Development of a simple high-performance capillary electrophoretic method with on-line mode in capillary derivatization for the determination of spermidine

A new high-performance capillary electrophoretic (HPCE) method with an on-l ine mode in-capillary derivatization (ICD)procedure for determinations of s ome amines using 20 mmol/L sodium dodecyl sulfate (SDS) - 2 mmol/L o-phthal aldehyde (OPA) - 2 mmol/L N-acetylcysteine (NAC) - 20 mmol/L phosphate-bora te buffer [9] has previously been shown. Although this technique offers dir ect fluorescence detection of free amines without any derivatization proced ures before or after HPCE separation, the presence of spermidine (Spd) is d ifficult to detect due to low fluorescence intensity. The purpose of this s tudy is to improve the detection sensitivity of Spd by reoptimizing this me thod with regard to the run buffer; the reoptimized method was applied to t he determination of Spd in human plasma. To enhance the fluorescence intens ity of the Spd signal, it is effective to use the run buffer in the presenc e of both beta-cyclodextrin (beta-CD: 8.8 mmol/L) and NAC at high concentra tion (16 mmol/L). By contrast, the intensity was remarkably decreased when SDS was used in the presence of beta-CD. After ultrafiltrating (UF) spiked human plasma with Spd, UF plasma was directly analyzed using the reoptimize d method. Spd peak was detected and separated from the other peaks of blank plasma. The present method gave good linearity (r = 0.999), reproducibilit y (3.85% coefficient of variation at 5 mu mol/L level; n = 10) and specific ity. The detection limit and lower limit of quantitation is for 0.2 mu mol/ L and 1 mu mol/L, respectively.