新型胰岛素增敏剂ZG02在大鼠肝微粒体中的代谢稳定性研究

Study on Metabolic Stability of Novel Insulin Sensitizer ZG02 in Rat Liver Microsomes

目的:建立测定大鼠肝微粒体孵育体系中新型胰岛素增敏剂ZG02质量浓度的方法,并探讨ZG02的体外代谢稳定性。方法:分别将ZG02溶解于还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)启动的大鼠肝微粒体Ⅰ相孵育体系、尿苷二磷酸葡萄糖醛酸(UDPGA)启动的Ⅱ相孵育体系以及二者联合启动的两相孵育体系中,置于37℃水浴中进行孵育,分别于孵育0、5、10、15、20、30、45 min时终止反应;以吲哚美辛为内标,采用超高效液相色谱-串联质谱法(UPLC-MS/MS)测定ZG02的质量浓度。色谱柱为Waters BEH C18,流动相为水(含0.01%甲酸)-乙腈(含0.01%甲酸)(35∶65,V/V),流速为0.4 mL/min,柱温为35℃,进样量为2μL;离子源为电喷雾离子源,以多反应监测模式进行负离子扫描,用于定量分析的离子对分别为m/z 458.115 9→323.073 0(ZG02)、m/z356.069 0→312.080 4(内标)。以孵育0 min时ZG02的质量浓度为参照,计算其在不同孵育体系中的剩余百分比、酶动力学参数和各代谢酶的贡献率。结果:ZG02检测质量浓度线性范围为1～400μg/L,定量下限为1μg/L,最低检测限为0.25μg/L;日内、日间RSD均小于10%,准确度为87.26%～94.58%,提取方法和基质效应均不影响待测物的测定。孵育45 min时,ZG02在Ⅰ相、Ⅱ相和两相孵育体系中的剩余百分比分别为(73.33±1.53)%、(50.63±3.42)%、(45.81±2.56)%,半衰期分别为67.28、30.26、25.57min,清除率分别为20.60、45.80、54.20μL/(mg·min),Ⅰ相、Ⅱ相代谢酶的贡献率分别为38.01%、84.50%。结论:本研究建立的UPLC-MS/MS法简便、快速、专属性强,可用于大鼠肝微粒体孵育体系中ZG02质量浓度的测定及体外代谢稳定性的研究。ZG02在Ⅰ、Ⅱ相孵育体系中的代谢稳定性中等,在两相孵育体系中的代谢稳定性较差,其代谢反应可能以Ⅱ相代谢酶介导的葡萄糖醛酸化结合反应为主。

OBJECTIVE:To establish a method for the concentration determination of novel insulin sensitizer ZG02 in liver microsomes of rats,and to study its metabolism stability in vitro.METHODS:ZG02 was respectively dissolved in phaseⅠincubation system of rat liver microsomes initiated by NADPH,in phaseⅡincubation system initiated by UDPGA and in two-phase incubation system initiated by NADPH and UDPGA.After incubated in 37℃water bath for 0,5,10,15,20,30,45 min,the reaction was terminated.Using indometacin as internal standard,UPLC-MS/MS method was used to determine the concentration of ZG02.The determination was performed on Waters BEH C18 column with mobile phase consisted of water(0.01%formic acid)-acetonitrile(0.01%formic acid)(35∶65,V/V)at the flow rate of 0.4 mL/min.The column temperature was 35℃,and the sample size was 2μL.The ion source was electrospray ion source and negative ion scanning carried out with multiple reaction monitoring mode.The ion pairs used for quantitative analysis were m/z 458.115 9→323.073 0(ZG02)and m/z 356.069 0→312.080 4(internal standard),respectively.The residual percentage and enzyme kinetic parameters of ZG02,contribution rates of metabolic enzymes in different incubation systems were calculated with the concentration of ZG02 incubated for 0 min as reference.RESULTS:The linear range of ZG02 was 1-400μg/L;the limits of quantitation and detection were 1μg/L and 0.25μg/L.RSDs of inter-day and intra-day were all lower than 10%,accuracies were 87.26%-94.58%.The extraction method and the matrix effect didn’t affect the determination of the tested substance.At 45 min of incubation,the residual percentages of ZG02 were(73.33±1.53)%in phaseⅠincubation system,(50.63±3.42)%in phaseⅡincubation system and(45.81±2.56)%in two-phase incubation system.Half-time period of phaseⅠincubation system,phaseⅡincubation system and two-phase system were 67.28,30.26,25.57 min;clearance rate were 20.60,45.80,54.20μL/(mg·min),respectively.The contribution rates of phaseⅠincubation system and phaseⅡincubation system were 38.01%and 84.50%.CONCLUSIONS:Established UPLCMS/MS method is simple,rapid and specific.It can be used for the concentration determination of ZG02 and in vitro metabolism stability study in rat liver microsomes incubation system.The metabolism stability of ZG02 is moderate in phaseⅠand phaseⅡincubation systems,but poor in two-phase incubation system.The main metabolic reaction may be phaseⅡmetabolic enzyme mediated glucuronization.