Determination of kynurenine levels in rat plasma by high-performance liquid chromatography with pre-column fluorescence derivatization.
Kynurenine (KYN), a tryptophan metabolite, is a crucial compound for modulating neurotransmission because it can be metabolized in vivo into both quinolinic acid and kynurenic acid, which are the agonist and antagonist, respectively, of N-methyl-d-aspartate receptor. For the highly sensitive detection of KYN by high-performance liquid chromatography (HPLC), a fluorescence derivatization of KYN with a benzofurazan-type fluorogenic reagent, 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was investigated in the present study. KYN was derivatized with DBD-F (DBD-KYN) at 60 degrees C for 30 min, and separated on an octadecylsilica column with a gradient elution of the mobile phase, which consists of 0.1% formic acid in acetonitrile/methanol/water. DBD-KYN was detected fluorimetrically at 553 nm with an excitation wavelength of 431 nm. The limits of detection and quantification were approximately 0.30 pmol [signal-to-noise ratio (S/N) 3] and 1.0 pmol (S/N, 10) on column, respectively. Plasma KYN levels were successfully determined using 10 microL of rat plasma with satisfactory precision and accuracy. Intra- and inter-day precisions and accuracies were 1.7-6.8%, and -10 to 9.6%, respectively. KYN levels in plasma of male Sprague-Dawley rats (7 weeks old) were approximately 2.4+/-0.32 micromol L(-1) (n=4). The proposed HPLC method was applied to determine KYN levels in the plasma of ketamine-treated rats--the animal model of schizophrenia.
Mitsuhashi
Shogo
S
Division of Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan.
Fukushima
Takeshi
T
Tomiya
Masayuki
M
Santa
Tomofumi
T
Imai
Kazuhiro
K
Toyo'oka
Toshimasa
T
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005453:Fluorescence; D007649:Ketamine / Q000494:pharmacology; D007737:Kynurenine / Q000097:blood*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012559:Schizophrenia / Q000097:blood
D007737:Kynurenine; D007649:Ketamine