HPLC法测定CYP 3A4酶活性的研究

目的建立一种快速、准确的以咪达唑仑作为＂探针药物＂评价或测定细胞色素酶（CYP3A4）酶活性的高效液相色谱（high-performance liquid chromatography,HPLC）-紫外检测（UV detection,VWD）方法。方法采用的色谱柱为依立特C18柱（4.6mm×100mm,5μm）,流速0.8ml/min,紫外检测波长254nm,柱温40℃。咪达唑仑（midazo-lam,MDZ）与大鼠肝微粒体温孵后,加入甲醇在低温下终止反应,再加入定量内标地西泮（diazepam,DZP）,涡旋后高速离心,取上清液50μl进样。结果1′-羟基咪达唑仑（1-hydroxymidazolam,1′-OHMDZ）与内标DZPtR分别为7.02min、10.51min,峰形对称且分离充分（R＆gt;1.5）,线性范围为15.6-780ng.ml-1,最低定量限（lower li mit of quantitation,LLOQ）为15.6ng.ml-1,高中低浓度提取回收率为106.3%、102.5%和102.6%,待测物的日内、日间相对标准偏差（rela-tive standard deviation, RSD）小于10％。肝微粒体Km=15μmol·L^-1，Vmax=0．08159nM·min^-1·mg^-1。结论温孵体系中的其他内源性物质不干扰测定。该方法快速、稳定、灵敏度高，适合体外DZP及其代谢产物1′-羟基咪达唑仑的测定，可用于体外CYP3A酶活性测定或酶动力学研究。

Determination of Cytochrome P450 3A4 Activity Using High Performance Liquid Chromatography

Objective To establish a rapid and accurate method with midazolam as a ＂probe drug＂ in order to evaluate or determine the activity of cytochrome（CYP3A）by the high-performance liquid chromatography（HPLC）-UV detection（VWD）.Methods The analytical column was used in accordance with Litas C18 column（4.6 mm × 100 mm,5 μm）,flow rate at 0.8 ml/min,UV detection at 254 nm,and column temperature at 40 ℃.Midazolam（MDZ）and rat liver microsomes after incubation were appended by methanol to terminate the reaction at low temperature, and then the quantitative internal standard diazepam （DZP）. After vortex and high-speed centrifugation, the supernatant from 50μl injection was extracted and analysed. Results 1-hydroxymidazolam （1′-OHMDZ） and the internal standard DZP retention times were 7. 02 min and 10. 51 min with symmetrical peak and full separation （R〉1.5）. The linear range was 15.6 780 ng·ml^-1 , and the lower limit of quantitation （LLOQ） was 15.6 ng·ml^-1. Extraction recovery at low, medium and high concentrations were 106. 3%, 102. 5% and 102. 6% respectively. The intra- and inter-day relative standard deviation （RSD） was less than 10%. For the liver microsomes Km was 15 μmol·L^-1 and Vmax was 0. 081 59 nM·min^-1·mg-1. Conclusion Other endogenous substances in the incubation system do not interfere with the determinatiorL The method is rapid, stable and highly sensitive. It＇s suitable for determination of DZP and its metabolites 1- hydroxymidazolam and can be used for in vitro determination of CYP 3A activity and research of enzyme kinetics.