Lin Ping-Ping, Chen Wei-Li, Yuan Fei, Sheng Lei, Wu Yu-Jia, Zhang Wei-Wei, Li Guo-Qing, Xu Hong-Rong, Li Xue-Ning
Department of Clinical Pharmacology, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
School of Pharmacy, Fudan University, Shanghai 201203, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Dec 1;1070:82-91. doi: 10.1016/j.jchromb.2017.10.047. Epub 2017 Oct 23.
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 N- Aβ1-38, N- Aβ1-40 and N- 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 15ng/mL were prepared. A "linear" regression (1/x weighting): y=ax+b was used to fit the calibration curves over the concentration range of 0.1-20ng/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 24h at 4°C, 4h 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.
脑脊液中的β淀粉样蛋白（Aβ）肽被广泛评估，以作为诊断生物标志物用于阿尔茨海默病（AD）的识别。不幸的是，其广泛应用受到Aβ自聚集倾向、与表面和基质蛋白非特异性结合的干扰，以及缺乏定量标准化的阻碍。在此，我们报告一种替代的超高效液相色谱 - 串联质谱（UHPLC-MS/MS）方法，该方法使用微洗脱固相萃取（SPE）同时测量脑脊液（CSF）中的人β淀粉样蛋白肽Aβ1-38、Aβ1-40和Aβ1-42。样品通过混合模式微洗脱固相萃取进行预处理，并使用电喷雾电离在正离子多反应监测（MRM）模式下进行定量。稳定同位素标记的Aβ肽N - Aβ1-38、N - Aβ1-40和N - Aβ1-42肽用作内标。含有5%大鼠血浆的人工脑脊液（ACSF）用作校准曲线的替代基质。制备了浓度为0.25、2和15ng/mL的质量控制（QC）样品。使用“线性”回归（1/x加权）：y = ax + b对所有三种肽在0.1 - 20ng/mL浓度范围内的校准曲线进行拟合。Aβ1-38的批内和批间测定变异系数（CV）均小于6.44%，Aβ1-40为6.75%，Aβ1-42为10.74%。三种分析物所有QC样品的精密度值均符合验收标准。低质量控制和高质量控制样品中Aβ1-38、Aβ1-40和Aβ1-42的萃取回收率均大于70.78%。稳定性评估表明，低水平和高水平的QC样品在4°C下至少可稳定24小时，在室温下可稳定4小时，并且经过三个冻融循环，而不会牺牲准确性或精密度。并且未观察到明显的残留效应。这种经过验证的UHPLC/MS/MS方法已成功应用于实际人脑脊液样品中Aβ肽的定量分析。我们的工作可能为同时定量脑脊液中的人Aβ1-38、Aβ1-40和Aβ1-42提供一种参考方法。
