# On-Line Preconcentration of Selected Kynurenine Pathway Metabolites and Amino Acids in Urine via Pressure-Assisted Electrokinetic Injection in a Mixed Micelle System

## TL;DR

A new method improves detection of kynurenine pathway metabolites in urine using advanced injection techniques.

## Contribution

A novel pressure-assisted electrokinetic injection method with mixed micelles enhances detection of trace metabolites.

## Key findings

- PAEKI increased signal intensity for kynurenines by 146-fold compared to hydrodynamic injection.
- The method achieved detection limits as low as 1.2 ng/mL for kynurenine in urine samples.
- The technique effectively analyzed kynurenine pathway metabolites and aromatic amino acids in complex matrices.

## Abstract

To enhance the signal intensity of kynurenines, which are present at trace concentrations in biological fluids, a novel analytical approach was developed, combining pressure-assisted electrokinetic injection (PAEKI) with a mixed micelle system based on sodium dodecyl sulfate (SDS) and Brij-35. The method was applied to key compounds of the kynurenine pathway, including L-tryptophan, kynurenine, 3-hydroxykynurenine, and kynurenic acid, as well as to the aromatic amino acids (AAs) L-tyrosine and L-phenylalanine. PAEKI was performed by electrokinetic injection for 2 min at −6.5 kV (reversed polarity) and 0.5 psi (3.45 kPa) using a fused silica capillary (50 cm in length, 50 µm inner diameter). The background electrolyte (BGE) consisted of 20 mM Na2B4O7 (pH 9.2), 2 mM Brij-35, 20 mM SDS, and 20% (v/v) methanol (MeOH). The limit of detection (LOD) using a diode array detector (DAD) was 1.2 ng/mL for kynurenine and ranged from 1.5 to 3.0 ng/mL for the other analytes. The application of PAEKI in conjunction with micellar electrokinetic capillary chromatography (MEKC) and solid-phase extraction (SPE) of artificial urine samples resulted in a 146-fold increase in signal intensity for kynurenines compared to that observed using the hydrodynamic injection (HDI) mode. The developed method demonstrates strong potential for determining kynurenine pathway metabolites in complex biological matrices.