大鼠肝微粒体中CYP2C9酶的活性及动力学研究

Activity and Pharmacokinetics of CYP2C9 in Rat Liver Microsomes

关键词:  高效液相色谱法  肝微粒体  双氯芬酸  4-羟基双氯芬酸  CYP2C9

Key words:  HPLC  liver microsomes  diclofenac  4-hydroxydiclofenac  CYP2C9

摘要:

目的 以双氯芬酸为探针药，建立HPLC测定大鼠肝微粒体CYP2C9酶活性的方法，并对其进行动力学考察。方法 采用Agilent ZORBAX SB-C₁₈色谱柱；流动相为乙腈-水-0.1%三氟乙酸，梯度洗脱；流速为1 mL·min^-1；柱温30 ℃；检测波长为278 nm。大鼠肝微粒体加入双氯芬酸钠孵育30 min后，用盐酸和乙酸乙酯终止反应，加入内标地西泮，涡旋后高速离心，取上层有机相吹干复溶进样检测；以Lineweaver-Burk作图计算Km和Vmax。结果 双氯芬酸、4-羟基双氯芬酸和内标分离良好且无内源性干扰。4-羟基双氯芬酸浓度在0.05~10 μmol·L^-1内线性关系良好(r=0.999 8)，定量下限为0.05 μmol·L^-1；日内、日间精密度均<10%，回收率>75%。动力学考察表明选择盐酸和乙酸乙酯作为终止试剂效果良好，测得大鼠肝微粒体中双氯芬酸羟化反应的Km为26.87 μmol·L^-1，Vmax为2.359 nmol·min^-1·mg^-1 pro。结论 该方法稳定，结果能准确反映CYP2C9酶的活性，可用于相关动力学研究。

Abstract:

OBJECTIVE To develop a high performance liquid chromatography method for the determination of the activity of CYP2C9 in rat liver microsomes using diclofenac as probe drug. METHODS The analytical column was packed with Agilent ZORBAX SB-C₁₈. The mobile phase was acetonitrile-water-0.1% trifluoroacetic acid using gradient elution and the flow rate was 1.0 mL·min^-1. The UV detection wavelength was 278 nm. The incubation was performed with diclofenac at 37 ℃ for 30 min, then the reaction was terminated by adding HCl solution and ethyl acetate. Then diazepam solution was added. After vortexing for 2 min, the samples were centrifuged at 13 000 r·min^-1 for 5 min. The organic phase was transferred into a clean tube and evaporated under nitrogen stream. The residue was dissolved in 100 μL of mobile phase. Then a 20 μL aliquot of the solution was injected into the HPLC system for analysis. RESULTS Diclofenac, 4-hydroxydiclofenac and diazepam were perfectly separated. Excellent liner relationship of 4-hydroxydiclofenac was obtained from 0.05-10.0 μmol·L^-1(r=0.999 8). RSD of inter- and intra-day preasion were <10% for diclofenac and 4-hydroxy diclofenac, and the method recoveries were >75%. The HCl and ethylacetate were selected as reagent to terminate the reaction. Km was 26.87 μmol·L^-1 and Vmax was 2.359 nmol·min^-1·mg^-1 pro. CONCLUSION The method is steady, accurate and suitable for assaying CYP2C9 activity which can be used to evaluate the pharmacokinetics of CYP2C9 in rat liver microsomes.