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