高效液相色谱-荧光检测法同时测定血清中的犬尿氨酸和犬尿喹啉酸

Simultaneous determination of kynurenine and kynurenic acid in serum by high performance liquid chromatography-fluorescence detection

建立了在线衍生、双波长高效液相色谱-荧光检测器同时检测血清中犬尿氨酸（kynurenine,Kyn）和犬尿喹啉酸（kynurenic acid,KYNA）含量的方法。血清标本经5%高氯酸溶液去除蛋白质后,上清液直接进样分析测定。采用的色谱柱为Hypersil C8柱;流动相为0.25mol/L醋酸锌-50mmol/L醋酸溶液（含3%乙腈）,流速为1.5mL/min。在0~10min时间段,在激发波长和发射波长分别为365nm和480nm时检测Kyn;10min后,在激发波长和发射波长分别变换为344nm和404nm时检测KYNA。Kyn的保留时间约为8.1min,线性范围为98~19 600nmol/L,最低检出浓度为50nmol/L,平均回收率为94.88%,日内、日间测定值的相对标准偏差（RSD）均低于4%。KYNA的保留时间约为13.0min,线性范围为2.62~1 047nmol/L,最低检出浓度为0.11nmol/L,平均回收率为102.72%,日内、日间测定的RSD均低于4%。苯丙氨酸、酪氨酸、色氨酸和5-羟色胺等物质对目标物的检测无干扰。71例健康成人血清中,Kyn和KYNA含量分别为（1.40±0.34）μmol/L和（24.22±8.67）nmol/L。该方法简便、快速、灵敏、特异,适于临床和科研应用。

A method of high performance liquid chromatography-fluorescence detection（HPLC-FLD） for the simultaneous determination of kynurenine（Kyn） and kynurenic acid（KYNA） in serum was developed.A 20 μL of the supernatant of a serum sample deproteinized by 5% perchloric acid solution was 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 at the beginning of the run,and 10 min later,the excitation and emission wavelengths were changed to 344 nm and 404 nm,respectively.The retention times of Kyn and KYNA were 8.1 min and 13.0 min,respectively.The linearities of the assay were from 98 to 19600 nmol/L for Kyn and from 2.62 to 1047 nmol/L for KYNA,the detection limits were 50 nmol/L for Kyn and 0.11 nmol/L for KYNA.The average recoveries were 94.88% for Kyn,and 102.72% for KYNA.The within-day and between-day relative standard deviations（RSD） were 3.87% and 3.94% for Kyn,and 3.79% and 4.71% for KYNA,respectively.The results indicated phenylalanine（Phe）,tyrosine（Tyr）,tryptophan（Trp）,5-hydroxytryptamine（5-HT）,and creatinine（Cr） had no interfering effects to the determination of Kyn and KYNA.Kyn and KYNA concentrations in the sera of 71 health people were（1.40±0.34） μmol/L and（24.22±8.67） nmol/L,respectively.The method is simple,rapid,accurate,convenient,and suitable for routine analysis.