SPE-LC-MS/MS法检测膳食羧甲基赖氨酸在大鼠脑组织中的沉积
首发时间:2014-12-30
摘要:建立了定性定量分析大鼠脑组织中羧甲基赖氨酸(CML)的固相萃取-高效液相色谱-串联质谱方法,并基于该方法考察了灌胃CML的高脂和普通膳食大鼠脑组织中CML的沉积情况。脑组织样品经正己烷脱脂、硼氢化钠还原、三氯乙酸沉淀蛋白、盐酸水解、Oasis MCX固相萃取柱净化,氨化甲醇(氨水:甲醇= 5:95,v/v)溶液洗脱,收集洗脱液,氮气吹干,以水定容,微孔滤膜过滤,LC-ESI-MS/MS检测,同位素内标法定量。结果表明,CML在82.43 ~ 2637.50 ng/mL浓度范围内线性关系良好,相关系数大于0.9996;检测限为13.10ng/mL(LOD,S/N=3),定量限为44.93ng/mL(LOD,S/N=10);样品中添加100,200和300ng/mL浓度水平的回收率为92.0%~115.4%,相对标准偏差为4.39% ~ 11.91%。采用此方法对普通饲料和高脂饲料喂养灌胃CML大鼠脑组织中CML的含量进行了测定,结果表明高脂膳食和CML的摄入都会加剧CML在脑组织中的蓄积。
关键词: 羧甲基赖氨酸 固相萃取-高效液相色谱-串联质谱法 脑组织?????
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Analysis of dietary N?-(carboxymethyl)lysine accumulation in the rat brain by Solid Pase Extraction-LC-MS/MS
Abstract:A method was developed to determine the content of N?-(carboxymethyl)lysine in the rat brain by SPE-LC-MS/MS, then the CML accumulationin in the brain of rats fed on two diets, high fat diet and normal diet after intragastric administration of CML was analyzed based on this method.The sample preparation of rat brain consists of defatting by hexane, reducing by 0.1mol/L sodium borohydride, precipitating protein by trichloroacetic acid, hydrolyzing by 6mol/L HCl, cleaning up by Oasis MCX SPE cartridge, eluting with 5% ammonia/methanol (v/v).Then the eluent was dried by a stream of nitrogen gas, dissolved in water, filtered by microporous membrane, finally detected by LC-ESI-MS/MS, internal standard method was used in the quantitative analysis.The results showed that in the concentration range of 82.43~2637.50 ng/mLng/mL it exhibited a good linear relationship, the correlation coefficient was greater than 0.9996.The detection limit was 13.10 ng/mL (LOD,S/N = 3), quantification limit was 44.93 ng/mL (LOD,S/N = 10).The average recovery at three spiked levels ranged from 92.0% to 115.4%, the relative standard deviation ranged from 4.39% to 11.91%.This method was used to determine the CML content in the brain of the rats fed on two diets after intragastric administration of CML, it showed that the intake of high-fat diet and CML both exacerbated CML accumulation in the brain tissue.
Keywords: N?-(carboxymethyl)lysine SPE-LC-MS/MS brain tissue?????
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SPE-LC-MS/MS法检测膳食羧甲基赖氨酸在大鼠脑组织中的沉积
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