In-capillary derivatization with 1-methoxycarbonylindolizine-3,5-dicarbaldehyde for high-performance capillary electrophoresis

Abstract

A simple, selective and reproducible high-performance capillary electrophoresis (HPCE) assay with in-capillary derivatization using 1-methoxycarbonylindolizine-3,5-dicarbaldehyde (IDA) as a derivatization reagent for the amino group has been developed. The method involves two modes of in-capillary derivatization, namely, ‘on-line’ and ‘sandwich’. The on-line mode involves derivatization and separation in a capillary filled with a run buffer containing IDA. The sandwich mode refers to a method in which the front end of a separation capillary for HPCE is used as a derivatization chamber, in which a sample buffer and reagent buffer are introduced into the injection end of a capillary as a ‘sandwich’, i.e., reagent buffer–sample solution–reagent buffer. In both modes, each sample was subsequently derivatized, electromigrated and detected (409 nm) by flow in the capillary. The assay reproducibility (n= 5) on the on-line mode, sandwich mode and pre-column method at 1 mmol l^–1 alanine (Ala) was 1.31, 12.26 and 3.72%, respectively. The detection limit (S/N = 3) obtained by both modes was 1 µmol l^–1 of Ala, which is nearly the same level as that obtained by the pre-column method. Calibration curves, for both modes, were constructed over the concentration range 10 µmol l^–1 to 10 mmol l^–1 Ala. The pH of the run buffer was limited to 10 for the on-line mode; for the sandwich mode a wide range of pH could be used. The present method was applied to the determination of 16-component amino acid solution, 4-component polyamine (cadaverin, histamine, spermidine and tyramine) solution, and 4-component aminocyclitol antibiotic (amikacin, arbekacin, micronomicin and netilmicin) solution.