Simultaneous determination of kynurenine and tryptophan concentrations in serum of RA patients by HPLC fluorescence detection

Abstract

Objective To explore the clinical significance of serum kynurenine and tryptophan by HPLC fluorescence detection in patients with rheumatoid arthritis ( RA). Methods Serum samples were deproteinized by equal volume of 5% ( V/V) perchloric acid. It employed a Hypersil C8 column and a mobile phase consisted of 0. 25 mol/L zinc acetate, 50 mmol/L acetic acid with 3% (V/V) acetonitrile at a flow rate of 1. 5 ml/min. The fluorescence excitation and emission wavelengths were operated at 365 nm and 480 nm respectively at the beginning of the run, and 10 minutes later, the excitation and emission wavelength changed to 254 nm and 404 nm. TRP and KYN of health group and RA group were determined by HPLC-FLD. Results The retention time of KYN was 8. 1 min;the linearity was from 0. 098 μmol/L to 19. 600 μmol/L with the detection limit of 0. 04 μmol/L , the recovery were 90. 8%-96. 2% , and the intra-day and inter-day variations were 3. 68% and 4. 97% respectively. The retention time of TRP was 11. 5 min, the linearity of the assay was from 4. 9 μmol/L to 196. 0 junol/L, the detection limits was 0. 005 μmol/L,the recovery of TRP was 92. 6% -106. 9% , the intraday and interday coefficients of variations were 3. 63% and 4.44% .respectively. Phenylalanine, tyrosine, serotonin, kynurenic acid and creatinine didn't interfere in the assay. Compared with healthy subjects, there were significantly increased concentration of serum KYN and K/T Ratio [(1.51 ±0.35) nol/L vs(2. 06 ±0. 38) ujnol/L,tf = 3 251. 0,P =0. 000; (32. 54 ± 9. 00) × 10~(-3) vs(55. 46 ±5. 81) × 10~(-3) ,t = 10 741 ,P =0. 000] and significantly decreased concentrations of TRP [(47.52±5.79) μmol/L vs (38.24±5.27) μmol/L; t = 10. 399,P =0. 000) ] in the patients with RA. The sensitivity, specificity of K/T ratio for diagnsis RA was 83. 6% (92/110) and 85. 8 ( 103/ 120)% respectively. Conclusions A new method is established for simultaneous determination of KYN and TRP in serum by HPLC-FLD with on-column derivatization. The method is simple and rapid, and its precision, sensitivity and specificity are satisfactory for clinical and scientific study. K/T Ratio is a supporting diagnosis biomarker of RA. Key words: Chromatography; high performance liquid; Kynurenine; Tryptophan; Rheumatoid arthritis