人血浆中血管紧张素Ⅱ候选参考方法的建立

Establishment of a candidate reference method for angiotensinⅡin human plasma

目的建立用同位素稀释液相色谱串联质谱(ID-LC/MS/MS)测定人血浆中血管紧张素Ⅱ的候选参考方法,并对方法性能进行评价。方法以[13C6-15N]-血管紧张素Ⅱ为内标,重量法准确称取血浆及一定量内标混合,同时加入酶抑制剂,经硫酸锌溶液蛋白沉淀、反相固相萃取板处理,用液相色谱串联质谱分析,质谱选择多反应离子监测模式检测血管紧张素Ⅱ及内标的特定离子碎片。按照ISO15193要求对所建方法进行线性、灵敏度、精密度、回收率等性能评估及不确定度的评价。结果血管紧张素Ⅱ在10~1000 pg/g范围内线性良好(r=0.9995),检测下限7.68 pg/g,加标回收率范围为97.14%~102.85%,批内不精密度≤3.21%,批间不精密度≤2.96%,总不精密度≤3.67%。结论用同位素稀释液相色谱串联质谱建立了血浆血管紧张素Ⅱ的检测方法,该方法准确可靠,有望成为血浆中血管紧张素Ⅱ测定的参考方法。

Objective To establish a candidate reference method for the determination of angiotensinⅡin human plasma by isotope dilution liquid chromatography tandem mass spectrometry(ID-LC-MS/MS)and to evaluate its performance.Methods Using[13C6-15N]-angiotensinⅡas the internal standard,the plasma was accurately weighed by gravimetric method and mixed with a certain amount of internal standard.At the same time,enzyme inhibitor was added.After zinc sulfate solution protein precipitation and reversed-phase solid-phase extraction plate treatment,it was analyzed by liquid chromatography tandem mass spectrometry.The multi reaction ion monitoring mode(MRM)was selected by mass spectrometry to detect specific ion fragments of angiotensinⅡand internal standard.The linearity,sensitivity,precision,recovery rate and uncertainty of the performance of the established method were evaluated according to ISO15193.Results AngiotensinⅡhad good linearity in the range of 10-1000 pg/g(r=0.9995),the lower limit of quantification was 7.68 pg/g,the analytical recoveries were 97.14%to 102.85%,intra-batch imprecision≤3.21%,inter-batch imprecision≤2.96%,and total imprecision≤3.67%.Conclusion A method for the determination of plasma angiotensinⅡwas established by ID-LC-MS/MS.The method is accurate and reliable,and is expected to be a reference method for the determination of plasma angiotensinⅡ.