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 700 μl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。

abstractsObjective:To establish and validate an LC-MS/MS method for simultaneous determination of Aβ 1-42, Aβ 1-40, and Aβ 1-38 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 labeled β-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 700 μl plate. At the positive ion mode, the multi-reaction ion monitoring mode based on electric spray ionization is chosen for the determination of CSF Aβ 1-42, Aβ 1-40, and Aβ 1-38. 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 Aβ 1-42 and Aβ 1-40 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 Aβ 1-42, 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 Aβ 1-42 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 Aβ 1-42 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 methods were remarkably different.Conclusion:We established and validated a robust method based on LC-MS/MS technology for simultaneous determination of Aβ 1-42, Aβ 1-40, and Aβ 1-38 in CSF. The method is accurate, simple, and suitable for clinical measurements. However, despite good correlations, there were substantial differences in the measurement results of Aβ 1-42 and Aβ 1-40 among different analytical platforms, indicating the need for further promotion of harmonization and standardization processes for AD classic biomarkers.