title: 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.
authors.affiliation: Department of Chemistry, Center of Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA.
authors.forename: Xiaochun
authors.initials: X
authors.lastname: Wang
authors.forename: Ian
authors.initials: I
authors.lastname: Davis
authors.forename: Aimin
authors.initials: A
authors.lastname: Liu
authors.forename: Shahab A
authors.initials: SA
authors.lastname: Shamsi
mesh_terms: D050561:3-Hydroxyanthranilate 3,4-Dioxygenase / Q000378:metabolism; D015095:3-Hydroxyanthranilic Acid / Q000032:analysis* / Q000378:metabolism; D002021:Buffers; D002262:Carboxy-Lyases / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D010848:Picolinic Acids / Q000032:analysis* / Q000378:metabolism; D011312:Pressure; D017378:Quinolinic Acid / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D002021:Buffers; D009097:Multienzyme Complexes; D010848:Picolinic Acids; D015095:3-Hydroxyanthranilic Acid; D050561:3-Hydroxyanthranilate 3,4-Dioxygenase; D002262:Carboxy-Lyases; D017378:Quinolinic Acid; C030614:picolinic acid