高效液相色谱-荧光检测法测定血清中的犬尿氨酸

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

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Hy-persil C8色谱柱（300mm×6.0mm,10μm）,流动相为0.25mol/L醋酸锌及50mmol/L醋酸溶液（含3%乙腈）,流速为1.5mL/min,荧光检测激发波长和发射波长分别为365nm和480nm。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8.3min,线性范围为0.098-19.6μmol/L,最低检出浓度为0.04μmol/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。

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 × 5.0 mm, 10μm） 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 355 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 19.5 μmol/L. The relative standard deviations of intraday and interday determinations were 3.8% and 4.5%, 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.