Improved method for the determination of kynurenic acid in rat plasma by column-switching HPLC with post-column fluorescence detection

Determination of rat brain kynurenic acid by column‐switching HPLC with fluorescence detection

“…This superior selectivity of the HPLC method was mainly based on the separation on two octadecylsilica (ODS) columns, which were connected via a six-port valve and the post-column formation of the fluorescence complex of KYNA with zinc ions. Moreover, using the proposed HPLC method, we were able to determine KYNA in 7.5 µL of normal human serum (28.4 ± 2.81 nM, n = 35), thereby demonstrating its clinical usefulness that can be attributed to its low detection limit of approximately 4.0 fmol on the column (signal-to-noise ratio 3; Fukushima et al, 2006). Thus, in the present study, the applicability of the column-switching HPLC was extended to determine brain KYNA, and a suitable pretreatment procedure was also examined for the determination of brain KYNA in rats.…”

“…In our previous studies, we developed a columnswitching HPLC method for the determination of KYNA (Mitsuhashi et al, 2006), and revealed that a single fluorescence peak originated from rat plasma KYNA was clearly observed using the HPLC after facile pretreatment of deproteinization with 50 mM ammonium acetate (AcONH 4 ) in methanol. This superior selectivity of the HPLC method was mainly based on the separation on two octadecylsilica (ODS) columns, which were connected via a six-port valve and the post-column formation of the fluorescence complex of KYNA with zinc ions.…”

Fluorimetric assay for D-amino acid oxidase activity in rat brain homogenate by using D-kynurenine as a substrate

“…As reported, KYNA emits fluorescence at 396-398 nm with an excitation wavelength at 251 nm (19,23) or 344-346 nm (24,25). An excitation wavelength of 346 nm was used in the present study to increase the signal to noise ratio and to avoid interference fluorescence stemming from endogenous substances included in the brain homogenate.…”

Utilization of Kynurenic Acid Produced from D-kynurenine in an in Vitro Assay of D-Amino Acid Oxidase Activity

“…KYNA itself emits fluorescence, especially in the presence of zinc ions; [23][24][25][26] therefore, the procedure described above can be used to evaluate in vitro DAAO activity. A test compound was added to the enzymatic reaction of DAAO with D-KYN.…”