de Souza Israel Donizeti, Queiroz Maria Eugênia Costa
Departamento de Química, Faculdade de Filosofia Ciências E Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901, Ribeirão Preto, São Paulo, Brazil.
Anal Bioanal Chem. 2023 Jul;415(18):4003-4021. doi: 10.1007/s00216-023-04631-9. Epub 2023 Mar 6.
Alzheimer's disease (AD), a neurological disorder, is a major public health concern and the most common form of dementia. Its typical symptoms include memory loss, confusion, changes in personality, and cognitive impairment, which result in patients gradually losing independence. Over the last decades, some studies have focused on searching for effective biomarkers as early diagnostic indicators of AD. Amyloid-β (Aβ) peptides have been consolidated as reliable AD biomarkers and have been incorporated into modern diagnostic research criteria. However, quantitative analysis of Aβ peptides in biological samples remains a challenge because both the sample and the physical-chemical properties of these peptides are complex. During clinical routine, Aβ peptides are measured in the cerebrospinal fluid by immunoassays, but the availability of a specific antibody is critical-in some cases, an antibody may not exist, or its specificity may be inadequate, leading to low sensitivity and false results. HPLC-MS/MS has been reported as a sensitive and selective method for determining different fragments of Aβ peptides in biological samples simultaneously. Developments in sample preparation techniques (preconcentration platforms) such as immunoprecipitation, 96-well plate SPME, online SPME, and fiber-in-tube SPME have enabled not only effective enrichment of Aβ peptides present at trace levels in biological samples, but also efficient exclusion of interferents from the sample matrix (sample cleanup). This high extraction efficiency has provided MS platforms with higher sensitivity. Recently, methods affording LLOQ values as low as 5 pg mL have been reported. Such low LLOQ values are adequate for quantifying Aβ peptides in complex matrixes including cerebrospinal fluid (CSF) and plasma samples. This review summarizes the advances in mass spectrometry (MS)-based methods for quantifying Aβ peptides and covers the period 1992-2022. Important considerations regarding the development of the HPLC-MS/MS method such as the sample preparation step, optimization of the HPLC-MS/MS parameters, and matrix effects are described. Clinical applications, difficulties related to analysis of plasma samples, and future trends of these MS/MS-based methods are also discussed.
阿尔茨海默病（AD）是一种神经疾病，是一个主要的公共卫生问题，也是最常见的痴呆形式。其典型症状包括记忆力减退、意识混乱、性格改变和认知障碍，这些症状会导致患者逐渐丧失独立生活能力。在过去几十年里，一些研究致力于寻找有效的生物标志物作为AD的早期诊断指标。淀粉样β（Aβ）肽已被确认为可靠的AD生物标志物，并已纳入现代诊断研究标准。然而，对生物样品中Aβ肽进行定量分析仍然是一项挑战，因为样品以及这些肽的物理化学性质都很复杂。在临床常规检测中，通过免疫测定法检测脑脊液中的Aβ肽，但特异性抗体的可用性至关重要——在某些情况下，可能不存在抗体，或者其特异性不足，导致灵敏度低和结果出现偏差。据报道，高效液相色谱-串联质谱法（HPLC-MS/MS）是一种用于同时测定生物样品中不同Aβ肽片段的灵敏且具选择性的方法。免疫沉淀、96孔板固相微萃取、在线固相微萃取和管内纤维固相微萃取等样品制备技术（预浓缩平台）的发展，不仅能够有效富集生物样品中痕量存在的Aβ肽，还能从样品基质中有效排除干扰物（样品净化）。这种高提取效率为质谱平台提供了更高的灵敏度。最近，已有报道称一些方法的最低定量限（LLOQ）值低至5 pg/mL。如此低的LLOQ值足以对包括脑脊液（CSF）和血浆样品在内的复杂基质中的Aβ肽进行定量。本综述总结了1992年至2022年期间基于质谱（MS）的Aβ肽定量方法的进展。描述了HPLC-MS/MS方法开发中的重要注意事项，如样品制备步骤、HPLC-MS/MS参数的优化以及基质效应。还讨论了这些基于MS/MS方法的临床应用、与血浆样品分析相关的困难以及未来趋势。
