摘要
目的：优化和建立大黄酸及其代谢活化产物检测方法,并应用于大黄酸肝微粒体代谢活化研究。方法：肝微粒体孵育样品以含3%甲酸的甲醇酸化并沉淀蛋白后,用API 4000LC-MS/MS对大黄酸和其活性代谢物大黄酸乙酰葡糖醛酸进行定性、定量分析。使用Agela Venusil XBP C18column（50×2.1 mm,3μm）色谱柱,流动相为0.1%甲酸水-乙腈梯度洗脱。ESI离子源在负离子模式下进行测定,多反应离子监测,用于定量分析的离子对为m/z 283.0→239.5（大黄酸）和459.0→283.0（大黄酸葡糖醛酸）。结果：大黄酸乙酰葡糖醛酸在PBS、甲醇中都不稳定,以含3%甲酸的甲醇处理后,大黄酸乙酰葡糖醛酸在样品溶液中稳定性良好。大黄酸和大黄酸乙酰葡糖醛酸的线性范围分别为100～25 000 nmol·L-1和10～5 000 nmol·L-1,日内和日间RSD均〈10.7%,准确度在95.8%～112%之间。最适宜孵育时间优化为40 min。结论：本实验所建立的肝微粒体体系中大黄酸及其代谢活化产物大黄酸乙酰葡糖醛酸的测定方法简便、可行,为进一步研究大黄酸的代谢活化提供了基础。
Objective： To optimize and establish an analytical method for determination of rhein and its activated metabolite,and further apply in the study of metabolic activation of rhein in liver microsomes. Methods：Incubation samples of liver microsomes was deproteinized by addition of methanol containing 3% formic acid. API4000 LC-MS / MS was used to qualitatively and quantitatively determine rhein and rhein acyl glucuronide. Samples were analyzed on an Agela Venusil XBP C18column（ 50 × 2. 1 mm,3 μm） adopting a gradient elution. MRM modein negative ionization was chosen and ion transitions were monitored as rhein,283. 0→239. 5; rhein glucuronides,459. 0→283. 0. Results： Rhein acyl glucuronide was not stable in PBS and methanol,but it was stable in the sample solution processed by methanol containing 3% formic acid. A good linearity of rhein and rhein acyl glucuronide was obtained in the range of 100-25 000 nmol·L-1and 10-5 000 nmol·L-1,respectively. Intraand inter-day variations were 〈 10. 7% and great accuracies of 95. 8%-112% were achieved at the concentrations examined. The incubation time was optimized as 40 min. Conclusion： The established method is stable and applicable,and can be used for the determination of rhein and rhein acyl glucuronidations in human liver microsomes（ HLM） and rat liver microsomes（ RLM）. Our study also provides experimental foundation for the further investigation of metabolic activation of rhein.
出处
《东南大学学报（医学版）》
CAS
北大核心
2016年第6期894-899,共6页
Journal of Southeast University(Medical Science Edition)
关键词
大黄酸
大黄酸乙酰葡糖醛酸
代谢活化
高效液相色谱-质谱联用
rhein
rhein acyl glucuronidations
metabolic activation
liquid chromathgraphy tandem mass spectrometry
