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A high‐performance liquid chromatography assay with a triazole‐bonded column for evaluation of d ‐amino acid oxidase activity
Author(s) -
Iwasaki Megumi,
Kashiwaguma Yoshiyuki,
Nagashima Chihiro,
Izumi Mao,
Uekusa Ayano,
Iwasa Sumiko,
Onozato Mayu,
Ichiba Hideaki,
Fukushima Takeshi
Publication year - 2016
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.3559
Subject(s) - chemistry , kynurenic acid , high performance liquid chromatography , chromatography , elution , ammonium formate , tryptophan , amino acid , biochemistry
Elution profiles of kynurenic acid (KYNA) and 7‐chlorokynurenic acid (Cl‐KYNA) were examined by high‐performance liquid chromatography (HPLC) using a triazole‐bonded stationary phase column (Cosmosil® HILIC) under isocratic elution of a mobile phase consisting of CH 3 CN–aqueous 10 m m ammonium formate between pH 3.0 and 6.0. The capacity factors of KYNA and Cl‐KYNA varied with both the CH 3 CN content and the pH of the mobile phase. The elution order of KYNA and Cl‐KYNA was reversed between the CH 3 CN‐ and H 2 O‐rich mobile phases, suggesting that hydrophilic interactions and anion‐exchange interactions caused retention of KYNA and Cl‐KYNA in the CH 3 CN‐ and H 2 O‐rich mobile phases, respectively. The present HPLC method using a triazole‐bonded column and fluorescence detection (excitation 250 nm, emission 398 nm) was applied to monitor in vitro production of KYNA from d ‐kynurenine ( d ‐KYN) by d ‐amino acid oxidase (DAO) using Cl‐KYNA as an internal standard. A single KYNA peak was clearly observed after enzymatic reaction of d ‐KYN with DAO. Production of KYNA from d ‐KYN was suppressed by the addition of commercial DAO inhibitors. The present HPLC method can be used to evaluate DAO activity and DAO inhibitory effects in candidate drugs for the treatment of schizophrenia. Copyright © 2015 John Wiley & Sons, Ltd.