Cardiovascular diseases (CVDs) represent a major concern in today's society, with more than 17.5 million deaths reported annually worldwide. Recently, five metabolites related to the gut metabolism of phospholipids were identified as promising predictive biomarker candidates for CVD. Validation of those biomarker candidates is crucial for applications to the clinic, showing the need for high-throughput analysis of large numbers of samples. These five compounds, trimethylamine N-oxide (TMAO), choline, betaine, l-carnitine, and deoxy-l-carnitine (4-trimethylammoniobutanoic acid), are highly polar compounds and show poor retention on conventional reversed phase chromatography, which can lead to strong matrix effects when using mass spectrometry detection, especially when high-throughput analysis approaches are used with limited separation of analytes from interferences. In order to reduce the potential matrix effects, we propose a novel fast parallel electromembrane extraction (Pa-EME) method for the analysis of these metabolites in plasma samples. The evaluation of Pa-EME parameters was performed using multi segment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Recoveries up to 100% were achieved, with variability as low as 2%. Overall, this study highlights the necessity of protein precipitation prior to EME for the extraction of highly polar compounds. The developed Pa-EME method was evaluated in terms of concentration range and response function, as well as matrix effects using fast-LC-MS/MS. Finally, the developed workflow was compared to conventional sample pre-treatment, i.e., protein precipitation using methanol, and fast-LC-MS/MS. Data show very strong correlations between both workflows, highlighting the great potential of Pa-EME for high-throughput biological applications.

中文翻译：

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高极性化合物的电膜萃取：血浆中心血管生物标志物的分析。

心血管疾病（CVD）是当今社会关注的主要问题，全世界每年报告的死亡人数超过1750万。最近，与磷脂的肠代谢有关的五种代谢物被鉴定为有前途的心血管预测生物标志物候选物。那些生物标志物候选物的验证对于临床应用至关重要，这表明需要对大量样品进行高通量分析。这五种化合物，即三甲胺N-氧化物（TMAO），胆碱，甜菜碱，左旋肉碱和脱氧左旋肉碱（4-三甲基氨丁酸），是高极性化合物，在常规反相色谱上的保留较差，这可能导致在使用质谱检测时产生强烈的基质效应，尤其是在使用高通量分析方法且分析物与干扰物分离程度有限的情况下。为了减少潜在的基质效应，我们提出了一种新颖的快速平行电膜萃取（Pa-EME）方法，用于分析血浆样品中的这些代谢产物。Pa-EME参数的评估是使用多段进样-毛细管电泳-质谱（MSI-CE-MS）进行的。回收率高达100％，变异性低至2％。总的来说，这项研究强调了在EME提取高极性化合物之前必须进行蛋白质沉淀的步骤。使用快速LC-MS / MS对开发的Pa-EME方法进行了浓度范围和响应函数以及基质效应方面的评估。最后，将开发的工作流程与常规样品预处理进行了比较，即使用甲醇和快速LC-MS / MS进行蛋白质沉淀。数据显示这两个工作流程之间具有很强的相关性，突出了Pa-EME在高通量生物应用中的巨大潜力。