中文摘要：

目的建立rCYP3A4中1’-羟基咪达唑仑含量测定方法,并对rCYP3A4体外孵育条件进行优化。方法反相高效液相色谱法RP-HPLC测定rCYP3A4中1’-羟基咪达唑仑的浓度,用单因素实验法对其体外孵育条件进行优化,用线性Lineweaver-Burk双倒数作图法研究rCYP3A4酶促动力学。结果1’-羟基咪达唑仑在18.20-1820.00ng·mL^-1内线性关系良好;体外优化的孵育条件为5μmol·L-1咪达唑仑、10pmolrCYP3A4、孵育10min;rCYP3A4酶促动力学参数Km为0.96μmol·L^-1,Vmax为0.78pmol·min^-1。结论RP-HPLC法操作简便、灵敏、快速,适用于rCYP3A4中1’-羟基咪达唑仑的浓度测定,rCYP3A4孵育条件优化及其酶促动力学研究为研究经CYP3A4代谢的药物相互作用及其他物质对CYP3A4酶的影响提供理论依据。

英文摘要：

Objective To establish a HPLC method for determining 1＇-hydroxymidazolam and optimize the incubated conditions for rCYP3A4.Methods 1＇-hydroxymidazolam was determined by RP-HPLC.Single effect experimental design was used to optimize the incubated conditions and the enzyme kinetics was evaluated by graphical analysis with Lineweaver-Burk double reciprocal plots（1/velocity vs.1/[substrate]）.Results Calibration curves of 1＇-hydroxymidazolam were linear in the range of 18.20-1 820.00 ng·mL^-1（R2= 0.999 9）.The optimal incubated conditions were 5 μmol·L-1,10 pmol and 10 min for midazolam concentration,rCYP3A4 and incubation time,respectively.The enzymic kinetic parameters of rCYP3A4 were 0.96 μmol·L-1 for Km and 0.78 pmol·min-1 for Vmax.Conclusion The HPLC method is simple,accurate and rapid,suitable for the determination of 1＇-hydroxymidazolam in rCYP3A4.The incubated conditions of rCYP3A4 and its kinetics can be applied for study on the drug interaction metabolized by CYP3A and effects of others on it.