞培养液),3mL去离子水洗涤,3mL甲醇淋洗,收集甲醇溶液,60℃氮气保护下吹干,用150ul流动相复溶,0.2umGHP滤膜过滤回收至进样瓶,进样分析。结果与讨论采用常规筛查方法对7种选择性雄激素受体调节剂类药物进行方法验证工作,最低检出限为0.01-2ng/ml,回收率均高于60%,日内及日间精密度均低于20%,符合世界反兴奋剂机构技术文件中方法验证要求。采用大鼠实验及人体干细胞体外实验对3种药物进行了代谢研究,分别发现了多种代谢物。结论本研究建立了针对7种选择性雄激素受体调节剂类药物的检测方法,方法稳定性好,符合WADA技术文件要求;并通过代谢实验对三种药物进行了系统的代谢研究,发现了多个代谢物,其中包括多个新代谢物。对200例样品进行了筛查工作,保证运动员干干净净参赛。
[Abstract]:Objective to study the determination and evaluation of selective hormone receptor modulators and their metabolites in human urine by liquid chromatography tandem mass spectrometry (liquid chromatography tandem mass spectrometry) and tandem time of flight mass spectrometry (SARMs). To prove the effectiveness of the detection method, it can ensure that the athletes abroad do not appear doping problem. Method using liquid liquid extraction (LLE) method: Taking 1ml urine with 10 mu L x 5 ppm as internal standard, 50 mu L glucuronase, 0.5 mL phosphate buffer salt, mixing, 1 h in 55 C water bath, 0.5 mL alkaline buffer salt, and 4 mL methyl tert butyl. Base ether, extraction, retention of organic phase and dry nitrogen air flow to dry, use initial flow phase to dissolve to 100 mu L, sample analysis; use solid phase extraction (SPE) method to study metabolites. Waters Sep-Pak C18 solid phase extraction column (6CC, 500mg) is activated by 3mL methanol, 3mL deionized water washing, 1ml rat urine (or cell culture), 3mL go away Subwater washing, leaching of 3mL methanol, collecting methanol solution, drying under 60 centigrade nitrogen gas protection, reusing 150ul liquid phase and recovering to sample bottle with 0.2umGHP filter membrane filtration, analysis. Results and discussion using conventional screening methods to verify 7 selective androgen receptor modulators, the minimum detection limit is 0.01-2ng/ml. The yield was higher than 60%, the intraday and daytime precision was less than 20%, which was in line with the requirements of the method verification in the World Anti Doping Agency technical documents. The metabolic study of 3 drugs was carried out in rats and human stem cells in vitro, and several metabolites were found. Conclusion this study was established for the regulation of 7 kinds of selective androgen receptor. The methods for the detection of drug drugs are stable and conform to the requirements of the WADA technical documents; and the metabolic study of three kinds of drugs is carried out through metabolic experiments. Several metabolites are found, including a number of new metabolites. 200 samples are screened to ensure the athletes to compete clean and clean.

【学位授予单位】：北京体育大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.12;O657.63

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