超高效液相色谱-荧光检测法测定人体尿液中酪氨酸、对羟苯基乳酸和对羟苯基乙酸

Determination of Tyrosine,4-Hydroxyphenyllactic Acid and 4-Hydroxyphenylacetic Acid in Human Urine by Ultra-Performance Liquid Chromatography Coupled with Fluorescence Detection

采用固相萃取技术,结合超高效液相色谱-荧光分析（UPLC-FLD）手段,建立了人体尿液中酪氨酸、对羟苯基乳酸和对羟苯基乙酸的同时测定新方法。系统探讨了不同机理下,应用固相萃取技术处理尿液的效果,最终选用Clean-Screen DAU混合固相萃取小柱处理尿液,成功地除去了大量干扰物质。在优化的实验条件下,上述三种化合物线性关系良好（R^2〉0.9996）,检测限均为0.016mg/L。健康人和癌症病人尿液三个添加水平的平均回收率在86.9%~107.9%之间,相对标准偏差（RSD）小于2.8%。该方法快速、灵敏度高、选择性好,可用于人体尿液中三组分的同时测定。

A sensitive,rapid and practical method for simultaneous determination of tyrosine（Tyr）,4-hydroxyphenyllactic acid（PHPLA）and 4-hydroxyphenylacetic acid（PHPAA）in human urine by ultraperformance liquid chromatography（UPLC）coupled with fluorescence detection（FLD）has been developed.The Clean-Screen DAU cartridge was employed to purify Tyr,PHPLA and PHPAA from human urine.The seperation of Tyr and its metabolites from other substances was carried on ZORBAX SB-C18 Rapid Resolution HD column with isocratic elution using 95∶5（V/V）mixture of 50 mmol/L ammonium formate buffer（pH=5.8,adjusted with formic acid）and acetonitrile at the flow rate of 0.20mL/min.Under the optimized condition,the limits of detection and limits of quantification for Tyr and its metabolites were 0.016 mg/L and 0.040 mg/L,respectively.Recoveries of Tyr and its metabolites in normal subjects and gastric cancer patients were in the range of 95.2%-107.8% and 86.9%-107.9%,respectively,with RSDs of 0.6%-2.8% and 0.5%-2.5%,respectively.The present method was successfully applied to the simultaneous determination of Tyr,PHPLA and PHPAA in human urine from healthy volunteers and gastric cancer patients.