College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China.
Key Laboratory of Marine Environment and Ecology, Ocean University of China, Ministry of Education, Qingdao 266100, China.
Toxins (Basel). 2022 Jun 2;14(6):387. doi: 10.3390/toxins14060387.
Neurotoxin β--methylamino-L-alanine (BMAA) is hypothesized as an important pathogenic factor for neurodegenerative diseases such as amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC). Comparative study on the accuracy of BMAA analyzed by the regular LC-MS/MS methods is still limited for different biological matrices. In this study, a free-BMAA sample of cyanobacterium and BMAA-containing positive samples of diatom, mussel, scallop, and oyster were extracted with varied extraction ratios (ER) ranging from 1:20 to 1:2000. These extracts were then purified by MCX cartridges. After SPE purification, these different biological samples were analyzed by two common LC-MS/MS analysis methods, a direct analysis without derivatization by a hydrophilic interaction liquid chromatography (HILIC)-MS/MS and pre-column 6-aminoquinolyl--hydroxysuccinimidyl carbamate (AQC) derivatization combined with a C18 column. The results suggested that the recoveries of BMAA spiked in the cyanobacterial sample were close to 100% in the total soluble form extracts with the ER of 1:100 (g/mL) and the precipitated bound form extracts with the ER of 1:500. The recommended ER for the precipitated bound form of BMAA in diatoms and the total soluble form of BMAA in mollusks are 1:500 and 1:50, respectively. The quantitative results determined by the AQC derivatization method were lower than those determined by the direct analysis of the HILIC method in diatom and mollusk samples. The results of the HILIC method without the derivatization process were closer to the true value of BMAA in cyanobacteria. This work contributes to the performance of the solid-phase extraction (SPE) purification protocol and the accuracy of BMAA analysis by LC-MS/MS in diverse biological samples.
神经毒素 β--甲基氨基-L-丙氨酸（BMAA）被假设为肌萎缩侧索硬化/帕金森病-痴呆症复合征（ALS-PDC）等神经退行性疾病的重要致病因素。用于不同生物基质的常规 LC-MS/MS 方法分析 BMAA 的准确性比较研究仍然有限。在这项研究中，使用从 1:20 到 1:2000 不等的不同提取比（ER）从蓝藻中提取游离 BMAA 样品和含有 BMAA 的硅藻、贻贝、扇贝和牡蛎阳性样品。然后，将这些提取物用 MCX 小柱纯化。SPE 纯化后，通过两种常用的 LC-MS/MS 分析方法，即亲水作用液相色谱（HILIC）-MS/MS 分析方法和无需衍生化的直接分析方法和预柱 6-氨基喹啉基--羟基琥珀酰亚胺基碳酸酯（AQC）衍生化与 C18 柱结合来分析这些不同的生物样品。结果表明，在总可溶形式提取物中，当 ER 为 1:100（g/mL）和沉淀结合形式提取物中，ER 为 1:500 时，添加到蓝藻样品中的 BMAA 的回收率接近 100%。推荐用于硅藻中沉淀结合形式的 BMAA 和贝类中总可溶形式的 BMAA 的 ER 分别为 1:500 和 1:50。在硅藻和贝类样品中，通过 AQC 衍生化法测定的定量结果低于通过 HILIC 法直接分析测定的结果。未经衍生化处理的 HILIC 方法的结果更接近蓝藻中 BMAA 的真实值。这项工作有助于固相萃取（SPE）净化方案的性能和 LC-MS/MS 在不同生物样品中分析 BMAA 的准确性。
