临床前大鼠药物代谢酶CYP2D6亚型HPLC技术研究

Studies on HPLC methods for preclinical research on rats CYP2D6 isoform

目的:建立测定临床前大鼠药物代谢酶2D6亚型活性的 HPLC 方法。方法:HPLC 法测定大鼠尿液及肝微粒体中探针药物右美沙芬(DM)、其代谢物去甲右美沙芬(DX)及内标物丁丙诺啡(BP)的含量。采用 Hypersil-C_6H_5柱(250mm×4.6mm,5μm),以0.01mol-L^(-1)磷酸二氢钾-0.01mol·L^(-1)已烷磺酸钠-乙腈-甲醇(33:33:80:54,v/v,pH4.0)为流动相,流速1.0mL·min^(-1),荧光检测器激发波长280nm,发射波长310nm。结果:本方法稳定性良好,尿样 DM 在0.04～1.20mg·L^(-1)内线性良好(r=0.9981),DX 在3.12～100.0mg·L^(-1)内线性良好(r=0.9971)。肝微粒体重组样品 DM 在30.0～180.0mmol·L^(-1)内线性良好(r=0.9980),DX 在5.0～50.0mg·L^(-1)内线性良好(r=0.9976),日内、日间样品 RSD 均小于10%,样品-20℃保存30d 后测定稳定良好,体外肝微粒体实验 RSD 小于10%。结论:本方法稳定、准确,可用于新药临床前药物代谢酶途径及药物相互作用研究。

Objective：To establish HPLC methods for determination of CYP2D6 activity in rat urine and liver microsomes for preclinical research. Methods： probe drug dextromethorphan （ DM ）, its metabolite dextrophan （ DX ） and internal standard buprenorphin（BP） were determined by HPLC in rat urine and liver microsomal reconstituted system to estimate activity of CYP2D6 by using Hypersil - C6 H5 analysis column （250 mm ×4. 6 mm,5 μm ）. The mobile phase of 0. 01mol·L^-1 potassium dihydrogen phosphate, sodium hexane sulfonate, acetonitrile, methanol （33： 33： 80： 54, V： V,pH4.0）was pumped at 1.0 mL·min^- 1 and the fluorescence detector was set at λex280 nm,λem310 nm. Results ：The methods were stable and linear in range of 0. 04 mg · L^-1 - 1.2 mg · L^-1for DM （r =0. 9981 ） ,3.12 mg· L^-1 - 100. 0 mg · L^-1for DX（ r =0. 9971 ） in urine samples;30. 0 mg · L^-1 - 180. 0 mg · L^ - 1 for DM （ r = 0. 9980 ）, 5.0 mg·L^- 1 - 50.0 mg·L^-1 for DX （ r = 0. 9976 ） in liver microsomes reconstiction samples respectively. The average recovery of within - day and between day for DM and DX were less than 10%. Samples kept in -20 ℃ for 30 days were stable for HPLC analysis. Conclusion The methods were stable and accurate for study on metabolite ways of new drug preclinical study and drug interactions.