高效液相色谱-二极管阵列法同时检测尿中内源性氢化可的松及其代谢产物

目的:建立高效液相色谱-二极管阵列法同时检测尿中内源性氢化可的松及其代谢产物的方法。方法:采用KromasilC18反相色谱柱(250mm×4·6mm,5μm),柱温为40℃,流动相为水-乙腈(78∶22,V/V,用磷酸调pH值至2·8,流速梯度洗脱),检测波长为238nm,尿液用固相萃取法处理。结果:6β-羟基氢化可的松和氢化可的松分别在20~1000、5~100μg/L浓度范围内线性关系良好,最低检测浓度分别为2、3μg/L,萃取回收率均大于80%,日内、日间相对标准差均小于5%。尿中6β-羟基氢化可的松与氢化可的松浓度的平均比值为0·66±0·37。结论:本方法专属性强、灵敏度高、简单可靠,可用于研究体内药物代谢机制及药物相互作用。

Simultaneous Determination of Endogenous Cortisol and Its Metabolite in Urine by HPLC-DAD

OBJECTIVE:To establish a HPLC-DAD method for simultaneous determination of endogenous cortisol and its metabolite in human urine.METHODS:The HPLC separation was performed on an Kromasil C 18 (250mm×4.6mm,5μm)reversion phase chromatography column at column temperature of40℃.Water-acetonitrile(78∶22,pH value regulated to2.8with phosphoric acid,flow-rate gradient elution)was used as mobile phase.Wave length was238nm.The urine was extracted with solid-phase extraction method.RESULTS:The calibration curves were linear in the range of20～1000μg/L for6β-hydroxycortisol and5～100μg/L for cortisol,respectively.The lowest detectable concentrations were2μg/L and3μg/L respectively.The average extraction recoveries for the two compounds were above80%.The intra-day and inter-day RSD were less than5%.The average ratio of concentration of endogenous6β-hydroxycortisol to cortisol in urine was0.66±0.37.CONCLUSION:The present method is highly specific,sensitive,simple and reliable,and can be used for the study of metabolic mechanism and interactions of drugs in vivo.