高效液相荧光法测定人尿液中儿茶酚胺含量

摘要

Objective To establish a method based on high performance liquid chromatography-fluorimetric detection to determine the concentrations of catecholamines ( norepinephrine, epinephrine and Dopamine) in human urine for diagnosis of pheochromocytoma. Methods The catecholamines in human urine samples were adsorbed by homemade activated alumina with Tris-buffer under alkaline conditions, using internal standard method for the determination of their concentrations.Separation was performed on a ZORBAX-SB (4.6 mm ×250 mm, 5 μm) colum.The mobile phase was consist of methanol-20 mmol· L-1 potassium dihydrogen phosphate with flow rate of 0.8 mL· min -1 , column temperature:25 ℃.The fluorescence intensity was measured at the excitation wavelengths of 286 nm and the emission wavelengths of 318 nm.Results The linear relationships were good within 1.56~1 000μg· L-1 for norepi-nephrine bitartrate, epinephrine hydrochloride, and dopamine hydrochloride (r=0.999 5, 0.999 7, 0.999 8);The intra-and inter-day coefficients of variation were between 0.31%and 8.02%;the mean analytical recoveries of high, medium and low concentrations were in the range of 94.22%to 108.69%.Conclusion The method is accurate, stable, and shows wide linear range, which can pro-vide a basis for the corresponding disease diagnosis and Perioperative treatment.%目的：建立高效液相色谱－荧光检测（ HPLC－FD）法同时测定人尿液中儿茶酚胺（去甲肾上腺素、肾上腺素、多巴胺）含量，用于嗜铬细胞瘤的诊断。方法自制活性氧化铝，在弱碱性Tris缓冲液条件下吸附尿液样本中儿茶酚胺，使用 ZORB-AX－SB （4．6 mm ×250 mm，5μm）色谱柱，以甲醇－20 mmol· L－1磷酸二氢钾溶液为流动相；流速：0．8 mL· min－1，柱温25℃；激发波长（EX）为286 nm，检测波长（EM）为318 nm。结果重酒石酸去甲肾上腺素、肾上腺素、盐酸多巴胺在1．56～1000μg· L－1浓度范围内线性关系良好（r＝0．9995、0．9997、0．9998）；日内及日间精密度RSD在0．31％～8．02％之间；高、中、低三个浓度的平均方法回收率在94．22％～108．69％之间。结论该方法准确，稳定性较好，线性范围广，为嗜铬细胞瘤的诊断及围手术期治疗提供依据。