色谱

色谱

• 研究论文 • 上一篇    下一篇

高效液相色谱-荧光检测法测定血清中的犬尿氨酸

罗昔波,唐爱国,皮兰敢,肖乐东,敖翔,卜艳红,王瑞

  

  1. Department of Clinical Laboratory, the Second Xiangya Hospital of Central
    South University, Changsha 410011, China
  • 收稿日期:2007-06-18 修回日期:2007-08-21 出版日期:2008-01-30 发布日期:1984-09-25
  • 通讯作者: 唐爱国

Determination of kynurenine in serum using high performance
liquid chromatography-fluorescence detection

LUO Xibo, TANG Aiguo, PI Langan, XIAO Ledong, AO Xiang, PU Yanhong, WANG Rui

  

  1. Department of Clinical Laboratory, the Second Xiangya Hospital of Central
    South University, Changsha 410011, China
  • Received:2007-06-18 Revised:2007-08-21 Online:2008-01-30 Published:1984-09-25
  • Contact: 唐爱国 TANG Ai-Guo

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摘要：

建立了高效液相色谱-荧光检测(HPLC-FLD)测定血清中犬尿氨酸(kynurenine,Kyn)含量的方法。采用Hypersil C8色谱柱(300 mm×6.0 mm,10 μm), 流动相为0.25 mol/L醋酸锌及50 mmol/L醋酸溶液(含3%乙腈),流速为1.5 mL/min, 荧光检测激发波长和发射波长分别为365 nm和480 nm。血清标本经5%(v/v)高氯酸溶液去除蛋白质后取上层清液直接进样, Kyn经流动相等度洗脱分离后,用FLD进行测定。研究结果表明Kyn保留时间约为8.3 min,线性范围为0.098～19.6 μmol/L,最低检出浓度为0.04 μmol/L,回收率为90.8%～96.2%,日内、日间测定的相对标准偏差均小于5%,苯丙氨酸、酪氨酸、色氨酸、5-羟色胺和犬尿喹啉酸等物质对犬尿氨酸的测定均无干扰。建立的方法简便、快速、灵敏、特异,适用于临床和科研应用。

关键词: 高效液相色谱法, 犬尿氨酸, 人血清 , 荧光检测

Abstract:

A sensitive method of high performance liquid chromatography-fluorescence detection (HPLC-FLD) for the determination of kynurenine (Kyn) in serum was developed. A 20 μL supernatant of a serum sample deproteinized by 5% perchloric acid solution was assayed and separated on a Hypersil C8 column (300 mm×6.0 mm, 10 μm) with an isocratic elution of 0.25 mol/L zinc acetate-50 mmol/L acetate containing 3% (v/v) acetonitrile. The fluorescence excitation and emission wavelengths were 365 nm and 480 nm, respectively. The limit of detection was approximately 0.04 μmol/L (signal-to-noise ratio of 3) and the linearity of the assay was from 0.098 μmol/L to 19.6 μmol/L. The relative standard deviations of intraday and interday determinations were 3.8%and 4.6%, respectively. The results indicated tryptophan (Trp), 5-hydroxytryptamine (5-HT), kynurenic acid (KYNA), phenylalanine (Phe), tyrosine (Tyr) and creatinine (Cr) had no interfering effects to the determination of Kyn. The method is of high accuracy, easy operation, satisfactory recoveries and good reproducibility, and can be used for routine analysis.

Key words: fluorescence detection (FLD), human serum
, kynurenine, high performance liquid chromatography (HPLC)