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

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Xtimate^TM C₁₈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5 μl分析。结果:16种胆汁酸在0.013~8.000 μmol·L^-1范围内,线性关系良好,定量下限浓度为0.013 μmol·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。

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

OBJECTIVE To determine concentrations of 16 bile acids in mice liver by LC-MS/MS.METHODS After added 70% acetonitrile into mice liver, protein precipitation with methanol was used for sample preparation. Liquid chromatographic separation was performed on a Xtimate^TM C₁₈ 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 5 μl. RESULTS The linear calibration curves were obtained in the concentration range of 0.013-8.000 μmol·L^-1. The lower limit of quantification was 0.013 μmol·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.