高效液相色谱-荧光法同时测定血清5-羟色胺和色氨酸方法的建立

Simultaneous Determination of 5-Hydroxytryptamine and Tryptophan in Serum by High Performance Liquid Chromatography-fluorescence Detection

目的建立一种同时测定血清中5-羟色胺(5-HT)和色氨酸(Trp)浓度的高效液相色谱-荧光检测法(HPLC-FD)。方法采用的色谱柱为Hypersil C18柱(250mm×4.6mmi.d.,7μm),流动相为0.1mol/L磷酸二氢钾/甲醇(85:15,V/V)溶液,流速1.0ml/min,荧光检测激发波长和发射波长在0～6min分别为278和338nm,6～10min转换为280和340nm。结果5-HT保留时间约为4.9min,线性方程为Y=4223461X-35303.9,最低检出浓度为0.001μmol/L,回收率为82.70%～95.40%,日内和日间RSD分别为2.03%和3.42%;Trp保留时间约为8.4min,线性方程为Y=421314X+38292.9,最低检出浓度为0.007μmol/L,回收率为83.67%～94.98%,日内和日间RSD分别为1.32%和3.31%;苯丙氨酸、酪氨酸、犬尿氨酸和犬尿喹啉酸等物质对本方法无干扰;健康成人(n=71)血清中5-HT和Trp的浓度分别为(544.8±156.8)nmol/L和(53.08±8.29)μmol/L。结论该方法简便、快速、灵敏,适于临床和科研应用。

Objective To establish a method for simultaneous determination of 5-hydroxytryptamine （5-HT） and tryptophan （Trp） in serum by high performance liquid chromatography-fluorescence detection （HPLC-FD）.Methods The Hypersil C18 column（250 mm×4.6 mm i.d.,7 μm）was used as analytical column with a mobile phase consisting of 0.1 mol/L potassium dihydrogen phosphate solution and methanol 85：15（V/V）,the flow rate was 1.0 ml/min,excitation and emission wavelengths of fluorescence detector were 278 nm and 338 nm in 0～6 min,280 nm and 340 nm in 6～10 min,respectively.Results The retention time of 5-HT was approximately 4.9 min,the linear equation was Y=4223461X-35303.9,and the limit of detection was 0.001 μmol/L,and the recoveries of 5-HT were from 82.70% to 95.40%,the intra-day and inter-day variations of RSD were 2.03% and 3.42%.The retention time of Trp was approximately 8.4 min,the linear equation was Y=421314X＋38292.9,and the limit of detection was 0.007 μmol/L,and the recoveries of Trp were from 83.67% to 94.98%.The intra-day and inter-day variations of RSD were 1.32% and 3.31%,respectively.Phenyalanine,tyrosine,kynurenine,and kynurenic acid did not interfere in the assay.The serum levels of 5-HT and Trp in 71 healthy subjects were 544.8±156.8 nmol/L and 53.08±8.29 μmol/L.Conclusions The method is simple,rapid,sensitive,and suitable for clinical application and scientific research.