[On-line sample stacking for determination of carnosine-related peptides by capillary electrophoresis]

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 x 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 micromol/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.