液-质联用测定Nrf2野生型和基因敲除型小鼠肝脏中16种胆汁酸

Determination of 16 bile acids in Nrf2 wild-type and knockout mice liver by using LC-MS/MS method

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 determine concentrations of 16 bile acids in mice liver by LC-MS/MS.METHODS After added70% acetonitrile into mice liver,protein precipitation with methanol was used for sample preparation.Liquid chromatographic separation was performed on a XtimateTMC18 column with gradient eluent consisted of methanol-ammonium acetate and formic acid buffer,at a flow rate of 0.25 ml·min^-1 and injection volume of 5μl.RESULTS The linear calibration curves were obtained in the concentration range of 0.013-8.000μmol·L^-1.The lower limit of quantification was 0.013μmol·L^-1.The recovery of the samples at low,middle and high concentrations were 94.9%-112.8%.The intra-day and inter-day RSD were less than 10.8%.CONCLUSION This method is accurate and sensitive for determination of bile acids in mice liver and can provide reference for determination of bile acid in biological samples.