反相高效液相色谱法测定尿液4种肾病标志物

Reversed-phase high performance liquid chromatography assay for determining 4 kinds of markers of diabetic nephro- pathy in urine

目的:建立糖尿病患者尿液中与肾病相关的非蛋白氮代谢产物(肌酸、肌酐、尿素和尿酸)的反相高效液相色谱分离分析方法.方法:采用AgilentC18(200mm×4.6mm,内径5μm)色谱柱,流动相含30%(体积比)甲醇和10mmol/L,pH6.86的磷酸缓冲溶液,流速0.9mL/min,检测波长200nm,柱温为室温.结果:在5min内可对患者尿液中上述4种物质同时进行测定,样品前处理简单,重现性较好(迁移时间和峰面积的RSD均≤3%),线性范围较宽(肌酸:5～300μmol/L,肌酐:10～200μmol/L,尿素:1～30mmol/L,尿酸:10～1500μmol/L).结论:此法完全适用于临床上患者尿液的日常分析和早期肾病的监测.

AIM： To establish a new reversed-phase high performance liquid chromatography （RP-HPLC） method for assay 4 kinds of non-protein-nitrogen markers of human diabetic nephropathy （DN）, creatine （Cr）, creatinine （Cn）, urea （U） and uric acid （Ua） in DN patients＇ urines. METHODS： The urine samples were assayed by RP-HPLC on C18 column （200 mm× 4.6 mm, id 5 μm） at the ambient temperature and a wave length of 200 nm, using a 10 mmol/L pH 6.86 phosphate buffer [ consisted 30% （V/V） methanol] as mobile phase with the flow rate of 0.9 mL/min. RESULTS： Baseline separation of the urine could be easily achieved in 5 min after a simple sample preparation. The method also displayed a high reproducibility （RSD values of retention time and peak area were no more than 3% ） and the calibration curves showed good linearities in the range of 5 -300 μmol/L, 10 -200 μmol/L, 1 -30 μmol/L, 10 - 1500μmol/L for Cr, Cn, U and Ua respectively. CONCLUSION： This method has great potential for routine assay of clinical urine samples and monitoring DN early.