Feng Chengya, Gao Jieying, Zhen Qianna, Fan Zimian, Zhu Mingsong, Yang Xiangchun, Ding Min
College of Laboratory Medicine, Chongqing Medical University, Key Laboratory of Clinical Laboratory Diagnostics of Ministry of Education, Chongqing 400016, China.
Se Pu. 2013 Jun;31(6):577-81. doi: 10.3724/sp.j.1123.2013.01054.
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 x 4.0 mm, 5 microm) was used for the analysis at 30 degrees C. 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 (lambda(ex))/343 nm (lambda(em)) for 5-Hiaa and 244 nm (lambda(ex))/400 nm (lambda(em)) 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 micromol/L to 320.00 micromol/L for Trp, 0.32 micromol/L to 15.36 micromol/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 micromol/L, 0.056 micromol/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 < 0.01). The method is simple and sensitive with good reproducibility, and it is suitable for clinical measurements.
建立了一种采用柱上衍生化的高效液相色谱 - 紫外/荧光检测法（HPLC - UV/FLD），用于同时测定色氨酸（Trp）、犬尿氨酸（Kyn）、5 - 羟吲哚乙酸（5 - Hiaa）和犬尿酸（Kyna）。使用Hypersil C - 18柱（250 mm×4.0 mm，5μm）在30℃下进行分析。以250 mmol/L醋酸锌（pH 5.5）和乙腈（95∶5，v/v）为流动相，流速为0.8 mL/min，以3 - 硝基酪氨酸作为内标（IS）进行分离。5 - Hiaa的激发（Ex）和发射（Em）波长分别设置为278 nm（λ(ex)）/343 nm（λ(em)），Kyna的为244 nm（λ(ex)）/400 nm（λ(em)），而紫外检测波长Kyn和IS设置为360 nm，Trp设置为302 nm。回收率在91.62%至114.17%范围内。Trp的线性范围为2.50 μmol/L至320.00 μmol/L，Kyn为0.32 μmol/L至15.36 μmol/L，5 - Hiaa为3.27 nmol/L至104.60 nmol/L，Kyna为14.00 nmol/L至464.80 nmol/L。Trp、Kyn、5 - Hiaa和Kyna的检测限分别为0.078 μmol/L、0.056 μmol/L、0.690 nmol/L和1.290 nmol/L。检测了30例正常孕妇的血浆样本和28例健康对照的血浆样本，结果显示正常孕妇血浆中Trp、Kyn和Kyna的浓度与对照组有显著差异（均P < 0.01）。该方法简单、灵敏，重现性好，适用于临床检测。
