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目的:建立测定小鼠肝脏中16种胆汁酸浓度的LC-MS/MS法,并将测定结果应用于比较Nrf2野生型和基因敲除型小鼠胆汁酸代谢谱。方法:小鼠肝脏样品加入70%乙腈匀浆,用甲醇沉淀匀浆液中的蛋白并高速离心,取上清液置进样瓶,用XtimateTMC18柱分离,以甲醇-醋酸铵及甲酸水溶液为流动相进行梯度洗脱,流速0.25 ml·min-1,进样5μl分析。结果:16种胆汁酸在0.013~8.000μmol·L-1范围内,线性关系良好,定量下限浓度为0.013μmol·L-1,肝脏匀浆液加样回收率在94.9%~112.8%之间,日内、日间RSD均小于10.8%。结论:该方法准确可靠,灵敏度高,处理简便,适用于肝脏中胆汁酸浓度的测定,为生物样品中胆汁酸含量测定提供参考。
OBJECTIVE: To establish a LC-MS / MS method for the determination of 16 bile acids in the liver of mice and to apply the results to compare the bile acid metabolism profiles of Nrf2 wild-type and knockout mice. Methods: The liver samples of mice were homogenized with 70% acetonitrile. The protein in the homogenate was precipitated with methanol and centrifuged at high speed. The supernatant was placed in a sample bottle and separated on a XtimateTMC18 column. The mobile phase consisted of methanol-ammonium acetate and formic acid solution Gradient elution, flow rate 0.25 ml · min-1, injection 5μl analysis. Results: The linear range of 16 bile acids in the range of 0.013-8.000 μmol·L-1 was good. The lower limit of quantification was 0.013 μmol·L-1, and the recovery rate of hepatic homogenates was between 94.9% and 112.8% , Daytime RSDs were less than 10.8%. Conclusion: The method is accurate, reliable, sensitive and easy to handle. It is suitable for the determination of bile acid in liver and provides a reference for the determination of bile acid in biological samples.