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

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.

Keywords: anionic compounds; capillary electrokinetic chromatography; kynurenine pathway; mixed micelle; on-line preconcentration; pressure-assisted electrokinetic injection.

Figure 1

(A) In the initial situation, a sample with low conductivity (0.01 mM NaOH) is present at the inlet of the capillary, while a micellar BGE is located at the outlet. Voltage is applied with reversed electrode polarity, simultaneously utilizing pressure that counterbalances the EOF, which is directed opposite to the applied pressure. The analytes preconcentrate at the sample/BGE interface, where a layer of counterions (cations) stabilizes it. (B,C) The sample at the capillary inlet is replaced by the BGE, and the electrode polarity is switched to normal, leading to the migration of accumulated anions. In this case, the EOF is directed toward the capillary outlet (cathode), and its value exceeds the migration velocities of the micelles. Mixed micelles of SDS and Brij-35, which have a net negative charge and an apparent migration direction toward the anode (note that V_EOF > V_micelle), interact with the analytes, enhancing the separation potential. (D) The separated and stacked analytes migrate through the detection window. SM—sample matrix.

Figure 2

The electrophoretic separation of kynurenines at increasing concentrations of surfactants in the BGE, (A) without SDS and Brij-35, (B) 20 mM SDS, (C) 2 mM Brij-35, (D) 2 mM Brij-35 and 5 mM SDS, (E) 2 mM Brij-35 and 10 mM SDS, (F) 2 mM Brij-35 and 20 mM SDS, (G) 2 mM Brij-35 and 30 mM SDS. Other conditions: BGE: 20 mM Na₂B₄O₇, 20% (v/v) MeOH; applied voltage 25 kV, HDI (5 s, 0.5 psi), capillary: 50 cm (length) × 50 µm. Analytical wavelength: 203 nm. Analyte concentration: each at 20 μg/mL. IS—internal standard (phenobarbital).

Figure 3

Signal enhancement effect for kynurenine pathway compounds using the PAEKI-MEKC technique and the mixed micelle system compared to standard HDI coupled with the MEKC separation. (A) PAEKI: 2 min, −6.5 kV, 0.5 psi, (B) HDI 5 s, 0.5 psi. Other conditions: BGE: 2 mM Brij-35, 20 mM SDS, 20 mM Na₂B₄O₇, 20% (v/v) MeOH; applied voltage: 25 kV; capillary 50 cm (length) × 50 µm (I.D.). Analytical wavelength: 203 nm. Analyte and IS concentration: (A) 1 μg/mL, (B) 20 μg/mL.

Figure 4

Representative electropherograms for random 5 mL artificial urine samples spiked with (A) 50 ng/mL each of analyte, (B) 5 ng/mL each of analyte, and (C) artificial blank urine samples. All samples were spiked with 100 ng/mL of the IS. Other conditions are the same as in Figure 3A.