HPLC-MS/MS法同时测定大鼠血浆中26种胆汁酸

目的：建立HPLC-MS/MS法同时定量检测大鼠血浆中26种胆汁酸的浓度。方法：大鼠血浆采用蛋白沉淀法处理，26种胆汁酸使用Symmetry C₁₈色谱柱分离。流动相为水（0.1%甲酸+10 mmol·L^-1乙酸铵）-甲醇（0.1%甲酸+10 mmol·L^-1乙酸铵），梯度洗脱，流速0.15 mL·min^-1，检测时间50 min。检测系统采用电喷雾离子源，负离子模式。结果：26种胆汁酸线性关系良好且色谱峰互不干扰，定量下限为0.010~0.200 μmol·L^-1，日内、日间回收率均在85.9%~113.8%之间，精密度均在2.0%~13.3%之间，提取回收率在65.7%~104.5%之间。正常SD大鼠血浆中26种胆汁酸浓度范围为0.0192~2.83 μmol·L^-1，其中猪去氧胆酸（HDCA）、去氧胆酸（DCA）、胆酸（CA）、甘氨胆酸（GCA）、β-鼠胆酸（β-MCA）和甘氨脱氧胆酸（GDCA）是SD大鼠血浆中的主要胆汁酸。结论：该方法准确有效，灵敏度高，操作简单，可在50 min内同时检测大鼠血浆中26种胆汁酸，并可为其他生物基质中的胆汁酸检测方法提供参考。

Objective:To establish a simultaneously quantitative analysis method of 26 bile acids in rat plasma by HPLC-MS/MS.Methods:Rat plasma was prepared by protein precipitation and analyzed on a Symmetry C₁₈ reverse-phase column with gradient elution of mobile phase consisting of water(0.1% formic acid+10 mmol·L^-1 ammonium acetate) -methanol(0.1% formic acid+10 mmol·L^-1 ammonium acetate) in 50 min.The flow rate was 0.15 mL·min^-1.The detection was carried out on MS/MS with ESI in the negative ionization mode.Results:The method showed excellent linearity on all 26 bile acids, the MRM signals of all analyzed bile acids were well separated, including the conformational isomers.The LOQ ranged from 0.010 to 0.200 μmol·L^-1.Intra-day and inter-day accuracy were between 85.9% and 113.8%.Intra-day and inter-day precision were between 2.0% and 13.3%.The average recovery rate for all the analyzed bile acids ranged from 65.7% to 104.5%.The concentrations of 26 bile acids ranged from 0.0192 to 2.83 μmol·L^-1.It showed that HDCA、DCA、CA、GCA、β-MCA and GDCA were the major bile acids in rat plasma.Conclusion:The HPLC-MS/MS method was validated with good accuracy, sensitivity, and high-efficiency sample preparation procedure.It enabled the simultaneous quantitation of 26 bile acids in rat plasma in 50 min, demonstrating the utility of such analysis on other biological matrices.