RP-HPLC法测定健康者尿液中6β-羟基氢化可的松和氢化可的松的含量

Determination of the contents of 6β-hydroxycortisol and hydrocortisone in healthy human's urine by RP-HPLC

目的建立RP-HPLC法同时检测人尿样中内源性氢化可的松及其代谢产物6β-羟基氢化可的松的含量。方法采用岛津Insertil ODS-3反相色谱柱(250 mm×4.6 mm,5μm)进行梯度洗脱,柱温为30℃,流动相为水-乙腈,检测波长240nm。尿样用液-液萃取法处理,萃取剂为乙酸乙酯。结果 6β-羟基氢化可的松和氢化可的松标准曲线分别为y=0.004 l x-0.119 2,r=0.999 0;y=0.0127x-0.050 5,r=0.997 8,两者回收率均>95%,日内、日间RSD均<5%。20名健康志愿者尿中6β-羟基氢化可的松与氢化可的松质量浓度分别为(151.90±94.05)和(26.38±20.07)μg·L^(-1),比值为6.32±2.36。结论本方法专属性强、灵敏度高、简单可靠,适用于大样本分析。

AIM To establish a method for determining the concentration of β-hydroxycortisol and hydrocortisone in human urine. METHODS The RP-HPLC separation using gradient elution was performed on a SHIMADZU Insertil ODS- 3（250 mm × 4.6 mm,5 μm）reversion phase chromatography column at column temperature of 30℃. The mobile phase consisted of water-acetonitrile, and the detection wave-length was at 240 nm. The urine was extracted with liquid- phase extraction method by ethyl acetate. RESULTS The linear regress equations for 6β-hydroxycortisol and hydrocorti- sone were y = 0.004 1x - 0.119 2, r = 0.999 0 and y = 0.012 7x - 0.050 5, r = 0. 997 8, respectively. The percent- ages of their average recoveries were both above 95 %, and the intra-day RSD and inter-day RSD were less than 5 %. The concentration of 6β-hydroxycortisol and hydrocortisone in 20 volunteers＇ urine was （ 151.90± 94.05） μg·L- 1 and （26.38 ±20.07） μg·L- 1 respectively and the ratio was 6.32 ±2.36. CONCLUSION The present method is highly specific, sensitive, simple and reliable, and can be used for the large sample analysis.