HPLC 法同时测定大鼠血浆中 CYP450酶探针药物氯唑沙宗、甲苯磺丁脲和咪达唑仑浓度

摘要

目的：建立同时测定大鼠血浆中细胞色素 P450（CYP450）酶探针药物氯唑沙宗、甲苯磺丁脲和咪达唑仑浓度的高效液相色谱法。方法色谱柱采用 Diamonsil C18（250 mm ×4．6 mm，5μm），流动相为甲醇与磷酸二氢铵缓冲液（61∶39，V ／V），流速1 mL·min －1，检测波长230 nm，带宽4 nm，柱温35℃，以地西泮为内标，同时检测氯唑沙宗、甲苯磺丁脲和咪达唑仑的大鼠血浆药物浓度。结果氯唑沙宗、甲苯磺丁脲和咪达唑仑均在0．1～50 mg·L －1（r ＝0．9995）的范围内线性关系良好；低、中、高3个浓度下，3种探针药物精密度试验中相对标准偏差为4．78％～9．78％，回收率为92．71％～109．79％。结论该方法操作简便，灵敏度高，重现性好，符合生物样品分析要求，适用于同时测定3种 CYP450酶亚型探针药物氯唑沙宗、甲苯磺丁脲和咪达唑仑的大鼠血浆药物浓度，可为 CYP450酶活性测定提供分析方法学参考。%Objective To establish an efficient,rapid,selective HPLC method for determining three CYP450s probe drugs,chlorzoxa-zone,tolbutamide and midazolam,in single run in rat plasma.Methods The three specific probe substrates of chlorzoxazone,tolbuta-mide,and midazolam,together with the internal standard diazepam,could be extracted using liquid-liquid extraction in rat plasma,fol-lowed by high-performance liquid chromatography (HPLC)using a C18 column (250 mm ×4.6 mm,5 μm).The mobile phase was con-stituted of a methanol and 0.05 mol·L -1 phosphate buffer (61∶39,V /V).Results All analytes were separated simultaneously in a single run .The detection limits ranged from 0.1 ~50 mg·L -1 for chlorzoxazone,tolbutamide and midazolam.The precisions for three probe substrates were 4.78% ~9.78%,the accuracy of three probe substrates ranged from 92.71% ~109.79%.The limit of quantifica-tion (LOQ)was 0.1 mg·L -1 for chlorzoxazone,tolbutamide and midazolam.Conclusions The present method provides a robust,fast analytical tool for the three-probe drug cocktail.Finally,the method was suitable for determining the plasma concentration of these com-pounds and evaluating the CYP2E1,CYP2C9 and CYP3A4 activities in the drug-drug interaction studies.