Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry: A High-Throughput Platform for Metabolomics with High Data Fidelity

A major constraint in large-scale mass spectrometry (MS)-based metabolomic initiatives is the low sample throughput associated with chromatographic or electrophoretic separations. Herein, we introduce multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) as a multiplexed separation platform for metabolomics that increases sample throughput up to one order of magnitude while improving overall data fidelity. We demonstrate that serial injection of seven or more discrete sample segments can be performed within a single capillary while maintaining isomeric resolution without ion suppression when using a high mass resolution time-of-flight-MS. Customized injection sequences can be devised to encode information temporally within a separation based on signal pattern recognition, which enables unambiguous identification and accurate quantification (mean bias <10%) of polar metabolites in human plasma with good reproducibility (CV ≈ 10%, n = 70). False discoveries are avoided when using a rigorous dilution trend filter to reject spurious signals and background peaks that comprise the majority (≈65%) of total detectable features. MSI-CE-MS offers an unprecedented approach to enhance sample throughput analogous to direct infusion-MS (≈3 min/sample) while delivering far greater selectivity, quantitative performance, and data quality since the same ion from different samples migrates into the ion source within a short time interval (≈2-6 min). These distinct analytical and bioinformatic merits are achieved without column switching, isotopic labeling, hardware modifications, or costly infrastructure investments.