建立并评价基于LC-MS/MS同时测定脑脊液Aβ1-42、Aβ1-40和Aβ1-38的检测方法  

Establishing and evaluating a robust method based on LC-MS/MS for simultaneous determination of Aβ1-42,Aβ1-40 and Aβ1-38 in cerebrospinal fluid

作　　者：邹雨桐 马晓丽 禹松林[1] 李倩倩 王丹晨 钟健 毛晨晖[3] 高晶[3] 邱玲[1] Zou Yutong;Ma Xiaoli;Yu Songlin;Li Qianqian;Wang Danchen;Zhong Jian;Mao Chenhui;Gao Jing;Qiu Ling(Department of Laboratory Medicine,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences,Beijing 100730,China;Waters Technology(Beijing)Co.,Ltd.,Beijing 102600,China;Department of Neurology,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences,Beijing 100730,China)

机构地区：[1]中国医学科学院北京协和医院检验科,北京100730 [2]沃特世科技(北京)有限公司,北京102600 [3]中国医学科学院北京协和医院神经科,北京100730

出　　处：《中华检验医学杂志》2023年第8期814-821,共8页Chinese Journal of Laboratory Medicine

基　　金：高水平医院临床科研业务费(2022-PUMCH-B-073,2022-PUMCH-A-138);国家重点研发计划(2020YFA0804500/2020YFA0804501);国家自然科学基金(82202651)。

摘　　要：目的建立并验证一种基于液相色谱串联质谱(LC-MS/MS)同时测定脑脊液Aβ1-42、Aβ1-40和Aβ1-38的检测方法,并评价该方法与3种主流检测方法间的一致性。方法方法建立、验证与一致性评价。以15N标记的β-淀粉样蛋白作为内标,采用Waters MCX 96孔固相萃取板进行抽提和萃取,收集洗脱液至QuanRecovery MaxPeak 700μl收集板。在正离子模式下,基于电喷雾离子化的多反应监测模式实现脑脊液Aβ1-42、Aβ1-40和Aβ1-38的同时测定。参考CLSI C62-A和EP-15A3指南对定量限、线性、回收率、精密度、正确度等性能进行方法学评价。收集57例临床检测剩余的脑脊液样本,采用INNOTEST ELISA试剂盒及Lumipulse G和Roche Elecsys全自动化学发光检测系统测定Aβ1-42和Aβ1-40的浓度,采用Passing-Bablok和Bland-Altman法进行不同检测系统间的比对及偏倚评估。结果该方法的分析时长为8 min,测定Aβ1-42、Aβ1-40和Aβ1-38的定量限分别为0.1、0.5、0.1 ng/ml,线性范围可覆盖临床实际样本浓度分布;回收率分别为86.2%~93.8%、100.9%~103.9%和103.3%~107.1%;总不精密度分别为4.7%~7.4%、3.5%~4.6%和5.2%~10.9%;Aβ1-42有证参考物质的测定值均在允许不确定度的范围内;携带污染率均≤0.11%。此外,基于该LC-MS/MS法测定的脑脊液Aβ1-42和Aβ1-40的结果与基于INNOTEST ELISA方法和Lumipulse G及Roche Elecsys全自动生化分析仪检测的结果相关性较好,相关系数r为0.920~0.970,但测定值间存在明显不同。结论本研究建立了一种基于LC-MS/MS同时测定脑脊液Aβ1-42、Aβ1-40和Aβ1-38的检测方法,该方法准确、简便,可应用于临床检测。质谱法检测Aβ1-42和Aβ1-40,特别是Aβ1-42,与其他检测系统间的相关性较好但测定值存在显著差异,提示阿尔茨海默症经典标志物检测的一致化和标准化有待改进。Objective To establish and validate an LC-MS/MS method for simultaneous determination of Aβ1-42,Aβ1-40,and Aβ1-38 in cerebrospinal fluid.Additionally,the consistency between this method and three mainstream detection methods was evaluated.Methods This study involved method establishment,validation,and consistency evaluation.The N15 labeledβ-amyloid protein was used as the internal standard.Extraction was performed using Waters MCX 96-wells solid phase extraction plate,and the eluent was collected to QuanRecovery MaxPeak 700μl plate.At the positive ion mode,the multi-reaction ion monitoring mode based on electric spray ionization is chosen for the determination of CSF Aβ1-42,Aβ1-40,and Aβ1-38.Referring to the CLSI C62-A and EP-15A3 guidelines,the method is evaluated and verified,including quantitation of limit(LOQ),linearity,recovery,precision,and accuracy.In addition,a total of 57 clinical residual CSF samples were collected and the concentrations of Aβ1-42 and Aβ1-40 were determined based on manual INNOTEST ELISA assay and Lumipulse G and Roche Elecsys fully automated biochemical analyzers.The comparison analysis and deviation evaluation were conducted by passing-bablok and Bland Altman methods.Results The analysis time of this method is 8 min,and the LOQ of Aβ1-42,Aβ1-40 and Aβ1-38 is 0.1 ng/ml,0.5 ng/ml,and 0.1 ng/ml,respectively,and the linear range can meet the needs of clinical detection.Respectively,the recovery is 86.2%-93.8%,100.9%-103.9%and 103.3%-107.1%;the total imprecision is 4.7%-7.4%,3.5%-4.6%and 5.2%-10.9%.The measured values of Aβ1-42 certified reference materials are all within the allowable uncertainty requirements.Moreover,the carryover rate of three analytes was all≤0.11%.In addition,the correlations of Aβ1-42 and Aβ1-40 in CSF between this LC-MS/MS method and the INNOTEST ELISA method,Lumipulse G and Roche Elecsys fully automated biochemical analyzers were all deemed good,with correlation coefficient(r)ranging from 0.920 to 0.970.However,the measured values between the four

关 键 词：色谱法 液相 串联质谱法 Β-淀粉样蛋白 脑脊液 

分 类 号：O657.63[理学—分析化学] R446.1[理学—化学]