Development of a CZE-ESI-MS assay with a sulfonated capillary for profiling picolinic acid and quinolinic acid formation in multienzyme system

Abstract

This article describes the development of a reliable CZE-ESI-MS method to simultaneously separate and quantitate three specific metabolites (3-hydroxyanthranilic acid (3-HAA), quinolinic acid (QA), and picolinic acid (PA)) of the kynurenine pathway (KP) of tryptophan catabolism. Using a covalently bonded sulfonated capillary, the parameters such as pH, type of background electrolyte, type of organic solvent, nebulizer pressure as well as both negative and positive ESI-MS modes were optimized to achieve the best Rs and S/N of three KP metabolites. The developed CZE-ESI-MS assay provided high resolution of PA/QA, high specificity, a total analysis time of 10 min with satisfactory intraday and interday repeatability of migration time and peak areas. Under optimized CZE-ESI-MS conditions, the calibration curves over a concentration range of 19-300 μM for 3-HAA and QA, and 75-300 μM for PA were simultaneously generated. The method was successfully applied for the first time to profile the concentrations of initial substrate, 3-HAA, and its eventual products, PA and QA, formed in the complex multienzyme system. As the ratio of two enzymes, 3-hydroxyanthranilate 3,4-dioxygenase (HAO) and α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) decreases, the concentration of QA approaches essentially zero indicating that all ACMS formed by the action of HAO is consumed by ACMSD rather than its spontaneous decay to QA.

Figure 1

Schematic representation of 3-HAA, PA and QA in the kynurenine (KP) pathway. The KP is very complex and the pathway shows only a few compounds relevant to this paper.

Figure 2

Effect of the buffer pH values on the separation of 3-HAA, PA and QA. CZE-ESI-MS conditions: 60 cm long (50 μm I.D.) BFS capillary. Running buffer, 15 mmol/L NH₄OAc. Applied voltage, +15 kV. Capillary temperature, 20°C. Injection, 5 mbar, 10 s; Spray champer parameters: nebulizer gas pressure at 3 psi; drying gas temperature at 200°C; dry gas flow at 5 L/min; capillary voltage at −3500 V; fragmentor at 65 V. Sheath liquid: 80:20 MeOH/water containing 20 mM NH₄OH, sheath liquid flow rate at 0.5 mL/min. Analytes: 0.5 mM for 3-HAA, 0.2 mM for PA and 0.3 mM for QA.

Figure 3

Effect of the nebulizer gas pressure on the separation and sensitivity of 3-HAA, PA and QA. CZE-ESI-MS conditions: Running buffer, 15 mmol/L NH₄OAc pH 7.0. Injection, 5 mbar, 100 s. Analytes: 0.4 mM for 3-HAA, 1 mM for PA and 1 mM for QA. Other conditions are the same as in Figure 2.

Figure 4

Comparison of negative and positive ion mode of CZE-ESI-MS. CZE-ESI-MS conditions: Running buffer, 15 mmol/L NH₄OAc pH 7.0. (A) Negative mode: sheath liquid: 80:20 MeOH/water containing 20 mM NH₄OH, capillary voltage at −3500 V; (B) Positive mode: sheath liquid: 80:20 MeOH/water containing 20 mM HOAc, capillary voltage at +3500 V. Analytes: 0.1 mM for 3-HAA, 0.8 mM for PA and 0.3 mM for QA. Other conditions are the same as in Figure 2.

Figure 5

CZE-ESI-MS of 3-HAA, PA and QA in the enzyme samples with different ratio of HAO:ACMSD. (A): Represented total ion chromatogram and extracted ion electrochromatograms of substrate 3-HAA incubated with the enzyme samples (HAO:ACMSD) in the ratio of 15:1. (B): Concentrations of substrate and products profile with different ratio of HAO:ACMSD. Other conditions are the same as Figure 4B.