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

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Abstract
A new high‐performance capillary electrophoretic (HPCE) method with an on‐line mode in‐capillary derivatization (ICD) procedure for determinations of some amines using 20 mmol/L sodium dodecyl sulfate (SDS) ‐ 2 mmol/L o‐phthalaldehyde (OPA) ‐ 2 mmol/L N‐acetylcysteine (NAC) ‐ 20 mmol/L phosphate‐borate buffer [9] has previously been shown. Although this technique offers direct fluorescence detection of free amines without any derivatization procedures before or after HPCE separation, the presence of spermidine (Spd) is difficult to detect due to low fluorescence intensity. The purpose of this study is to improve the detection sensitivity of Spd by reoptimizing this method with regard to the run buffer; the reoptimized method was applied to the determination of Spd in human plasma. To enhance the fluorescence intensity of the Spd signal, it is effective to use the run buffer in the presence of both β‐cyclodextrin (β‐CD: 8.8 mmol/L) and NAC at high concentration (16 mmol/L). By contrast, the intensity was remarkably decreased when SDS was used in the presence of β‐CD. After ultrafiltrating (UF) spiked human plasma with Spd, UF plasma was directly analyzed using the reoptimized method. Spd peak was detected and separated from the other peaks of blank plasma. The present method gave good linearity (r = 0.999), reproducibility (3.85% coefficient of variation at 5 μmol/L level; n = 10) and specificity. The detection limit and lower limit of quantitation is for 0.2 μmol/L and 1 μmol/L, respectively.