采用UPLC-MS/MS法研究树豆酮酸A在不同种属肝微粒体中的代谢差异

建立测定肝微粒体孵育体系中树豆酮酸A(CAA)质量浓度的方法,并比较其在不同种属肝微粒体中的代谢特征。方法:分别将CAA溶解于由还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)启动的大鼠、比格犬、人肝微粒体孵育体系中,置于37℃水浴中进行孵育,分别于孵育的0、5、10、15、30、45、60 min时用乙腈终止反应,以染料木素为内标,采用超高效液相色谱-串联质谱法(UPLC-MS/MS)检测各孵育体系中CAA的质量浓度。色谱柱为Waters BEH C18,流动相为水(含0.1%甲酸)-乙腈(含0.1%甲酸)(45∶55,V/V),流速为0.25 mL/min,柱温为30℃,进样量为2μL;采用电喷雾离子源,以选择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 353.14→309.11(CAA)、m/z 269.86→224.11(内标)。以孵育0 min时CAA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和酶动力学参数。结果:CAA检测质量浓度的线性范围为0.05~20μg/mL,定量下限为0.05μg/mL,最低检测限为0.01μg/mL;日内、日间RSD均小于10%,相对误差为-4.83%~8.94%,提取方法和基质效应均不影响待测物的测定。孵育60 min时,CAA在大鼠、比格犬、人肝微粒体中的剩余百分比分别为(62.79±9.99)%、(64.07±11.59)%、(96.66±5.71)%;在大鼠、比格犬肝微粒体中的半衰期(72.19、68.61 min)均显著短于人肝微粒体(364.74 min),清除率0.019 2、0.020 2 mL/(min·mg)]均显著高于人肝微粒体0.003 8 mL/(min·mg)](P<0.05)。结论:本研究建立的UPLC-MS/MS法简便、快速、专属性强、灵敏度高,可用于肝微粒体孵育体系中CAA质量浓度的测定及体外代谢稳定性的研究。CAA在大鼠、比格犬肝微粒体中的代谢稳定性均差于人肝微粒体。

Study on Metabolic Differences of Cajanonic Acid A in Different Species of Liver Microsomes by UPLC-MS/MS

OBJECTIVE:To establish a determination method for the concentration of cajanonic acid A(CAA) in liver microsome incubation system, and to compare the metabolism characteristics of it in different species of liver microsomes.METHODS:CAA was dissolved in liver microsome incubation system of rat,Beagle dog and human initiated by reduced nicotinamide adenine dinucleotide phosphate(NADPH),and was incubated in water at 37 ℃. The reaction was terminated with acetonitrile at 0,5,10,15,30,45 and 60 min,respectively. Using genistein as internal standard,the concentration of CAA in different incubation systems was determined by UPLC-MS/MS. The determination was performed on Waters BEH C18 column with mobile phase consisted of water(containing 0.1% formic acid)-acetonitrile(containing 0.1% formic acid)(45 ∶ 55,V/V)at the flow rate of 0.25 mL/min. The column temperature was 30 ℃,and the sample size was 2 μL. The electrospray ionization source was used to the select reaction monitoring mode for negative ion scanning. The ion pairs for quantitative analysis were m/z 353.14→309.11(CAA),m/z 269.86→224.11(internal standard)respectively. The residual percentage and enzymatic kinetic parameters of CAA in different incubation systems were calculated according to the mass concentration of CAA at 0 min. RESULTS:The linear range of CAA was 0.05-20 μg/mL;the limit of quantitation was 0.05 μ g/mL,and the lowest detection limit was0.01 μg/mL. RSDs of intra-day and inter-day were lower than10%;relative errors ranged-4.83%-8.94%;extraction method and matrix effect did not affect the determination of the substance to be measured. At 60 min of incubation,residual percentages of CAA in rat,Beagle dog and human liver microsomes were(62.79 ± 9.99)%,(64.07 ± 11.59)%,(96.66 ± 5.71)%,respectively. The half-life period(72.19,68.61 min)of CAA in rat and Beagle dog liver microsomes were significantly shorter than human liver microsome(364.74 min). The clearance rates 0.019 2,0.020 2 mL/(min·mg)] were significantly higher than human liver microsome 0.003 8 mL/(min·mg)](P<0.05). CONCLUSIONS:Established UPLC-MS/MS method is simple,rapid,specific and sensitive,and can be used for the determination of CAA concentration in liver microsome incubation system and the study of metabolism stability in vitro. The stability of CAA metabolism in rat and Beagle dog liver microsomes are poorer than human liver microsome.