Simultaneous Determination of Kynurenine and Kynurenic Acid in Serum by High Performance Liquid Chromatography-Fluorescence Detection

A method of high performance liquid chromatography-fluorescence detection (HPLC-FLD) for the simultaneous determination of kynurenine (Kyn) and kynurenic acid (KYNA) in serum was developed. A 20 microL of the supernatant of a serum sample deproteinized by 5% perchloric acid solution was separated on a Hypersil C8 column (300 mm x 6.0 mm, 10 microm) with an isocratic elution of 0.25 mol/L zinc acetate-50 mmol/L acetate containing 3% (v/v) acetonitrile. The fluorescence excitation and emission wavelengths were 365 nm and 480 nm at the beginning of the run; and 10 min later, the excitation and emission wavelengths were changed to 344 nm and 404 nm, respectively. The retention times of Kyn and KYNA were 8.1 min and 13.0 min, respectively. The linearities of the assay were from 98 to 19,600 nmol/L for Kyn and from 2.62 to 1047 nmol/L for KYNA; the detection limits were 50 nmol/L for Kyn and 0.11 nmol/L for KYNA. The average recoveries were 94.88% for Kyn, and 102.72% for KYNA. The within-day and between-day relative standard deviations (RSD) were 3.87% and 3.94% for Kyn; and 3.79% and 4.71% for KYNA, respectively. The results indicated phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), 5-hydroxytryptamine (5-HT), and creatinine (Cr) had no interfering effects to the determination of Kyn and KYNA. Kyn and KYNA concentrations in the sera of 71 health people were (1.40 +/- 0.34) micromol/L and (24.22 +/- 8.67) nmol/L, respectively. The method is simple, rapid, accurate, convenient, and suitable for routine analysis.