毛细管电动色谱分离手性氨基酸及其在生物样品分析中的应用  

Chiral Analysis of Amino Acids in Biological Samples by Micellar Electrokinetic Chromatography with Laser-induced Fluorescence Detection

作　　者：付煜荣[1] 颜流水[1] 罗国安[1] 陈翠真[2] 王义明[1] 张兰桐[3] 

机构地区：[1]清华大学分析中心 [2]北京市眼科研究所,北京100005 [3]河北医科大学药学院,石家庄050017

出　　处：《分析化学》2004年第12期1575-1579,共5页Chinese Journal of Analytical Chemistry

基　　金：国家973项目资助(No.2001CB510306)

摘　　要：建立了MEKC法分离丙氨酸(Ala)、谷氨酸(Glu)和天门冬氨酸(Asp)三对手性氨基酸,并对大鼠正常组和白内障组晶体和血清中DAla、LAla、DGlu、LGlu、DAsp和LAsp进行定量分析。利用FITC柱前衍生,以β环糊精(βCD)作为手性添加剂,激光诱导荧光检测(LIF),优化电泳条件为气压进样5s,分离温度25℃,分离电压为20kV,运行缓冲溶液为40mmol/L硼砂缓冲溶液(pH9.3),其中包含50mmol/LSDS和13mmol/LβCD。考察了硼砂缓冲溶液浓度、pH值、分离电压、β环糊精浓度对分离的影响。在优化电泳条件下,上述3种氨基酸对映体均得到基线分离,并可对大鼠的晶体和血清进行定量分析。本方法操作简单,检测灵敏度高,可用于药品质量监测以及生物样品的分析。A micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection (LIF) for chiral analysis of amino acids in biological samples was established. These amino acids, including D or L-alanine (Ala), D or L-glutamic acid (Glu) and D or L-aspartic acid (Asp), were derivatized with fluorescein isothiocyanate (FITC). The optimized conditions in electrophoresis were that uncoated fused-silica capillary (50 mum x 37 cm, 30 cm to detector) was used, running voltage was 20 kV, running buffer was 40 mmol/L borate buffer (pH 9.3) containing 50 mmol/L sodium dodecyl sulfate (SDS) and 13 mmol/L beta-cyclodextrin (beta-CD), the sample solution was injected 5 s with pressure mode, separated at 25degreesC, and monitored with a LIF detection. The baseline separation of these amino acid enantiomers was achieved. The linear ranges of six FITC-derivatives was about 0.050 similar to 2.000 mg/L, and the determination limits (S/N = 3) were 0.005 similar to 0.050 mg/L. The average recoveries (n = 6) and RSD of the six FITC-derivatives were 96.33% similar to 102.0% and 3.6% similar to 5.2% from rat serum, 97.59% similar to 102.8% and 3.5% similar to 5.9% from rat lens. The within-day precisions (n = 9) and between-day precisions (n = 5) of them were lower than 5.0% and 6.5% from rat serum, lower than 5.0% and 5.5% from rat lens, respectively.

关 键 词：血清 大鼠 分离电压 L-ASP 正常 天门冬氨酸 白内障 手性氨基酸 进样 晶体 

分 类 号：O657.7[理学—分析化学]