Capillary Electrophoresis-Mass Spectrometry at Trial by Metabo-Ring: Effective Electrophoretic Mobility for Reproducible and Robust Compound Annotation

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (μeff) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The μeff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the μeff for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.毛细管区带电泳-质谱（Capillary zone electrophoresis-mass spectrometry, CE-MS）是一种成熟的分析工具，可实现（高度）极性与可电离化合物的高效轮廓分析。然而，与其他分离技术相比，CE-MS在代谢组学（metabolomics）中的应用仍较为匮乏，因为该分析方法仍被认为技术门槛较高、重现性欠佳，尤其在迁移时间方面。后者是实现代谢谱可靠比对，以及与高分辨质谱/质谱（high resolution MS/MS）互补的未知生物标志物鉴定的核心参数。本研究展示了一项代谢环试验（Metabo-ring trial）的结果，该试验覆盖两大洲的13家实验室，共涉及16台CE-MS分析平台。本试验旨在以有效电泳迁移率（effective electrophoretic mobility, μeff）作为核心参数，对比相对迁移时间（relative migration time, RMT）方法，评估CE-MS的重现性与鉴定能力。为此，研究团队制备了代表性的水相阳离子代谢物混合液、预处理人血浆，以及掺入相同代谢物混合液的尿液样本，并分发给所有参与实验室进行分析。针对每个样本中掺入的所有代谢物，均测定了其有效电泳迁移率。所有参与实验室均按照统一方案制备并使用背景电解质（background electrolyte, BGE）。其余所有参数（包括毛细管、接口、进样体积、电压梯度、温度、毛细管活化与冲洗流程等）均由各参与实验室自主确定。结果显示，尽管13家实验室的实验条件与分析平台存在显著异质性，但21种模型化合物中有20种的有效电泳迁移率重现性低于3.1%，而相对迁移时间的重现性仅为10.9%。总体而言，此项代谢环试验证实，CE-MS是一种适用于代谢组学研究的可行且重现性良好的分析方法。