摘要
目的建立超高效液相色谱-串联质谱法(UHPLC-MS/MS)的各亚型CYP450酶探针底物定量分析方法,并用于药物对代谢酶活性影响的评价。方法采用超高效液相色谱仪-三重四极杆串联质谱仪,选用Waters HSS T3(1.8μm,10×2.1 mm)色谱柱,以0.1%甲酸水(A)-乙腈(B)为流动相进行梯度洗脱。采用CYP450酶特异性探针药物法,根据FDA推荐选择7种特异性探针药物(CYP1A2、CYP2B6、CYP2C8、CYP2C9、CYP2D6、CYP2E1和CYP3A4)进行测定。采用电喷雾离子源(ESI),在正离子扫描下,采用多反应检测模式(MRM)进行测定。采用体外共孵育法比较柴胡多炔对酶活性的抑制率。结果7种特异性探针底物可在6 min内准确测定;空白孵育基质中的内源性物质对待测物及内标的测定无干扰;待测物最低定量限最低达0.32μmol/L,线性良好,相关系数均>0.99;精密度≤14.1%,准确度≤11.2%;提取回收率和基质效应均符合分析要求。柴胡多炔(RB-2与RB-4)对CYP1A2有较微弱的抑制(底物减少率分别为38%和24%),而对其他代谢酶活性均无明显抑制作用。结论该方法分析时间短、专属性强、准确度好、精密度高、基质效应小,适用于7种代谢酶活性的测定。
Objective To establish a quantitative method of enzyme probe substrates of CYP450 isoforms by ultra high performance liquid chromatography-tandem mass spectrometry（ UHPLC-MS/MS）,and evaluate the effect of drug on metabolic enzyme activity.Methods An ultra performance liquid chromatograph-triple quadrupole tandem mass spectrometer was used to detect compounds with Waters HSS T3（ 1. 8 μm,10 mm × 2. 1 mm） column and the mobile phase consisted of 0. 1% formic acid in water（ A）-acetonitrile（ B）. Seven kinds of specific probe drugs（ CYP1 A2,CYP2 B6,CYP2 C8,CYP2 C9,CYP2 D6,CYP2 E1 and CYP3 A4） were selected according to the FDA,and measured by CYP450 enzyme-specific probe drug method. The electrospray ionization source（ ESI） was used for the determination by multi-reaction detection mode（ MRM） under positive ion scan. The inhibition rate of polyacetylenes to the enzyme activity was compared by the method of co-incubation in vitro. Results The results showed that the seven specific probe substrates were accurately determined within 6 min. The endogenous substances in the blank incubation matrix showed no interfere for the determination of the analytes and the internal standard. The lowest limit of quantification of the analytes was 0. 32 μmol/L,and the linearity was good. The correlation coefficients were all more than 0. 99. The RSD of precision was less than 14. 1% and the RSD of accuracy was less than 11. 2%. The extraction recovery and matrix effect were all in accordance with the analysis requirements. The polyacetylenes had no significant inhibition to the activities of metabolic enzymes,except that the inhibition rates of RB-2 and RB-4 to CYP1 A2 were 38% and 24%,respectively. Conclusion The method has the advantages of short analysis time,good special specificity,good accuracy,high precision and small matrix effect,and can be suitable for the determination of seven metabolic enzyme activities.
作者
吴莉
张峰
王鹏
赵芳
秦雪梅
高晓霞
WU Li;ZHANG Feng;WANG Peng;ZHAO Fang;QIN Xueme;GAO Xiaoxia(Modern Research Center for Traditional Chinese Medicine, Shanxi University , Taiyuan 030006, China;College of Chemistry and Chemical Engineering, Shanxi University)
出处
《山西医科大学学报》
CAS
2018年第5期492-497,共6页
Journal of Shanxi Medical University
基金
国家自然科学基金资助项目(81473415)
山西省高等学校优秀青年带头人项目
山西省应用基础研究计划优秀青年基金项目(201701D211009)
山西省科技创新重点团队项目(201605D13 1045-18)
山西省重点实验室地产中药功效物质研究与利用项目资助(201605D111004)
