摘要
目的:采用LC-MS/MS法测定细胞培养液中6个酶代谢底物对应代谢产物(1-7二甲基黄嘌呤、7-羟基香豆素、4-羟基美芬妥英、1-羟基咪达唑仑、6-羟基氯唑沙宗、4-羟基甲苯磺丁脲)的浓度。方法:生物样品用液-液萃取法进行预处理;采用Agilent ZORBAX SB-C18(2.1 mm×150 mm,5μm)色谱柱,以甲醇-水(75∶25)为流动相,流速为0.3 mL·min^-1,柱温为35℃,硝西泮为内标,通过电喷雾离子化四极杆串联质谱,分别以正、负离子选择反应监测方式(SRM)检测。结果:6个酶代谢产物的线性范围均为1~500 ng·mL^-1,定量下限均为1 ng·mL^-1;日内和日间RSD分别小于9.9%和10.7%;准确度在92.9%~110.0%;提取回收率为71.9%~98.6%;基质效应为90.1%~106.5%;稳定性良好。结论:该方法灵敏、准确、简单、快速,可用于细胞培养液中6个CYP450酶代谢产物的测定。
Objective:To develop an LC-MS/MS method for the determination of six enzyme metabolites in cell culture fluid,including 1,7-dimethyl-xanthine,7-hydroxy coumarin,4-hydroxy mephenytoin,1-hydroxy midazolam,6-hydroxy chlorzoxazone and 4-hydroxy tolbutamide.Methods:Biological samples were pretreated by liquid-liquid extraction,and separated on an Agilent ZORBAX SB-C18 column(2.1 mm×150 mm,5μm).The mobile phase consisted of methanol and water(75∶25)at a flow rate of 0.3 mL·min-1.The column temperature was 35℃and the internal standard was nitrazepam.Analytes were determined using selective reaction monitoring in the positive and negative electrospray ionization modes by LC-MS-MS,and(SRM).Results:The culture solution standard curves of six enzyme metabolites were linear in the concentration range of 1-500 ng·mL-1.The limit of quantification was 1 ng·mL-1.The intra-and inter-day RSDs were less than 9.9%and 10.7%,respectively.The intra-and inter-day accuracy range was 92.9%-110.0%.The extraction recovery was 71.9%-98.6%and matrix effect was 90.1%-106.5%.Stability was good.Conclusion:A sensitive,accurate,simple and rapid LC-MS/MS was successfully developed and validated for the determination of six cytochrome P450 enzyme metabolites in cell culture fluid.
作者
吴丽颖
张运好
薛改
闫成
WU Li-ying;ZHANG Yun-hao;XUE Gai;YAN Cheng(Department of Pharmacy,Joint Logistics Support Force 980th Hospital,Chinese People’s Liberation Army,Shijiazhuang 050082,China;Sports Human Sciences Department,Shijiazhuang University,Shijiazhuang 050035,China)
出处
《药物分析杂志》
CAS
CSCD
北大核心
2020年第9期1565-1574,共10页
Chinese Journal of Pharmaceutical Analysis
