高效液相色谱—电化学法测定尿液中单胺类神经递质代谢物含量

目的:建立快速、准确测定人体尿液中香草扁桃酸(VMA)、高香草酸(HVA)、5-羟吲哚乙酸(5-HIAA)的高效液相色谱—电化学法,为实施无创性采样运动训练监控研究提供新的实验室检测手段。方法:采用ISO-3100高效液相色谱仪系统,BM钛系统泵、WPS 3000自动进样器、WAX-1阴离子交换色谱柱(3μm,3×150 mm),5011A双通道库仑电化学检测器;流动相由20 mmol/L磷酸缓冲盐配制成盐溶液并调节pH值=5,然后与乙腈分别按照75:20(v/v)和80:25(v/v)配制成流动相A和流动相B,超声20 min后使用。流速为0.4 ml/min,进样量为5μl。检测器采用E1=100 mV和E2=500 mV两种电势条件。采用以上色谱条件分别测定标准品和成年人24 h尿样的VMA、HVA、5-HIAA含量,并进行统计和分析。结果:VMA、HVA、5-HIAA的线性关系分别为y=55.387x-0.2327(r=0.9998),y=55.31x+0.3247(r=0.9996),y=59.752x-0.1938(r=0.9987);最低检测限分别为5 ng/ml、5 ng/ml和7 ng/ml;批内变异系数(CV)分别为2.7%、2.4%、1.6%,批间变异系数分别为3.2%、3.1%、4.1%;所测成年人24h尿的VMA、HVA和5-HIAA含量分别为(1.65±0.08)mg/day、(2.67±0.24)mg/day、(1.79±0.13)mg/day,均分别符合正常成年人24 h尿中VMA、HVA、5-HIAA的水平。结论:本研究所采用的新型WAX阴离子交换色谱柱特异性好,对阴离子化合物的分离效果显著。本方法测定结果良好,线性关系、精密度以及检测限均满足定量要求,值得在研究中推广使用。

Application of High Performance Liquid Chromatography-Electrochemical Method in Determination of Monoamine Neurotransmitter Metabolites Content in Urine

Objective To establish a rapid and accurate technique for determination of vanilmandelic acid(VMA),homovanillic acid(HVA),and 5-hydroxyindoleacetic acid(5-HIAA) in human urine.Methods ISO-3100 high-performance liquid chromatography,BM titanium system pump,WPS 3000 autosampler,WAX-1 anion exchange column(3μm,3 × 150 mm),and 5011A dual channel Coulomb electrochemical detector were used in this study.Mobile phases A and B were prepared with 20 mmol/L phosphate buffered saline solution and acetonitrile at the ratios of 75:20(v/v) and 80:25(v/v),respectively.VMA,HVA,and 5-HIAA contents in adult 24h urine were determined.Results The regression equation for VMA,HVA,and 5-HIAA was y = 55.387x-0.2327(r = 0.9998),y = 55.31x+ 3247(r = 0.9996),and 5-HIAA y = 59.752x-0.1938(r = 0.9987),respectively.The detection limit was 5 ng/ml for VMA,5 ng/ml for HVA,and 7 ng/ml for 5-HIAA,respectively.The coefficient of variation(CV) was 2.7% for VMA,2.4% for HVA,and 1.6% for 5-HIAA,and the inter-assay coefficient of variation(CV) was 3.2% for VMA,4.1% for HVA,and 3.1% for 5-HIAA;and the levels of VMA,HVA,5-HIAA in adult 24h urine were 1.65±0.08 mg/day,2.67±0.24 mg/day,1.79±0.13 mg/day,respectively.All of them were accorded to adult normal levels.Conclusion The technique used in this study demonstrates extremely high specificity and effectiveness for separation of anionic compounds.