高效液相色谱法测定人肝细胞色素P4503A4转基因细胞中氨氯地平

Determination of amlodipine in CYP3A4 cDNA-expressed cells by HPLC

目的 :研究氨氯地平经人肝细胞色素 P4 5 0 3A4 (CYP3A4 )的代谢 ,建立反相高效液相色谱的测定人肝转基因细胞 CYP3A4酶 S9孵育液中氨氯地平浓度的方法。方法 :与人肝转基因细胞提取的 S9上清液孵育之后的样品 ,用 3倍量的甲醇沉淀后离心 ,取上清液用 0 .4 5μm微孔滤膜滤过后进样。采用 Hypersil C18柱 ,以乙腈 -磷酸盐缓冲液 (4 5∶ 5 5 ,v/ v,p H4 .5 )为流动相 ,普萘洛尔为内标 ,在 2 5 0 nm波长处测定。结果 :氨氯地平浓度在 0 .2～ 30 .0μg/ ml范围内线性关系良好 ,r=0 .9993,方法平均回收率为 (98.2± 2 .4 ) % (n=5 ) ,日内和日间相对标准偏差(RSD)均小于 10 % ,检测限为 2 0 ng/ ml,定量限为 0 .2 μg/ ml(回收率为 10 4 .0 % ,RSD为 11.4 % ,n=5 )。结论 :建立的反相高效液相色谱方法简便、准确 ,可用于研究氨氯地平在人肝 CYP3A4转基因细胞中的代谢。

Objective: To establish a RP-HPLC method for the determination of amlodipine after metabolism by cytochrome P450 cDNA-expressed cells. Methods: The determination was performed on a C 18 reversed phase co-lumn with a mobile phase composed of acetonitrile phosphates buffer (45∶55,v/v,pH 4.5) with UV detection (λ250nm).Propranolol was used as the internal standard. Results: The standard curve was linear over the concentration range of 0.2~30.0 μg/ml (r=0.9993), and the limits of determination was 20 ng/ml (S/N≥3),the limits of quantity was 0.2 μg/ ml(recovery 104.0%,RSD 11.4%,n=5)?The recovery for this assay was (98.2±2.4)%,precision for inter-assay and intra-assay was <10% and 6%,respectively. Conclusion: The HPLC method established is simple,accurate and suitable for the determination of amlodipine in cytochrome p450 cDNA-expressed cells.