Amyloid beta (Aβ) peptides in cerebrospinal fluid are extensively estimated for identification of Alzheimer’s disease (AD) as diagnostic biomarkers. Unfortunately, their pervasive application is hampered by interference from Aβ propensity of self-aggregation, nonspecifically bind to surfaces and matrix proteins, and by lack of quantitive standardization. Here we report on an alternative Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method for simultaneous measurement of human amyloid beta peptides Aβ1-38, Aβ1-40 and Aβ1-42 in cerebrospinal fluid (CSF) using micro-elution solid phase extraction (SPE). Samples were pre-processing by the mixed-mode micro-elution solid phase extraction and quantification was performed in the positive ion multiple reaction monitoring (MRM) mode using electrospray ionization. The stable-isotope labeled Aβ peptides ¹⁵N₅₁- Aβ1-38, ¹⁵N₅₃- Aβ1-40 and ¹⁵N₅₅- Aβ1-42 peptides were used as internal standards. And the artificial cerebrospinal fluid (ACSF) containing 5% rat plasma was used as a surrogate matrix for calibration curves. The quality control (QC) samples at 0.25, 2 and 15 ng/mL were prepared. A “linear” regression (1/x² weighting): y = ax + b was used to fit the calibration curves over the concentration range of 0.1–20 ng/mL for all three peptides. Coefficient of variation (CV) of intra-batch and inter-batch assays were all less than 6.44% for Aβ1-38, 6.75% for Aβ1-40 and 10.74% for Aβ1-42. The precision values for all QC samples of three analytes met the acceptance criteria. Extract recoveries of Aβ1-38, Aβ1-40 and Aβ1-42 were all greater than 70.78%, both in low and high QC samples. The stability assessments showed that QC samples at both low and high levels could be stable for at least 24 h at 4 °C, 4 h at room temperature and through three freeze-thaw cycles without sacrificing accuracy or precision. And no significant carryover effect was observed. This validated UHPLC/MS/MS method was successfully applied to the quantitation of Aβ peptides in real human CSF samples. Our work may provide a reference method for simultaneous quantitation of human Aβ1-38, Aβ1-40 and Aβ1-42 from CSF.

广泛评估了脑脊液中的淀粉样蛋白（Aβ）肽，以鉴定阿尔茨海默氏病（AD）作为诊断生物标志物。不幸的是，它们的普遍应用受到Aβ自聚集倾向的干扰，与表面和基质蛋白的非特异性结合以及缺乏定量标准化的阻碍。在这里，我们报告了另一种超高效液相色谱-串联质谱法（UHPLC–MS / MS），该方法同时使用以下方法测量脑脊髓液（CSF）中人淀粉样β肽Aβ1-38，Aβ1-40和Aβ1-42微洗脱固相萃取（SPE）。通过混合模式微洗脱固相萃取对样品进行预处理，并使用电喷雾电离以正离子多反应监测（MRM）模式进行定量。¹⁵ Ñ ₅₁ - Aβ1-38，¹⁵ Ñ ₅₃ - Aβ1-40和¹⁵ Ñ ₅₅ - Aβ1-42肽用作内标。含有5％大鼠血浆的人工脑脊液（ACSF）用作校正曲线的替代矩阵。制备了0.25、2和15 ng / mL的质量控制（QC）样品。“线性”回归（1 / x ²权重：y = ax + b用于拟合所有三种肽在0.1–20 ng / mL浓度范围内的校准曲线。批内和批间测定的变异系数（CV）分别小于Aβ1-38的6.44％，Aβ1-40的6.75％和Aβ1-42的10.74％。三种分析物的所有QC样品的精密度值均符合验收标准。在低和高QC样品中，Aβ1-38，Aβ1-40和Aβ1-42的提取物回收率均大于70.78％。稳定性评估表明，低水平和高水平的QC样品在4°C，24°C室温和经过三个冻融循环后至少可稳定24 h，而不会影响准确性或精密度。并没有观察到明显的残留效应。这种经过验证的UHPLC / MS / MS方法已成功应用于定量真实人CSF样品中的Aβ肽。我们的工作可能为同时定量脑脊液中人Aβ1-38，Aβ1-40和Aβ1-42提供参考方法。