Simultaneous Determination Of Tryptophan And Its Metabolites In Plasma By High-performance Liquid Chromatography With On-column Derivatization

Objective:1. To establish a method for simultaneous determination of plasmatryptophan (Trp), kynurenine (Kyn),5-hydroxyindole acetic acid (5-Hiaa)and kynurenic acid (Kyna) by HPLC with on-column derivatization.2. The developed method was applied to determine the concentrationsof plsma Kyn, Trp, Kyna and5-Hiaa in normal pregnant women, diabetics,cirrhotic patients and their respective controls. Investigate the metabolismof Trp more comprehensive in these people.Methods:1. A Hypersil C18column (250mm×4.0mm;5μm) was used for theanalysis using3-nitro-tyrosine (3-N-Tyr) as internal standard (IS) at30℃.The separation was carried out with the mobile phase consisting of250mmol/L zinc acetate (pH5.5) and acetonitrile (95:5, V: V) at a flowrate of0.8ml/min. The ultraviolet (UV) detector was set at360nm from0.00to13.00min and302nm from13.01to17.00min. The fluorescenceexcitation (EX) and emission (Em) wavelengths, respectively, were278nm and343nm from0.00to10.00min,244nm and400nm from10.01to14.00min,302nm and400nm from14.01to17.00min.2. The concentrations of plasma Trp,5-Hiaa, Kyna and Kyn in normalpregnant women (n=70), diabetics (n=101), cirrhotic patients (n=68) andtheir respective controls were determined with the developed method.Results:1. The retention time of5-Hiaa, Kyn, IS, Kyna and Trp were6.7,9.0,11.4,13.5and16.1min, respectively. The mean recoveries were in therange of91%to113%. The linearities were from2.5to320.0μmol/L forTrp,0.32to15.36μmol/L for Kyn,3.27to104.60nmol/L for5-Hiaa,14.0to464.8nmol/L for Kyna. The detection limits were0.54μmol/L,0.048μmol/L,1.22nmol/L and1.29nmol/L for Trp, Kyn,5-Hiaa andKyna, respectively. The within-day CVs were less than5％.2. The concentration of Kyn,5-Hiaa, Kyna,5-Hiaa/Trp and Kyn/Trpin plasma of diabetics were significant higher than that of the controls(p<0.05).3. The concentration of Kyn, Kyna and Kyna/Kyn in plasma of normalpregnant women were significantly lower than that of the controls(p<0.05).4. The concentration of Kyn,5-Hiaa, Kyna,5-Hiaa/Trp and Kyn/Trpin plasma of cirrhosis were significant higher than that of the controls,while the concentration of Trp were remarkably lower than that of the controls (p<0.05). In cirrhosis, the concentration of albumin was positivelycorrelated with the concentration of Kyn; The concentration of AST waspositively correlated with the concentration of5-Hiaa; And the positivecorrelation was observed between the concentration of Trp and ratio of A/G.Conclusions:1. We have developed a new method to simultaneously determine Trp,Kyna,5-Hiaa and Kyn in plasma by HPLC with on-column derivatization.The analytical method is simple, rapid, sensitive, accurate and is wellsuitable for clinical routine measurement and scientific research.2. In this research, we investigated the metabolism of Trp in diabetics,normal pregnant women and cirrhotic patients. The data showed that therewas severe metabolic disorder in the patients we studied.