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目的建立大鼠肝微粒体CYP1A2和CYP2D1酶活性的快速测定方法并考察两种CYP酶反应动力学。方法利用CYP1A2和CYP2D1选择性探针底物即非那西丁与右美沙芬进行温孵实验,采用超高压液相色谱与质谱联用(UPLC-MS)的方法建立相应代谢产物的含量测定方法。结果非那西丁代谢产物对乙酰氨基酚的线性范围为0.409~10.227μmol·L-1(r2=0.999 1),LOD和LOQ值分别为0.067和0.267μmol·L-1;右美沙芬代谢产物去甲右美沙芬为0.020~5.051μmol·L-1(R2=0.998 0),LOD和LOQ值分别为0.002和0.007μmol·L-1。方法回收率分别为95.9%~104.0%和102.1%~107.1%,精密度以RSD值表示均低于5%。CYP酶活性测试及反应动力学表明,CYP1A2和CYP2D1的米氏常数(Km值)分别为(28.4±2.7)和(13.9±1.3)μmol·L-1;其代谢活性(底物浓度为10μmol·L-1时)分别为(1.47±0.12)和(3.98±0.09)nmol·mg-1。结论本实验建立的UPLC-MS分析方法快速、灵敏、准确、可靠,适用于药物代谢研究中CYP1A2和CYP2D1体外活性测定及其反应动力学分析。
Objective To establish a rapid method for the determination of CYP1A2 and CYP2D1 activities in rat liver microsomes and investigate the kinetics of the two CYP enzymes. Methods CYP1A2 and CYP2D1 selective probe substrate phenacetin and dextromethorphan incubation experiment, the use of high pressure liquid chromatography and mass spectrometry (UPLC-MS) method to establish the corresponding metabolite content determination method . Results The linear range of phenacetin metabolites was 0.409-10.227μmol·L-1 (r2 = 0.999 1), the values of LOD and LOQ were 0.067 and 0.267μmol·L-1, respectively. The metabolites of dextromethorphan Dextromethorphan was 0.020-5.051μmol·L-1 (R2 = 0.998 0). The values of LOD and LOQ were 0.002 and 0.007μmol·L-1, respectively. The recoveries were 95.9% -104.0% and 102.1% -107.1%, respectively. The precision was lower than 5% with RSD values. CYP enzyme activity test and reaction kinetics showed that the Km values of CYP1A2 and CYP2D1 were (28.4 ± 2.7) and (13.9 ± 1.3) μmol·L-1, respectively. The metabolic activity (substrate concentration was 10μmol · L-1) were (1.47 ± 0.12) and (3.98 ± 0.09) nmol · mg-1, respectively. Conclusion The UPLC-MS analysis method established in this experiment is rapid, sensitive, accurate and reliable and is suitable for the in vitro activity determination and reaction kinetics analysis of CYP1A2 and CYP2D1 in drug metabolism research.