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Capillary electrophoresis–mass spectrometry (CE-MS) is a high efficiency separation platform for metabolomic studies that is ideal for the analysis of volume-restricted biological specimens. However, major technical hurdles prevent more widespread use of CE-MS technology, including poor method robustness that is supported by long-term validation studies. We outline efforts towards developing a more robust CE-MS method that offers significant improvements in sample throughput and data fidelity as required for large-scale clinical and epidemiological studies. In this chapter, multisegment injection (MSI)-CE-MS is demonstrated as a multiplexed separation platform for high throughput metabolite profiling in various biological samples with quality assurance. Careful attention to capillary preparation while using standardized operating protocols is critical for successful operations, including rigorous inter-method comparisons and batch-correction algorithms to adjust for system drift. MSI-CE-MS offers a versatile platform using serial injection formats for temporal encoding of mass spectral data, which allows for unambiguous identification and reliable quantification of both polar and non-polar ionic metabolites of clinical significance. Recent data workflows for accelerating biomarker discovery will be discussed, including new advances in population-based screening for early detection of in-born errors of metabolism, validation of lifestyle intervention studies that promote human health and comprehensive drug surveillance given the worldwide opioid crisis.

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