Determination of serum tryptophan metabolites in patients with systemic lupus erythematosus by high performance liquid chromatography-fluorescence detection / 临床检验杂志

ABSTRACT

Objective A method was developed for the simultaneous determination of tryptophan(Trp) and its metabolites kynurenine(Kyn) and kynurenic acid(Kyna) by high performance liquid chromatography with fluorescence detection(HPLC-FD),and testing serum levels of Trp metabolites in systemic lupus erythematosus(SLE) patients.Methods Serum samples were deproteinized by equal volume of 0.624 mol/L perchloric acid.The analytical column was Hypersil C18 column,and the mobile phase was 0.20 mol/L zinc acetate,8.3 mmol/L acetic acid,and 2.5% acetonitrile;flow rate was 1.5 ml/min.The excitation and emission wave length of fluorescence detector were 365 nm and 480 nm in 0~11 min,344 nm and 404 nm in 11~15.5 min,254 nm and 404 nm in 15.5~20 min,respectively.Results The linear range of Trp was 0.610~196 ?mol/L,the detection limit was 0.005 ?mol/L,and the average recovery was 103.71%.The linear range of Kyn was 0.049~98 ?mol/L,the detection limit 0.025 ?mol/L,and the average recovery was 97.45%.The linear range of Kyna was 1.050~1047 ?mol/L,the detection limit was 0.050 nmol/L,and the average recovery was 100.60%.Inter-and intra-day precisions were both less than 5%.Phenylalanine,tyrosine,and 5-hydroxytryptamine had no interference.The assay was employed to analyze serum samples of SLE patients.The result showed significant difference in Trp,Kyn,and Kyna content,Kyn/Trp ratio between SLE patients and control group.Conclusions A new method was established for simultaneous determination of Trp,Kyn,and Kyna in serum.The method is simple,fast,sensitive,specific,and suitable for applicability to clinical measurement.