肌肽类生物活性肽的毛细管电泳在线富集技术

On-Line Sample Stacking for Determination of Carnosine-Related Peptides by Capillary Electrophoresis

建立了毛细管区带电泳（CZE）在线富集3种肌肽类活性肽（肌肽、鹅肌肽和高肌肽）的两种简便有效的方法。一种是大体积进样反向压力排除基体富集（LVSRP）技术，即通过流体动力学进样，在不改变电源极性的条件下，利用反向压力排除样品基体，电堆积富集后进行CZE分离；另一种是大体积进样电渗流排除基体富集（LVSEP）技术，即通过流体动力学进样，于运行缓冲液中加入溴化十六烷基三甲基铵（CTAB）动态修饰毛细管表面，通过电渗流排除样品基体，改变电源极性后进行CZE分离。与常规CZE相比，LVSRP技术和LVSEP技术使检测灵敏度提高了40-60倍。对影响两种富集过程的一些因素进行了研究，在最优富集条件下考察本方法的线性范围为0．080—5．0umol／L。对3种生物活性肽的检测限（S/N=3）分别为LVSRP41—58nmol／L，LVSEP35—43nmol／L。

Two on-line sample stacking methods in capillary electrophoresis, large volume sample stacking using reversed pressure as a pump （LVSRP） and large volume sample stacking using electroosmotic flow （EOF） pump （LVSEP）, were developed for the separation and determination of carnosine, anserine and homocarnosine. The principles of the two stacking methods are described. Various parameters affecting capillary electrophoresis separation and sample stacking were studied and optimized. The optimal stacking conditions for LVSRP were as follows： hydrodynamic sample injection at 41.4 kPa （60 s）, then injection of a short plug of buffer at 13.8 kPa × 15 s, stacking of sample at a voltage of 10 kV with a back pressure of 34.5 kPa, and separation in phosphate buffer （pH 5.63, 100 mmol/L） at a voltage of 15 kV. The optimal stacking conditions for LVSEP were as follows： hydrodynamic sample injection at 48.3 kPa （60 s）, stacking at a voltage of 20 kV and separation at a voltage of - 20 kV in a buffer of 100 mmol/L phosphate-0.5 mmol/L cetyltrimethylammonium bromide （ CTAB ） （ pH 5.31 ）. About 40 - 60 fold improvement of sensitivity was achieved without loss of separation efficiency by LVSRP and LVSEP, when compared with the normal CZE method. Under the optimum conditions, excellent linear responses were obtained in the concentration range of 0.080 -5.0 umol/L. The relative standard deviations （RSDs） of peak height were 2.4% -2.7% and 0.6% - 4.0% for LVSRP and LVSEP, respectively. The concentration limits of detection （ defined as signal-to-noise ratio of 3 ） of carnosine, anserine and homocarnosine obtained for LVSRP and LVSEP were 41 -58 nmol/L and 35 -43 nmol/L, respectively.