在线衍生-高效液相色谱法同时测定血浆中的色氨酸及其代谢物

Simultaneous determination of tryptophan and its metabolites in plasma by high performance liquid chromatography with on-column derivatization

建立了醋酸锌在线衍生高效液相色谱法同时测定血浆中色氨酸(Trp)、犬尿氨酸(Kyn)、5-羟吲哚乙酸(5-Hiaa)和犬尿喹啉酸(Kyna)的方法。以3-硝基酪氨酸为内标(IS),采用Hypersil C-18柱(250 mm×4.0 mm,5μm),以250 mmol/L醋酸锌溶液(pH 5.5)-乙腈(95∶5,v/v)为流动相,流速为0.8 mL/min,柱温30℃。荧光检测波长设定:5-Hiaa为278 nm(λex)/343 nm(λem),Kyna为244 nm(λex)/400 nm(λem);紫外检测波长设定:Kyn和IS为360 nm,Trp为302 nm。4种物质的回收率在91.62%～114.17%之间;线性范围分别为2.50～320.00μmol/L(Trp),0.32～15.36μmol/L(Kyn),3.27～104.60 nmol/L(5-Hiaa),14.00～464.80 nmol/L(Kyna);检出限分别为0.078μmol/L(Trp),0.056μmol/L(Kyn),0.690 nmol/L(5-Hiaa),1.290 nmol/L(Kyna)。利用该方法对30例正常孕妇和28例女性健康志愿者的血浆进行测定,结果表明两组间Trp,Kyn和Kyna含量有显著性差异。该方法操作简便,重复性好,灵敏度高,适合于临床检测。

A high performance liquid chromatography-ultraviolet/fluorescence detection（HPLC-UV/FLD） with on-column derivatization was established to simultaneously determine tryptophan（Trp）,kynurenine（Kyn）,5-hydroxyindole acetic acid（5-Hiaa） and kynurenic acid（Kyna）.A Hypersil C-18 column（250 mm×4.0 mm,5 μm） was used for the analysis at 30 ℃.The separation was carried out with the mobile phase consisting of 250 mmol/L zinc acetate（pH 5.5） and acetonitrile（95∶5,v/v） at a flow rate of 0.8 mL/min using 3-nitrotyrosine as internal standard（IS）.The excitation（Ex） and emission（Em） wavelengths were set at 278 nm（λex）/343 nm（λem） for 5-Hiaa and 244 nm（λex）/400 nm（λem） for Kyna,while the wavelengths of ultraviolet detection were set at 360 nm for Kyn and IS,302 nm for Trp.The recoveries were in the range of 91.62% to 114.17%.The linearities were from 2.50 μmol/L to 320.00 μmol/L for Trp,0.32 μmol/L to 15.36 μmol/L for Kyn,3.27 nmol/L to 104.60 nmol/L for 5-Hiaa,and 14.00 nmol/L to 464.80 nmol/L for Kyna.The detection limits were 0.078 μmol/L,0.056 μmol/L,0.690 nmol/L and 1.290 nmol/L for Trp,Kyn,5-Hiaa,and Kyna,respectively.Thirty plasma samples of normal pregnant women and 28 plasma samples of healthy controls were tested,and the results exhibited that the concentrations of Trp,Kyn and Kyna in the plasma of the normal pregnant women were significantly different from those of the control group（all P ＆lt; 0.01）.The method is simple and sensitive with good reproducibility,and it is suitable for clinical measurements.