Determination of Kynurenine and Tryptophan in Human Plasma by Stacking-Micellar Electrokinetic Chromatography

Abstract

Kynurenine and tryptophan are important biomarkers for disorders in nervous and immune systems, while the low content of kynurenine in human plasma makes the determination by routine approaches difficult. In this work, an on-line preconcentration method for capillary electrophoresis was developed for the quantification of kynurenine and tryptophan. A concomitant sweeping phenomenon was observed due to the combination of micellar electrokinetic chromatography and stacking. The poor sensitivity in capillary electrophoresis was improved so that the determination of low concentrations of kynurenine became possible. Parameters that may affect the efficiency of the on-line concentration were systematically investigated and optimized. A large volume sample injection was employed for sampling at 100 mbar for 112 sec. A voltage of −20 kV was applied, and the ionic sample matrix was removed with stacking while analytes were kept in the capillary. The voltage was switched to +18 kV and micellar electrokinetic chromatography was performed for separation and determination of kynurenine and tryptophan. Based on peak heights, the concentration factors for kynurenine and tryptophan were 33 and 31, respectively. The analytical performance of the established method was studied; good linearity of kynurenine was observed in the range of 0.3–30 µM (R2 = 0.9988), and 0.3–60 µM (R2 = 0.9963) was obtained for tryptophan. LODs of 0.15 µM for kynurenine and 0.30 µM for tryptophan were obtained. The optimum method was successfully tested in human plasma; kynurenine and tryptophan were well identified, which suggests that this method has potential for the analysis of biological samples.