HPLC-MS/MS法测定人血中15种胆汁酸浓度及不同抗凝剂对检测结果的对比分析

目的：建立同时测定人血浆中15种胆汁酸的高效液相色谱-串联质谱联用（HPLC-MS/MS）方法，并分析不同抗凝剂对检测结果的影响。方法：血浆样本采用乙腈沉淀蛋白；采用Xterra RP18（4.6 mm&#215;150 mm，5 μm）色谱柱，以乙腈-10 mmol&#183;L^-1醋酸铵溶液为流动相，梯度洗脱，流速1 mL&#183;min^-1，进样10 μL，采用电喷雾离子源（ESI）-三重四极杆质谱仪于负离子模式下进行多反应检测（MRM）。在该测定方法下，比较血清、肝素锂抗凝血浆及EDTA-K₂抗凝血浆中15种胆汁酸的测定浓度。结果：15种胆汁酸在定量范围内线性关系良好，日内和日间精密度均<10%，低、中、高浓度提取回收率为71.2%~93.5%。用该方法同时检测血清、肝素锂抗凝血浆及edta-k_{2抗凝血浆中15种胆汁酸时，6名体检者血清、肝素锂抗凝血浆及EDTA-K2抗凝血浆中胆汁酸RE范围分别为-12.11%~4.67%、-13.08%~2.08%和-0.26%~15.46%。结论：该方法灵敏度高，专属性好，适用于人血中15种胆汁酸浓度的测定。建议使用血清或肝素锂抗凝血浆开展胆汁酸分型检测。}

Objective:To establish an HPLC-MS/MS method for simultaneous determination of 15 bile acids in human plasma and to investigate the potential impact from various anticoagulants. Methods:Proteins in plasma samples were precipitated with acetonitrile and the supernatant was fractionated on a Xterra RP 18(4.6 mm&#215;150 mm,5 μm) column. A injection of 10 μL analytes were gradient eluted with acetonitrile and aqueous solution containing 10 mmol&#183;L^-1 ammonium acetate at 1 mL&#183;min^-1 flow rate,followed by detection in electrospray triple-quadrupole mass spectrometry by multiple reaction monitoring in negative ion mode. The concentrations of 15 bile acids in serum,heparin anticoagulative plasma and EDTA anticoagulative plasma derived samples were determined and compared. Results:The calibration curves of bile acids showed to be linear in the quantitative range. All RSDs of intra-day and inter-day precision results were within 10%. The average extraction recoveries of the control samples at low,middle and high concentrations were between 71.2%-93.5%. RE ranges of the bile acids in 6 physical examinees' serum,heparin anticoagulative plasma and EDTA-K₂ anticoagulative plasma were -12.11%-4.67%,-13.08%-2.08% and -0.26%-15.46%,respectively. Conclusion:The method was proved sensitive and selective,and could be useful for determination of 15 bile acids in human plasma. Moreover,in clinical application,serum and heparin anticoagulative plasma would be preferred for this method.

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