血浆中血管紧张素Ⅱ液相色谱-串联质谱测定方法的建立

Quantitation of Angiotensin Ⅱ in plasma by liquid chromatography-mass spectrometry

目的:建立液相色谱串联质谱法检测血浆中的血管紧张素Ⅱ(AngⅡ),并研究不同抗凝剂对检测结果的影响。方法:2018年7月,在北京安贞医院住院的20例冠心病患者,分为乙二胺四乙酸(EDTA)抗凝组、枸橼酸钠抗凝组,和血清组。以血清组作为对照,对比EDTA组、枸橼酸钠组和血清中的AngⅡI浓度。用蛋白沉淀法进行样本前处理,利用TSQ quantum ultra三重四极杆质谱仪,在电喷雾离子源正离子模式下,质谱检测模式为选择反应监控,液相系统为Ultimate 3000 UPLC,流动相是含0.1%甲酸的水相和甲醇,色谱梯度为5 min。并对该方法的线性范围、灵敏度、精密度、提取回收率和基质效应等进行评价。结果:该方法的定量检出下限是50 ng/L,线性范围为50~5 000 ng/L,批内和批间准确度和精密度变异系数均<20%。提取回收率在92.2%~101.0%,基质效应在107.8%~111.1%。不同抗凝剂对AngⅡ检测结果有影响。结论:检测血浆中的AngⅡ的液相色谱串联质谱法,该方法色谱梯度时间短,前处理方法简单,易操作;并建议采用血清中检测AngⅡ浓度。

Objective: To establish a method to detect Angiotensin Ⅱ by liquid chromatography-mass spectrometry, and evaluate the effects of different collection tubes on the Angiotensin Ⅱ result. Methods: Samples of 20 coronary heart disease inpatients from Beijing Anzhen Hospital in July 2018 were obtained in three different collection tubes: EDTA plasma, sodium citrate plasma, and serum. EDTA and sodium citrate were compared with serum as the reference. Samples were extracted by protein precipitation. The detection was carried out by selected reaction monitoring using a TSQ Quantiva tandem quadrupole mass spectrometer under positive electrospray ionization conditions. Chromatography was performed on an Ultimate 3000 UPLC system under chromatographic conditions of water(0.1% formic acid) and methanol, giving a total chromatographic analysis time of 5 min. The linear range, sensitivity, precision, recovery and matrix effect of the method were evaluated. Results: The method was found to be linear over the analytical range of 50-5 000 ng/L giving the limit of detection of 50 ng/L. The coefficients of variation of intra-and inter-assay accuracies and precisions were less than 20%. The extraction recovery ranged from 92.2%-101.01% and the matrix effect varied from 107.8-111.11%. The result of Angiotension Ⅱ were distinct in different collection tubes. Conclusions: We developed a liquid chromatography-tandem mass spectrometry method for the quantitation of Angiotensin Ⅱ with shorter chromatographic analysis time and easier operation. It is suggested to detect the concentration of angiotensin Ⅱ in serum.