Simultaneous determination of tryptophan and kynurenine in plasma by HPLC with UV detection / 中华检验医学杂志

ABSTRACT

ObjectiveTo establish an accurate method for simultaneous determination of plasma Kyn and Trp by HPLC-UV detection.Methods Kyn and Trp were separated on Agilent Hypersil ODS column using 3-nitrotyrosine as internal standard.The mobile phase consisted of 15 mmol/L sodium acetateacetic acid (pH 5.5)acetonitrile 94∶ 6(v/v) at a rate of 0.8 ml/min.The chromatographic separation was performed at 25 ℃.The eluate was monitored with programmed wavelength setting at 360 nm from 0 to 4 min for Kyn and at 302 nm from 4 to 5 min for Trp.The method was applied to determination of plasma Kyn and Trp in 8 chronic glomerulonephritis,10 idiopathic thrombocytopenic purpura,15 chronic hepatitis B virus patients and 15 healthy controls from September to December in 2010.The differences were compared using ANOVA and SNK methods.Results The retention time of Kyn and Trp were 2.9 min and 4.4 min,respectively.For Kyn,the assay was linear from 0.44 μmol/L to 18.30 μmol/L.For Trp,the linearity was from 3.67 μmol/L to 470.00 μmol/L.The detection limits were 0.014 μmol/L for Kyn and 0.122 μmol/L for Trp,respectively.The within-day CVs were ＜ 3％ and the between-day CVs were ＜ 4％.The mean recoveries yield were in the range of 92.29 to 104.40.The plasma concentrations of Kyn were ( 1.59 ± 0.28),(2.73 ± 0.56),(2.69 ± 0.44) and ( 1.54 ± 0.48 ) μmol/L,the plasma concentrations of Trp were (59.8 ± 10.0),(46.1 ± 11.7),(58.5 ±8.0) and (41.4±13.1) μmol/L,the Kyn/Trp were (0.027 4±0.007 5),(0.061 6 ±0.016 5),(0.046 7 ±0.009 1) and (0.038 3 ±0.007 5)in controls,chronic glomerulonephritis patients,idiopathic thrombocytopenic purpura patients and chronic hepatitis B virus patients,respectively.There were significance difference of Kyn,Trp and Kyn/Trp amony the four groups (F=23.734,8.463,20.921,all P＜0.01).Conclusion The method is simple,fast,and suitable for applicability to clinical measurement.