柱上样本堆积毛细管电泳法同时检测细胞内氨甲蝶呤及其六种代谢产物  

Simultaneous determination of methotrexate and its six metabolites in cells by on-line sample stacking capillary electrophoresis

作　　者：李道静[1] 何晓东[1] 李明[1] 孙利[1] 许维东[1] 沈佐君[1] 

机构地区：[1]安徽医科大学附属省立医院安徽省临床检验中心,安徽合肥230001

出　　处：《检验医学》2011年第10期667-671,共5页Laboratory Medicine

基　　金：国家自然科学基金资助项目(30672011);安徽省自然科学基金资助项目(050430902);安徽省人才开发资金资助项目(2005Z040)

摘　　要：目的建立柱上堆积高效毛细管电泳法同时检测细胞内氨甲蝶呤(MTX)及其6种代谢产物氨甲蝶呤多聚谷氨酸链(MTXPG)的方法。方法采用高效毛细管电泳技术,以75 mmol/L磷酸盐缓冲液(pH值7.40)作为分离介质,用水和乙腈(V/V=1∶1)作溶剂重组残留样本的方法进行柱上样本堆积,0.5 psi压力进样,进样时间50 s,在分离电压28 kV、检测波长300 nm条件下对MTX和MTXPG进行分离。在此基础上,检测细胞内MTX及其代谢产物MTXPG的含量。结果通过不断改变分离缓冲液中异丙醇(5%、8%、10%,V/V)和羟丙基-β-环糊精(hp-β-CD)浓度(20、25、30 mmol/L),发现在含8%异丙醇、25 mmol/L hp-β-CD的缓冲液条件下,30 min内实现7种组分的完全分离。用水和乙腈(V/V=1∶1)作溶剂重组残留样本进行柱上样本堆积的方法可使检测灵敏度提高10倍。结论建立柱上样本堆积高效毛细管电泳法可同时检测细胞内MTX及其代谢产物MTXPG。该方法具有高效、快速、简便、高灵敏度的特点。Objective To establish a method for simultaneous determination of methotrexate（MTX） and its 6 metabolites methotrexate polyglutamate（MTXPG） in cells by on-line sample stacking capillary electrophoresis. Methods The eletrophoresis buffer was 75 mmol/L Na2HPO4（pH=7.40）.The voltage of 28 kV was applied and the injection time was 50 seconds at 0.5 psi.The separation of MTX and MTXPG was performed at a wavelength of 300 nm,and sample stacking was achieved by using distilled water and acetonitrile（V/V=1∶1） as dissolvent.Under optimal condition,intracellular MTX and MTXPG levels were detected by capillary electrophoresis.Results The optimal condition was made by adjusting isopropanol concentration（5%,8%,10%,V/V） and hp-β-CD concentration（20,25,30 mmol/L）.Seven substances were completely separated by capillary electrophoresis in 30 min.A 10 fold improvement in the amount of distilled water and acetonitrile（V/V=1∶1） could be achieved by on-line sample stacking method.Conclusions MTX and MTXPG in cells can be simultaneously separated by high-performance capillary electrophoresis efficiently,rapidly,conveniently and super-sensitively.

关 键 词：氨甲蝶呤 氨甲蝶呤多聚谷氨酸链 毛细管电泳法 柱上样本堆积 

分 类 号：R979.1[医药卫生—药品]