以睾酮为探针测定大鼠CYP3A酶活性的实验研究  

Determination of Cytochrome P450 3A Activity in Rats with Testosterone Probe

作　　者：黄红兵[1] 刘韬[1] 邓多[2] 周峰[2] 林子超[1] 陈倩超[1] 陈杰[3] 赵立子[2] 毕惠嫦[2] 

机构地区：[1]华南肿瘤学国家重点实验室,中山大学附属肿瘤医院,广州510060 [2]中山大学药学院,广州510080 [3]中山大学附属第一医院,广州510080

出　　处：《中国现代应用药学》2008年第5期379-382,共4页Chinese Journal of Modern Applied Pharmacy

基　　金：广东省医院药学研究基金资助项目(编号2008A007)

摘　　要：目的建立一种简便、快速、可靠的以睾酮作为探针药物,评价大鼠CYP3A酶活性的高效液相色谱检测方法。方法色谱柱为HypersilBDSC18柱(4.6mm×150mm,5μm),柱温为25℃,检测波长为254nm,内标为氢化可的松,流动相为甲醇-10mmol·L-1Na2HPO4水溶液(pH8.25)(55∶45),流速为1mL·min-1。睾酮以大鼠肝微粒体温孵后,用乙酸乙酯萃取,离心取上清液挥干后用甲醇-水(1∶1)复溶进样分析。结果睾酮和氢化可的松的保留时间分别为17.74min和6.05min;睾酮的孵育产物6β-羟基睾酮的保留时间为4.72min,回归方程为Y=0.0756X-0.0214(r=0.9980),线性范围为0.3125～20μg·mL-1,检测限下限质量浓度为(0.078±0.016)μg·mL-1(S/N≥3),提取回收率为79.3%～95.1%,方法回收率为98.0%～104.0%,低、中、高三个浓度日内、日间RSD均小于10.4%;温孵体系中其他内源性物质不干扰测定。结论所建立的HPLC稳定、高效,适合睾酮及其代谢产物6β-羟基睾酮的测定,可应用于药物对大鼠CYP3A酶活性的评价。OBJECTIVE To establish an HPLC method for evaluating CYP3A activity using testosterone in vitro. METHODS A Hypersil BDS C18 column （4.6 mm ×150 mm, 5 μm） at 25 ℃ was used, The mobile phase was methanol-10 mmol ·L^-1 Na2HPO4 solution （ pH 8.25 ） （55：45 ） and the flow rate was 1 mL·min^-1. The UV wave length was set at 254 nm. The internal standard was hydrocortisone. Testosterone was incubated with rat liver microsome in vitro. Testosterone and its metabolism were extracted by acetic ether and dissolved by methanol-water （ 1 ： 1 ） and further analyzed by HPLC. RESULTS The retention time of testosterone and hydrocortisone were 17.74 and 6.05 min. The retention time of 6β-hydroxylaled testosterone was 4.72 min, and the regression equation was Y = 0. 075 6X - 0. 021 4 （ r = 0. 998 0 ）. The linear range was 0.312 5 - 20 μg·mL^-1 , and the minimum detectable concentration was （0. 078 ±0. 016） μg ·mL^-1 （ S/N≥ 3）. The abstraction recoveries were from 79.3% to 95.1% and the relative recoveries were from 98.0% to 104.0%. The Intra-day RSD and Inter-day RSD of low, middle and high concentrations were 3.1% , 3, 6% , 1.90% and 9.8% , 10.4% ,1. 60% , respectively. Others endogenous metabolins didn＇ t interfere the detection of 6β- hydroxylated testosterone. CONCLUSION The method is stable, accurate and suitable for the determination of 6β-hydroxylated testosterone and evaluation of CYP3A activity.

关 键 词：睾酮 6β-羟基睾酮 肝微粒体 CYP3A 高效液相色谱法 

分 类 号：Q55[生物学—生物化学] R917.794[医药卫生—药物分析学]