A high‐performance liquid chromatography assay with a triazole‐bonded column for evaluation of d‐amino acid oxidase activity

Abstract

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 CH3 CN-aqueous 10 mm ammonium formate between pH 3.0 and 6.0. The capacity factors of KYNA and Cl-KYNA varied with both the CH3 CN content and the pH of the mobile phase. The elution order of KYNA and Cl-KYNA was reversed between the CH3 CN- and H2 O-rich mobile phases, suggesting that hydrophilic interactions and anion-exchange interactions caused retention of KYNA and Cl-KYNA in the CH3 CN- and H2 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.

Determination of l‐tryptophan and l‐kynurenine derivatized with (R)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole by LC‐MS/MS on a triazole‐bonded column and their quantification in human serum

“…A clear mechanism for the unique retention behaviour on the triazole-bonded column has not been elucidated to date. Our previous study suggested that hydrophilic interactions, rather than ionic interactions, may occur in the triazole-bonded column when a CH 3 CN-rich mobile phase is used (Iwasaki et al, 2015). Recently, some thermodynamic considerations have been employed to understand retention behaviour and separation mechanisms during chromatography; among them, van't Hoff plots have often been used (Qiu et al, 2011(Qiu et al, , 2013Yan and Row 2008).…”

Chromatographic analysis of tryptophan metabolites

“…Determination of ᴅ‐Kyn in tissues can be also performed with HPLC–FD. This method employs enzymatic oxidation of ᴅ‐Kyn by ᴅ‐amino acid oxidase and subsequent fluorescent detection of the generated Kyna .…”

Fluorimetric determination of the enantiomers of vigabatrin, an antiepileptic drug, by reversed‐phase HPLC with a novel diastereomer derivatization reagent

“…Kynurenate undergoes a post‐column reaction with Zn 2+ ions to form a fluorescent complex, Zn 2+ ‐KYNA. Although KYNA itself is fluorescent because of its quinoline structure, which is a π ‐electron‐rich resonance system with a plain structure, its fluorescence intensity is weak at wavelengths below 400 nm (Iwasaki et al, 2016). Therefore, it is necessary to perform a post‐column reaction of KYNA with Zn 2+ ions to form Zn 2+ ‐KYNA, a fluorescent complex, to increase its detection sensitivity.…”