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Development of a CZE ‐ ESI ‐ MS assay with a sulfonated capillary for profiling picolinic acid and quinolinic acid formation in multienzyme system
Author(s) -
Wang Xiaochun,
Davis Ian,
Liu Aimin,
Shamsi Shahab A.
Publication year - 2013
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201200679
Subject(s) - chemistry , chromatography , picolinic acid , quinolinic acid , capillary electrophoresis , kynurenine , tryptophan , mass spectrometry , biochemistry , amino acid
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 R s 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 .