摘要
目的建立一种高效液相色谱（HPLC）-荧光法（FD）同时测定血清色氨酸（Trp）和酪氨酸（Tyr）的方法。方法色谱条件：Megres C18色谱柱（250 mm×4.6 mm,内径为5μm）,流动相为10%乙腈溶液,流速为1.2 mL/min,Tyr和Trp的荧光检测激发波长和发射波长分别为λexTyr=228 nm、λexTrp=285 nm和λemTyr=306 nm、λemTrp=353 nm。血清样本经5%高氯酸溶液去除蛋白质后取上层清液直接进样进行分析测定。且对样本的保存方法进行了探讨。结果 Tyr的保留时间为3.4 min,线性范围为0.275~275μmol/L,最低检出浓度为0.004μmol/L,回收率为90.5%~108.8%。Trp的保留时间为7.6 min,线性范围为0.490~196μmol/L,最低检测浓度为0.005μmol/L,回收率为88.8%~97.2%。Tyr和Trp的日内、日间测定的相对标准偏差均〈5%,苯丙氨酸、5-羟色胺、犬尿喹啉酸、犬尿氨酸和肌酐等物质对该法均无干扰。样本应-20℃冰冻保存。结论该方法简便、快速、敏感、特异,可同时测定血清Tyr和Trp,适合于临床和科研应用。
Objective To develop a method for determination of tryptophan（Trp） and tyrosine（Tyr） in serum by high performance liquid chromatography-fluorescence detection（HPLC-FD）.Methods The chromatography conditions were a Megres C18 column（250 mm×4.6 mm,internal diameter： 5 μm）,mobile phase（10% acetonitrile）,flow rate： 1.2 mL/min and the excitation and emission wave lengths of Tyr and Trp： λexTyr=228 nm,λexTrp=285 nm and λemTyr=306 nm,λemTrp=353 nm.The deproteinized serum samples by 5%（v/v） perchloric acid solution were separated and determined.The storage condition of sample was also studied.Results The retention times of Tyr and Trp were 3.4 min and 7.6 min.The linearity ranges were 0.275-275 μmol/L and 0.490-196 μmol/L.The detection limits were 0.004 μmol/L and 0.005 μmol/L.The recoveries were 90.5%-108.8% and 88.8%-97.2% for Tyr and Trp respectively.The relative standard deviations of intraday and interday were 5%.The phenylalanine,5-hydroxytryptamine,kynurenic acid,kynurenine and creatinine had no interfering effects to the determination of Trp and Tyr.The samples should be stored at-20 ℃.Conclusions The HPLC-FD method is simple,rapid,sensitive and specific.It can determine Tyr and Trp simultaneously,which is suitable for clinical and scientific study.
出处
《检验医学》
CAS
北大核心
2010年第10期756-760,共5页
Laboratory Medicine
基金
湖南省科技计划项目(05SK3026)
湖南省医药卫生科研计划资助项目(C2005019)
关键词
色氨酸
酪氨酸
高效液相色谱法
荧光检测
Tryptophan
Tyrosine
High performance liquid chromatography
Fluorescence detection
