小鼠血清中胆汁酸的液相色谱串联质谱分析方法的建立

Establishment of a Liquid Chromatography-Tandem Mass Spectrometry Method for Determination of Bile Acids in Mouse Serum Samples

本研究旨在建立基于同位素内标的液相色谱串联质谱(LC-MS/MS)分析方法,以同步检测小鼠血清中28种胆汁酸。样本用甲醇沉淀蛋白质后,采用Waters BEH C18超高效液相色谱柱(1.7μm,2.1mm×100 mm)分离,以0.1%甲酸-水和0.1%甲酸-乙腈作为流动相进行梯度洗脱,流速0.5 mL/min,采用负离子模式进行质谱检测。结果显示:胆汁酸在定量范围内线性关系良好,检测限在0.2~1.5 ng/mL,样品中胆汁酸同位素内标的加标回收率均在83.5%~108.4%,变异系数均小于14.9%。采用该方法检测了小鼠血清样品中的各胆汁酸含量,所有样品均检测出了25种胆汁酸。结果表明,本研究所建立的LC-MS/MS法操作简捷,灵敏度高,多靶标同步分析能力强,能够用于小鼠血清胆汁酸谱的检测。

A liquid chromatography-tandem mass spectrometry(LC-MS/MS)method based on isotope internal standard was established for simultaneous determination of 28 bile acids in mouse serum samples.Samples were extracted with methanol and separated on a Waters BEH C18 ULTRA-high performance liquid chromatography(1.7μm,2.1 mm×100 mm)column.Gradient elution was performed with 0.1%formic acid-water and 0.1%formic acid-acetonitrile as mobile phases at the flow rate of 0.5 mL/min.Negative ion mode was used for mass spectrometry.The results showed that the calibration curves of 28 bile acids were linear in the quantitative range,the detection limit was 0.2-1.5 ng/mL.The average recovery ratios of bile acid isotopes ranged from 83.5%to 108.4%,and the coefficients of variation were all less than 14.9%.The contents of bile acids in mouse serum samples were determined by this method,and 25 bile acids were detected in all mouse serum samples.Thus,the LC-MS/MS method established in this study has the advantages of simple operation,high sensitivity and strong multiplexing detection capability,and facilitates the detection of serum bile acid profile in mice.